Helicobacter pylori and Gastric Cancer (Current Topics in Microbiology and Immunology, 444) 3031473302, 9783031473302

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Current Topics in Microbiology and Immunology

Steffen Backert   Editor

Helicobacter pylori and Gastric Cancer

Current Topics in Microbiology and Immunology Volume 444

Series Editors Rafi Ahmed, School of Medicine, Rollins Research Center, Emory University, Atlanta, GA, USA Shizuo Akira, Immunology Frontier Research Center, Osaka University, Suita, Osaka, Japan Arturo Casadevall, W. Harry Feinstone Department of Molecular Microbiology & Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA Jorge E. Galan, Boyer Center for Molecular Medicine, School of Medicine, Yale University, New Haven, CT, USA Adolfo Garcia-Sastre, Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA Rino Rappuoli, GSK Vaccines, Siena, Italy Marcio L. Rodrigues, Oswaldo Cruz Foundation, Carlos Chagas Institute, Curitiba, Brazil Olaf Weber, Universitätsklinikum Bonn, University of Bonn, Bonn, Germany

The reviews series Current Topics in Microbiology and Immunology publishes cutting-edge syntheses of the latest advances in molecular immunology, medical microbiology, virology and biotechnology. Each volume of the series highlights a selected timely topic, is curated by a dedicated expert in the respective field, and contains a wealth of information on the featured subject by combining fundamental knowledge with latest research results in a unique manner.

Steffen Backert Editor

Helicobacter pylori and Gastric Cancer

Editor Steffen Backert Department of Biology Division of Microbiology University of Erlangen-Nuremberg Erlangen, Bayern, Germany

ISSN 0070-217X ISSN 2196-9965 (electronic) Current Topics in Microbiology and Immunology ISBN 978-3-031-47330-2 ISBN 978-3-031-47331-9 (eBook) https://doi.org/10.1007/978-3-031-47331-9 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland Paper in this product is recyclable.

Foreword

The story of how Helicobacter pylori is related to gastric cancer evolved in its own particular way, at the intersection of biology and clinical medicine. Although pathologists found in the late 19th century that there were microbes present in everyone’s stomach, no one could isolate those organisms in pure culture, so they were ignored and ultimately forgotten for long time. A dogma developed that because the stomach was so acidic, microbes could not live there. That was in the era before medical scientists realized that microbes can live in geothermal vents at high temperatures, in volcanoes, or in salt flats with high osmolarity, and even in rock. Thus, the discovery in 1982 of the organisms we now call Helicobacter pylori came as a great surprise! When Robin Warren identified microbes in the stomach (again) in the 1970s, and he and Barry Marshall first isolated them in pure culture in 1982, a new age began (Warren and Marshall 1983; Marshall and Warren 1984). Since, unlike in the 19th century, there now were substantial numbers of people worldwide who carried or did not carry the organisms, medical scientists could determine their association with host conditions. Isolating the organisms allowed scientists to classify them, and their name sequentially evolved from Gastric Campylobacter-like organisms (GCLO) to Helicobacter pylori, when it became clear that the organisms were sufficiently distinct from their intestinal cousins to create a new genus (Goodwin et al. 1989). Importantly, other various mammals carry other Helicobacter species, consistent with the concept of a conserved group of organisms colonizing parallel gastric ecological niches (Haesebrouk et al. 2009). Although the mammalian stomach can produce an extremely harsh acidic environment, over the long span of bacterial life on earth, organisms have evolved to take advantage of the resources present in myriad niches, and in these cases persist over their host’s lifetime. An important point relevant to any discussion of H. pylori is that these organisms have had a long association with humans. Multiple independent studies provide evidence that humans have been carrying H. pylori in their stomach since pre-history (Ghose et al. 2002; Falush et al. 2003; Linz et al. 2007). Recent studies based on mathematical inferences from genomic analyses have pushed the clock of our association back to 300,000 years ago (Tourrette et al. 2023). The conserved relationships between helicobacters and acidic stomachs have led to postulates that our ancestors v

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carried these gastric organisms since before they were fully human, millions of years ago. The concept of an ancient organism, nearly universal in their hosts, with little or no early life fitness cost is most consistent with bacterial commensalism (Blaser 2006). By that argument, H. pylori has been the dominant resident of the human gastric microbiome, in parallel to how a distinct group of organisms, the lactobacilli, dominate the vagina of reproductive-age women. These oligo-colonization models contrast with the highly diverse microbiota colonizing the human colon and the human skin, for example. However, the incontrovertible finding, unknowingly first made by Marshall and Warren, is that H. pylori no longer is universal, and it is becoming increasingly less common around the world (Kosunen et al. 1997). While this process appears to have begun earlier in some places (e.g. the high income industrialized countries) than others, it is happening worldwide; H. pylori is progressively disappearing from the human stomach (Blaser and Falkow 2009). This is a major ecological shift in human biology, whose full consequences we still do not understand. But since there now are large numbers of people with or without the organism, it becomes possible to untangle the consequences of its presence (and its absence). If everyone smoked, we could never find the association with lung cancer; to uncover the real relationships, it is necessary to compare cancer rates in smokers and nonsmokers. So it is with H. pylori and clinical manifestations. Following its discovery in 1982, investigation of the clinical roles of H. pylori advanced quickly. First came the association of the presence of the organisms with the infiltration of the gastric mucosa with immune and inflammatory cells, termed by pathologists as “gastritis” (Warren and Marshall 1983). This is a histological finding that, by and large, is not associated with symptoms per se. Since H. pylori is the ancestral and formerly ubiquitous organism colonizing the human stomach, it also may be termed “the physiological response to a dominant microbiota colonizer.” What we call it must ultimately be based on the consequences of its presence in that niche; the present book (“Helicobacter pylori and gastric cancer”) highlights its clearly pathogenic role causing gastric cancer. The next important association of H. pylori was with peptic ulcer disease (Marshall and Warren 1984). Prospective studies showed that those who had the organism were significantly more likely to develop ulcers in the subsequent years (Nomura et al. 1994). Treatment studies showed that among those who had ulcers, taking an antibiotic-based regimen that removed H. pylori changed the natural history of the ulcer, essentially curing it (Hentschel et al. 1993). This was a major finding that electrified the world and changed the practice of medicine. Next, and of central importance here, came the association of H. pylori with adenocarcinoma of the stomach. By the early 1990s, the strong association with cancer was clear (Forman et al. 1991; Parsonnet et al. 1991; Nomura et al. 1991; Talley et al. 1991), and it was exhilarating to find evidence of such a strong relationship that could one day be actionable. Along with age, H. pylori was the leading risk factor for the development of these important cancers and was classified as a type 1 carcinogen (IARC 1994). Just as the prolonged carriage of Hepatitis B virus or Human Papilloma Virus (HPV) can lead to hepatocellular cancer, and cervical cancer in a proportion of

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their hosts, respectively, so can H. pylori, especially specific genotypes (e.g. cagA+ ) lead to gastric cancer in a decades-long process (Blaser et al. 1995; Kuipers et al. 1995; Parsonnet et al. 1997). The chapters in this book are the continuation from that frontier. Professor Steffen Backert, the Editor, has invited an eminent group of scholars to report on advances in our understanding of how H. pylori interacts with host cells and tissues and with other microbiota, and how the tumors might be treated. These chapters dissect in detail the mechanisms by which H. pylori can lead to gastric cancer. In its breadth, depth and precision, the work is elegant, using many sophisticated tools in biomedicine to address the scientific questions. These studies reflect the state-of-the-art with findings that in many ways were unimaginable 30 years ago. These reflect the power of scientific investigation. Yet despite these advances, several important mysteries remain unsolved to this day, which indicate the need to keep probing. First, only a fraction of people with long-term H. pylori colonization develop gastric cancer. Understanding the underlying biology that marks a minority for a terrible malignancy is a major scientific question, whose solution can have profound clinical implications related to defining risks and prevention. Exploring developmental relationships, even decades prior to the malignancy might have value (Blaser et al. 2007). Second, although gastric cancer usually takes decades to develop with risk strongly age-related, there are some individuals who develop it decades earlier than most (Bergquist et al. 2019). Understanding that particular host-microbe biology is especially important because it is increasing in the population (Kehm et al. 2019). Third, there is wide geographic variation in gastric cancer rates that is not explained by differences in H. pylori prevalence rates (Goshal et al. 2010). As above, the presence of H. pylori is not sufficient for cancer development; there must be important co-factors that modulate risk that must be investigated. Understanding these determinants can also help in defining risk and leading to preventive and therapeutic strategies. Fourth, there are important sex differences in gastric cancer incidence. Although males and females in most populations carry the organism at similar rates, overall gastric cancer rates have historically been much higher in males. It is not so simple because in males, gastric cancer rates gradually accelerate with age, but in women, the rates are relatively flat until menopause and then increase in parallel to men; the incidence curve is the same as in men but is shifted to the right by about 25–30 years (Sipponen et al. 1998). Just as reproductive age protects women against atherosclerosis, so it does against gastric cancer (until recently), but we do not understand the mechanisms underlying these differences. Fifth, the risk of gastric cancer, duodenal ulcer, and gastric ulcer all are promoted by the presence of H. pylori (Hansson et al. 1996). Yet compared to people without the organism, those with gastric ulcer have about twice the rate of gastric cancer, and those with duodenal ulcer have about half the rate. These epidemiologic patterns must reflect important underlying biological differences that are not yet understood.

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Solving any of these five major problems would be a great advance in the field. They are most worthy of the deep investigative efforts reflected in this book; these may indeed provide the pathways and mechanisms that lead to their solution. In closing, gastric cancer caused by H. pylori is deeply important, because of the significance of the disease, and for what we are learning about host-microbial interactions, oncogenesis, and immunobiology. Yet, this still is only part the story. Just as investigation has provided evidence about the injuries that H. pylori can cause, a parallel body of work has identified the injuries that occur when H. pylori is absent, now increasingly prevalent. We also need to explore the ways in which H. pylori protects against diseases of the esophagus, up to and including adenocarcinomas centered at the gastro-esophageal junction (Corley et al 2008; Rubinstein et al. 2014; Chow et al 1998; Whiteman et al. 2010), and against childhood asthma and allergic diseases (Reibman et al 2008; Chen et al. 2008; Zuo et al. 2021; Arnold et al. 2011), and new forms of gastric cancer that are arising in populations that are becoming free of H. pylori (Anderson et al. 2010; Anderson et al. 2018; Blaser and Chen 2018; Song et al 2021). This half of the mechanistic work remains to be done. But the pathways and relationships discovered about how H. pylori interacts with host epithelial and immune cells to promote gastric cancer may also be useful in understanding how it protects against other diseases. This is yet another challenge for the scientists who have contributed to this encyclopedic book about gastric cancer. I wish them well! Martin J. Blaser Center for Advanced Biotechnology and Medicine Rutgers University Piscataway, NY, USA [email protected]

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Kuipers EJ, Pérez-Pérez GI, Meuwissen SGM, Blaser MJ. (1995) Helicobacter pylori and atrophic gastritis: importance of the cagA status. J Natl Cancer Inst 87(23):1777–1780. https://doi.org/ 10.1093/jnci/87.23.1777 Linz B, Balloux F, Moodley Y, Manica A, Liu H, Roumagnac P, Falush D, Stamer C, Prugnolle F, van der Merwe SW, Yamaoka Y, Graham DY, Perez-Trallero E, Wadstrom T, Suerbaum S, Achtman M. (2007) An African origin for the intimate association between humans and Helicobacter pylori. Nature 445(7130):915–8. https://doi.org/10.1038/nature05562 Marshall BJ, Warren JR. (1984) Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet 323(8390):1311–5. https://doi.org/10.1016/S0140-6736(84)918 16-6 Nomura A, Stemmerman GN, Chyou P-H, Kato I, Pérez-Pérez GI, Blaser MJ. (1991) Helicobacter pylori infection and gastric carcinoma in a population of Japanese-Americans in Hawaii. N Engl J Med 325(16):1132–1136. https://doi.org/10.1056/NEJM199110173251604 Nomura A, Stemmerman GN, Chyou P-H, Pérez-Pérez GI, Blaser MJ. (1994) Helicobacter pylori infection and the risk for duodenal and gastric ulceration. Ann Intern Med 120(12):977–81. https://doi.org/10.7326/0003-4819-120-12-199406150-00001 Paragomi P, Dabo B, Pelucchi C, Bonzi R, Bako AT, Sanusi NM, Nguyen QH, Zhang ZF, Palli D, Ferraroni M, Vu KT, Yu GP, Turati F, Zaridze D, Maximovitch D, Hu J, Mu L, Boccia S, Pastorino R, Tsugane S, Hidaka A, Kurtz RC, Lagiou A, Lagiou P, Camargo MC, Curado MP, Lunet N, Vioque J, Boffetta P, Negri E, La Vecchia C, Luu HN. (2022) The association between peptic ulcer disease and gastric cancer: results from the Stomach Cancer Pooling (StoP) Project Consortium. Cancers (Basel) 14(19):4905. https://doi.org/10.3390/cancers14194905. Parsonnet J, Friedman GD, Orentreich N, Vogelman H. (1997) Risk for gastric cancer in people with CagA positive or CagA negative Helicobacter pylori infection. Gut. 40(3):297–301. https://doi. org/10.1136/gut.40.3.297. Parsonnet J, Friedman GD, Vandersteen DP, Chang Y, Vogelman JH, Orentreich N, Sibley RK. (1991) Helicobacter pylori infection and the risk of gastric carcinoma. N Engl J Med 325(16):1127–31. https://doi.org/10.1056/NEJM199110173251603 Reibman J, Marmor M, Filner J, Fernandez-Beros ME, Rogers L, Pérez-Pérez GI, Blaser MJ. (2008) Asthma is inversely associated with Helicobacter pylori status in an urban population. PLoS ONE 3(12):e4060. https://doi.org/10.1371/journal.pone.0004060 Rubenstein JH, Inadomi JM, Scheiman J, Schoenfeld P, Appelman H, Zhang M, Metko V, Kao JY. (2014) Association between Helicobacter pylori and Barrett’s esophagus, erosive esophagitis, and gastroesophageal reflux symptoms. Clin Gastroenterol Hepatol 12(2):239–45. https://doi. org/10.1016/j.cgh.2013.08.029. Schistosomes, liver flukes and Helicobacter pylori IARC onographs on the evaluation of carcinogenic risks to humans. (1994) IARC Monogr Eval Carcinog Risk Hum 61; 1–241. Schistosomes, Liver Flukes and Helicobacter pylori. IARC Monographs on the Evaluation of Sipponen P, Hyvarinen H, Seppala K, Blaser MJ. (1998) Pathogenesis of the transformation from gastritis to malignancy. Aliment Pharmacol Ther 12 (Supplement) 1: 61–72. https://doi.org/10. 1111/j.1365-2036.1998.00005.x Song M, Camargo MC, Katki HA, Weinstein SJ, Männistö, Albanes D, Surcel H-M, Rabkin CS. (2022) Association of antiparietal cell and anti-intrinsic factor antibodies with risk of gastric cancer. JAMA Oncol 8(2):268–274. https://doi.org/10.1001/jamaoncol.2021.5395 Talley NJ, Zinsmeister AR, DiMagno EP, Weaver A, Carpenter HA, Pérez-Pérez GI, Blaser MJ. (1991) Gastric adenocarcinoma and Helicobacter pylori infection. J Natl Cancer Inst 83(23):1734–1739. https://doi.org/10.1093/jnci/83.23.1734 Tourrette E, Torres RC, Svensson SL, Matsumoto T, Miftahussurur M, Fauzia KA, Alfaray RI, Vilaichone R-K, Tuan VP. HeliobacterGenomicsConsortium, Wang D, Yadegar A, Olsson LM, Zhou Z, Yamaoka Y, Thorell K, Falush D. (2023) An ancient ecospecies of Helicobacter pylori found in indigenous populations and animal adapted lineages. bioRxiv https://doi.org/10.1101/ 2023.04.28.538659v3 Warren JR, Marshall B. (1983) Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet 1(8336):1273–5. https://doi.org/10.1016/S0140-6736(83)92719-8

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Contents

Gastric Stem Cell Biology and Helicobacter pylori Infection . . . . . . . . . . . . Jonas Wizenty and Michael Sigal Clinical Pathogenesis, Molecular Mechanisms of Gastric Cancer Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lydia E. Wroblewski and Richard M. Peek Jr Mitochondrial Function in Health and Disease: Responses to Helicobacter pylori Metabolism and Impact in Gastric Cancer Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Javier Torres and Eliette Touati Immune Biology and Persistence of Helicobacter pylori in Gastric Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sonja Fuchs, Ruolan Gong, Markus Gerhard, and Raquel Mejías-Luque

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Pathogenomics of Helicobacter pylori . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 Yoshio Yamaoka, Batsaikhan Saruuljavkhlan, Ricky Indra Alfaray, and Bodo Linz Gastric Cancer: The Microbiome Beyond Helicobacter pylori . . . . . . . . . . 157 Melissa Mendes-Rocha, Joana Pereira-Marques, Rui M. Ferreira, and Ceu Figueiredo Helicobacter pylori-Induced Host Cell DNA Damage and Genetics of Gastric Cancer Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 Steffen Backert, Bodo Linz, and Nicole Tegtmeyer Gastric Epithelial Barrier Disruption, Inflammation and Oncogenic Signal Transduction by Helicobacter pylori . . . . . . . . . . . . 207 Michael Naumann, Lorena Ferino, Irshad Sharafutdinov, and Steffen Backert

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Impact of the Helicobacter pylori Oncoprotein CagA in Gastric Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239 Masanori Hatakeyama Bacterial Proteases in Helicobacter pylori Infections and Gastric Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259 Silja Wessler and Gernot Posselt Clinical Management of Gastric Cancer Treatment Regimens . . . . . . . . . 279 Juliette Boilève, Yann Touchefeu, and Tamara Matysiak-Budnik

Abbreviations

α-KG ~P ΔΨ m 14-3-3 4E-BP1 8-nitro-G 8-OHdG 8-oxo-dGTP 8-oxo-G A20 Abl ABO ACE ACLY ACSS1 ADAM10 ADP ADP-heptose AGC1/2 AID AIM2 AJ AKAP AKT/PKB ALDH1 AlpA AlpB ALPK1 ALR AmiE AmiF

α–Ketoglutarate Phosphate group Transmembrane potential Phospho-serine/phospho-threonine binding protein Eukaryotic translation initiation factor 4E-binding protein 1 8-nitroguanine 8-oxo-7, 8-dihydro-2’-deoxyguanosine 8-oxo-7, 8-dihydro-2’-deoxyguanosine-5’-Triphosphate 7, 8-dihydro-8-oxo-2’-deoxyguanine Deubiquitinylase Abelson kinase Blood group antigens Angiotensin-converting enzyme ATP-citrate lyase Acetyl-CoA synthetase short chain family member 1 A Disintegrin and Metalloproteinase 10 Adenosine diphosphate ADP-glycero-β-D-manno-heptose Aspartate-glutamate carrier 1/2 Activation-induced cytidine deaminase Absent in melanoma 2 Adherens junction A-kinase-anchoring protein Protein kinase B Aldehyde dehydrogenase 1 Adherence-associated lipoprotein A Adherence-associated lipoprotein B Alpha-protein kinase 1 AIM2-like receptor Amidase Formidase xv

xvi

AMP AMPK AP-1 Apaf-1 APC APCs APE1 aPKC APOBEC AQP5 ARF ARF-BP1 Arg2 ARHGAP6 ARHGAP26 ARID1A ASC ASK1 ASPP2 ATF2 ATF4 ATM ATP ATR ATRIP AUC B2M BabA BabB BAK Barx1 BATF3 BAX BCL2 BCL2A1 BE BER BHLHA15 BIRC2 BIRC3 BMDC BMI1 BMP BRCA1

Abbreviations

Antimicrobial peptide 5’ Adenosine monophosphate-activated protein kinase Activator protein-1 Apoptotic protease activating factor 1 Adenomatous polyposis coli Antigen-presenting cells Apurinic/apyrimidinic endonuclease 1 Atypical protein kinase C Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like Aquaporin 5 ADP-ribosylation factor Alternative reading frame-binding protein 1 Arginase II Gene name for: Rho GTPase activating protein 6 Gene name for: Rho GTPase activating protein 26 AT-rich interactive domain-containing protein 1A Adult stem cell Apoptosis signal-regulating kinase 1 Apoptosis-stimulating protein of p53 2 Activating transcription factor 2 Activating transcription factor 4 Ataxia telangiectasia mutated kinase Adenosine triphosphate ATM and Rad3 related kinase ATR-interacting protein Area under the curve Beta-2-microglobulin Blood group antigen binding adhesin A Blood group antigen binding adhesin B Bcl-2 homologous antagonist BarH-like homeobox 1 Basic leucine zipper ATF-like transcription factor 3 Bcl-2-associated X protein B-cell lymphoma 2 B-cell lymphoma 2-related protein A1 Barrett’s esophagus Base excision repair Basic helix-loop-helix family member a15 Baculoviral IAP repeat containing 2 Baculoviral IAP repeat containing 3 Bone marrow derived cell B lymphoma Mo-MLV insertion region 1 homolog Bone morphogenic protein Breast Cancer 1

Abbreviations

BRCA2 BrdU Bregs C57BL/6 c-Abl CAF cag cagA cagL cagPAI cagT4SS cagY CAMP cAMP Cas9 CASP-1 CCKBR CCKR2 CCL/CXCL CCL20 CCND1 CCNE1 CD Cdc42 Cdc6 CDH1 CDK1 CDKN Cdx1 Cdx2 CEA CEACAM c-Fos CFU CGD CGRP CGT CHK2 CHRDL1 CI c-IAP1 c-IAP2 CIITA CIMP CIN

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Breast Cancer 2 Bromodeoxyuridine Regulatory B cells C57 black six inbred laboratory mouse strain Cellular Abelson kinase Cancer-associated fibroblast Cytotoxin-associated gene Cytotoxin-associated gene A Cytotoxin-associated gene L cag pathogenicity island cag type IV secretion system Cytotoxin-associated gene Y Cationic antimicrobial peptides Cyclic adenosine monophosphate CRISPR associated protein 9 Caspase-1 Cholecystokinin B receptor (also called CCK2 ) Cholecystokinin 2 receptor Chemokine ligand Chemokine ligand 20 Gene name for: Cyclin D1 Gene name for: Cyclin E1 Cluster of differentiation Cell division cycle 42 Cell division control protein 6 Gene name for: E-cadherin Cyclin-dependent kinase 1 Cyclin-dependent kinase inhibitor gene Caudal type homeobox 1 Caudal type homeobox 2 Carcinoembryonic antigen Carcinoembryonic antigen-related cell adhesion molecule Proto-oncogene (part of AP-1 transcription factor) Colony forming unit Chronic granulomatous disease Calcitonin gene-related peptide Cholesterol glycosyl-transferase Checkpoint kinase 2 Chordin-like 1 Confidence interval Cellular inhibitor of apoptosis protein-1 Cellular inhibitor of apoptosis protein-2 Class II major histocompatibility complex transactivator CpG island methylator phenotype Chromosomal instability

xviii

c-Jun CK1 CLD18 Clp CLR CM CME c-Met CMV CMW Cox1 Cox2 CpG CREB CRISPR Crk CRPIA CS CSC CSE CSF/MCSF CSFR C-SH2 Csk CSMD CSN1 c-Src CT CTLA4 CTN CtsC CXCL1 CXCL2 CXCL8 DAMPs DC DC-SIGN DDR DFMO DKK DLBCL DLL1 DNA

Abbreviations

Proto-oncogene (part of AP-1 transcription factor) Casein kinase 1 Claudin 18 Caseinolytic protease C-type lectin receptor CagA multimerization sequence East Asian CagA-specific CM motif Hepatocyte growth factor receptor Cytomegalovirus Western CagA-specific CM motif Cyclooxygenase 1 Cyclooxygenase 2 Cytosine-guanine repeats cAMP responsive element binding protein Clustered regularly interspaced short palindromic repeats Chicken tumor virus Regulator of Kinase Conserved repeat responsible for phosphorylation-independent activity Citrate synthase Cancer stem cells Cystathionine γ lyase Colony-stimulating factor Colony-stimulating factor receptor C-terminal SH2 domain of SHP2 Carboxy-terminal Src kinase CUB and sushi multiple domains Constitutive photomorphogenesis 9 signalosome subunit 1 Cellular Sarcoma kinase Computerized tomographies Cytotoxic T lymphocyte associated antigen 4 Catenin Cathepsin C C-X-C motif chemokine ligand 1 C-X-C motif chemokine ligand 2 C-X-C motif chemokine ligand 8 (also called IL-8) Damage-associated molecular patterns Dendritic cell Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin DNA damage response Difluoromethylornithine Dickkopf 1 Diffuse large B cell lymphoma Delta-like canonical Notch ligand 1 Deoxyribonucleic acid

Abbreviations

DSB dsDNA DU DupA EAC EBER EBNA EBNA-LP EBV ECL ECM eEF2 EGF EGFR EMT EPIYA ER ERBB2(HER2) ERCC ERK ERK1/2 ESC ETC FAK FAP FasL FBXO24 FDA FGC FGF-10 FGFR2 Fic FlaA FOXO1 FOXO3 FoxP3 Fra-1 Fur GAP GATA6 gB GC GDH GEC GEF

xix

DNA double-strand break Double-stranded DNA Duodenal ulcer Duodenal ulcer promoting gene A Esophageal adenocarcinoma EBV encoded small RNA EBV nuclear antigens EBNA-leader protein Epstein-Barr virus Enterochromaffin-like Extracellular matrix Eukaryotic elongation factor 2 Epidermal growth factor Epidermal growth factor receptor Epithelial mesenchymal transition Glu-Pro-Ile-Tyr-Ala sequence motif Endoplasmic reticulum Erb-b2 receptor tyrosine kinase 2 Excision repair cross-complementing gene Extracellular signal-regulated kinase Extracellular signal-regulated kinase 1/2 Embryonic stem cell Electron transport chain Focal adhesion kinase Familial adenomatous polyposis Fas ligand F-box protein 24 Food and Drug Administration Familial gastric cancer Fibroblast growth factor-10 Fibroblast growth factor receptor 2 Filamentation-induced by cAMP Flagellin A Forkhead Box-O-1 Forkhead Box-O-3 Forkhead box P3 Fos-related antigen 1 Ferric uptake regulator GTPase-activating protein GATA binding protein 6 EBV glycoprotein B Gastric cancer Glutamate dehydrogenase Gastric epithelial cell Guanine exchange factor

xx

GEJC GERD GES-1 GF GGT gH GI GIF GIN GIST GITR GKN3 gL GLOBOCAN GLS GM-CSF GMDS GOT2 GPCR Grb2 GREM1 Gro-α GS GSH GSII GSK GSK-3β GWAS GyrA GyrB H. pylori H2AX H2 RA H2 S hBD hBD1 hBD2 hBD3 HBP HBV HDGC HDGF HER2/neu HGF HHI

Abbreviations

Gastroesophageal junction carcinomas Gastro-esophageal reflux disease Gastric epithelial cell line Germ-free Gamma-glutamyl transferase EBV glycoprotein H Gastrointestinal Gastric intrinsic factor Gastric intraepithelial neoplasia Gastrointestinal stromal tumors Glucocorticoid-induced TNFR-related protein Gastrokine-3 EBV glycoprotein L Global Cancer Statistics Glutaminase Granulocyte macrophage-colony stimulating factor GDP-mannose 4,6-dehydratase Glutamic-oxaloacetic transaminase 2 G protein-coupled receptor Growth factor receptor-bound protein 2 Gremlin 1 Growth-regulated protein alpha Glutamine synthetase Glutathione (reduced form) Griffonia simplicifolia lectin GS-II Glycogen synthase kinase Glycogen synthase kinase 3 beta Genome wide associated study Subunit A of DNA gyrase Subunit B of DNA gyrase Helicobacter pylori Histone H2A variant X H2 -receptor antagonist Hydrogen sulfide Human beta-defensin Human beta-defensin 1 Human beta-defensin 2 Human beta-defensin 3 Heptose-1,7-bisphosphate Hepatitis B virus Hereditary diffuse gastric cancer Hepatoma-derived growth factor Human epidermal growth factor receptor 2 Human gastric fibroblast cell line H. pylori HtrA inhibitor

Abbreviations

HIF-1 HIV HLA HLA-B HLA-II HMD2 HMG HNF4α HNPCC Hop HopP HopQ HopS HopZ Hor HPV HR HTLV-1 HtrA HU Hupki IAP IARC IBD ICPi ID IDO IFNGR1 IFN-U IgA IGFBP7 IGFIIR IGHV IHF IKKα IKKβ IL IL-1 IL-17A IL-1RA IL-1RI IL-1β IL-6 IL-8 ILCs

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Hypoxia-inducible factor-1 Human immunodeficiency virus Human leukocyte antigen class II Histocompatibility complex class I Human leukocyte antigen II Human double minute 2 Hydroxymethylglutaryl Hepatocyte nuclear factor alpha Hereditary non-polyposis colorectal cancer Helicobacter Outer Membrane protein Helicobacter Outer Membrane protein P Helicobacter Outer Membrane protein Q Helicobacter Outer Membrane protein S Helicobacter Outer Membrane protein Z Hop-related proteins Human papillomavirus Homologous recombination Human T-cell lymphotropic virus type 1 High temperature requirement A Histone-like protein Human TP53 knock-in Inhibitor of apoptosis protein International agency for research on cancer Inflammatory bowel disease Immune checkpoint inhibitors Small insertions and deletions Indoleamine 2, 3- dioxygenase Interferon-U receptor 1 Interferon-U Immunoglobulin A Insulin growth factor binding protein 7 Insulin-like growth factor II receptor Immunoglobulin heavy chain variable region Integration host factor IκB kinase α IκB kinase β Interleukin Interleukin-1 Interleukin-17 receptor A Interleukin-1 receptor antagonist Interleukin-1 receptor type I Interleukin-1β Interleukin-6 Interleukin-8 Innate lymphoid cells

xxii

IM IMC INF-γ iNOS INS-GAS INSR IP iP IP3 IPD iPSC IQGAP3 IRAK IRF ISR ITLN1 iTRAQ ITS IκBα JAK JAM-A JCV JNK JUP KCNQ1 KLF Kras LBP Leb LEfSe LES LFA-1 Lgr4 Lgr5 LL37 LMP LPS LRIG1 Lrp LRR LytM M1 m1/m2 M2 m4C

Abbreviations

Intestinal metaplasia Inner membrane complex Interferon gamma Inducible nitric oxide synthase Insulin-gastrin Insulin receptor Immunoprecipitation Inorganic phosphate Inositol triphosphate Interpulse duration Induced pluripotent stem cells IQ motif-containing GTPase-activating protein 3 IL-1 receptor-associated kinase Interferon-regulatory factor Integrated stress response Intelectin 1 Isobaric tags for relative and absolute quantitation Internal transcribed spacer Inhibitor of NF-κB alpha Janus kinase Junctional adhesion molecule A John Cunningham virus c-Jun N-terminal kinase Junction plakoglobin Potassium voltage-gated channel subfamily Q member 1 Krüppel-like factor Kirsten rat sarcoma oncogene Lipopolysaccharide binding protein Lewis b antigen Linear discriminant analysis Effect Size Lower esophageal sphincter Lymphocyte function-associated antigen 1 Leucine-rich-repeat-containing G-protein coupled receptor 4 Leucine-rich-repeat-containing G-protein coupled receptor 5 37-residue amphipathic α-helical cathelicidin Latent membrane protein Lipopolysaccharide Leucine-rich repeats and immunoglobulin-like domains 1 Global regulatory protein Leucine-rich repeat Lysostaphin/peptidase M23 domain Type 1 macrophage 2 allelic forms of the vacA gene Type 2 macropahge N4-methylcytosine

Abbreviations

m5C m6A MAGI-1 MALT MAMP MAP MAPK MAPKK MAPKKK MARK2 MCP-1 MDC MDC1 MDCK MDM2 MDSC MEK1 MEK2 MEKK3 MERC MFN1/2 MGC MGMT MHC MIM Mincle MIPα miRNA Mist1 MKI MLC MLCK MLH1 MLN MLS MMEJ MMP10 MMP3 MMP7 MMP9 MMR MNC MOI MOM

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N5-methylcytosine N6-methyladenine Membrane-associated guanylate kinase with inverted orientation 1 Mucosa-associated lymphoid tissue Microorganism-associated molecular patterns Mitogen-activated protein Mitogen-activated protein kinase Mitogen-activated protein kinase kinase Mitogen-activated protein kinase kinase kinase Microtubule affinity regulating kinase 2 Monocyte chemoattractant protein-1 Mitochondrial-derived compartment Mediator of DNA damage checkpoint protein 1 Madin-Darby canine kidney Mouse double minute 2 homolog Myeloid-derived suppressor cell Mitogen-activated protein kinase 1 Mitogen-activated protein kinase 2 Mitogen-activated protein kinase kinase 3 Mitochondria-ER contact Mitofusin 1/2 Mucosal gastric cells 6-O-methylguanine-DNA transferase Major histocompatibility complex Mitochondrial inner membrane Macrophage inducible C-type lectin Macrophage inflammatory protein alpha Non-coding micro RNA Basic helix-loop-helix family member a15 MARK Kinase inhibitor Myosin light chain Myosin light chain kinase Human homolog of MMR1 from Escherichia coli Mesenteric lymph nodes Mitochondrial leading sequence Microhomology-mediated end-joining Matrix metalloprotease 10 Matrix metalloprotease 3 Matrix metalloprotease 7 Matrix metalloprotease 9 DNA mismatch repair Mitonuclear communication Multiplicity of infection Mitochondrial outer membrane

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MOMP MPC Mre11 MRN mRNA MS MSH2 MSH6 MSI MSI-H MSI-L MSS Mtase mtDNA MTHFR mTOR mTORC1 MUC6 MukB MUPP MyD88 Myh9 MZB NADPH NapA NCI NCI-N87 nDNA NEIL NEMO NER NF-κB NFAT NFP NGS NH3 NH4 + NHEJ NIK NK NKG2DL NLR NLRP3 NLS

Abbreviations

Mitochondrial outer membrane permeabilization Mitochondrial pyruvate carrier Meiotic recombination 11 homolog MRE11-RAD50-NBS1 complex Messenger ribonucleic acid Multiple sclerosis MutS homolog 2 MutS homolog 6 Microsatellite instability High microsatellite instability Low microsatellite instability Microsatellite stable Methyltransferase Mitochondrial DNA Methylenetetrahydrofolate reductase Mammalian target of rapamycin Mechanistic target of rapamycin complex 1 Mucin-6 SMC homolog Multi-PDZ domain protein Myeloid differentiation primary response gene 88 Myosin heavy chain 9 Marginal zone B Nicotinamide adenine dinucleotide phosphate Neutrophil-activating protein A (also called HP-NAP) National Cancer Institute of the United States Epithelial cell line Nuclear DNA Nei-like protein NF-κB essential modifier Nucleotide excision repair Nuclear factor of kappa light polypeptide gene enhancer in B-cells Nuclear factor of activated T-cells N-formyl peptide Next generation sequencing Ammonia Ammonium ion Non-homologous end joining NF-κB-inducing kinase Natural killer Natural killer group 2, member D ligand NOD-like receptor NOD-, LRR- and pyrin domain-containing protein 3 Nuclear localization signal

Abbreviations

NO NOD NOG NOS2 NOTCH2 NOX NOXA1 NSAIDs N-SH2 NTF NTH1 NUMT OCT1 ODC OGG OGG1 OipA OMP OMV ONOOONOOCO2OPA1 OR ORC1 ORF OXPHOS p120ctn p38 p53 p53BP1 PAI PAK1 PALB2 PAMP Par1b Par3 Par6 ParA ParB PARK PARP1 ParS PC PD-1 pDC

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Nitric oxide Nucleotide binding oligomerization domain Noggin Nitric oxide synthase 2 Neurogenic locus notch homolog protein 2 NAPDH oxidase NADPH oxidase activator 1 Non-steroidal anti-inflammatory drugs N-terminal SH2 domain N-terminal fragment (E-cadherin ectodomain) Endonuclease III homolog 1 Nuclear mitochondrial DNA segment Octamer transcription factor1 Ornithine decarboxylase Oxoguanine DNA glycosylase 8-oxo-guanine-DNA glycosylase 1 Outer inflammatory protein A Outer membrane protein Outer membrane vesicle Peroxinitrite Nitrosoperoxycarbonate Optic atrophy protein 1 Odds ratio Origin recognition complex subunit 1 Open reading frame Oxidative phosphorylation system p120 catenin p38 MAP Kinase Tumor protein 53 p53 binding protein 1 Pathogenicity island p21 activated kinase Partner and localizer of BRCA2 Pathogen-associated molecular pattern Partitioning-defective kinase 1b Protease-activated receptor 3 Protease-activated receptor 6 Chromosome partitioning protein ParA Chromosome partitioning protein ParB Parkin gene Poly [ADP-ribose] polymerase 1 Centromere-like sequence Pyruvate carboxylase Programmed death protein 1 Plasmacytoid dendritic cell

xxvi

PDCD1LG2 PDHC PDK1 PD-L1 Pdx1 PDZ PG PGAM5 PGC PGC1 PGE2 PGE-M PI3K PIP2 PIP3 PKB1 PKC PLC PMN POLQ PPI PPs PqqE PRK2 PRR PS PSC PSCA PTEN PTPN11 PUD PUMA qPCR Rac1 Rag1 RASAL2 REG3γ RelA/p50 RelB/p52 RET RGD RGDLXXL RGM1 RhoA

Abbreviations

Programmed cell death 1 ligand 2 Pyruvate dehydrogenase complex 3-phosphoinositide dependent protein kinase-1 Programmed cell death ligand 1 Pancreatic and duodenal homeobox 1 Postsynaptic density protein, Drosophila disc large tumor suppressor and Zonula occludens-1 protein Peptidoglycan Phosphoglycerate mutase 5 Pepsinogen C Peroxisome-proliferator activated receptor coactivator 1 Prostaglandin E2 Prostaglandin E2 metabolite Phosphatidylinositol 3-kinase Phosphatidylinositol-4,5-biphosphate Phosphatidylinositol-3,4,5-triphosphate Protein kinase B 1 Protein kinase C Phosphoinositide phospholipase C Polymorphonuclear neutrophil DNA polymerase q (theta) Proton pump inhibitor Peyer’s Patches Zinc-dependent metalloprotease Protein kinase C-related kinase 2 Pattern recognition receptor Phosphatidylserine Pluripotent stem cells Prostate stem cell antigen Phosphatase and tensin homolog Protein tyrosine phosphatase non-receptor type 11 Peptic ulcer disease p53 upregulated modulator of apoptosis Quantitative PCR Ras-related C3 botulinum toxin substrate 1 Recombination-activating gene 1 RAS protein activator like 2 Regenerating family member 3 gamma REL proto-Oncogene, NF-κB Subunit A REL proto-Oncogene, NF-κB Subunit B Reverse electron transfer Arg-Gly-Asp sequence motif Arg-Gly-Asp-Leu/Met-X-X-Leu/Ile sequence motif Gastric cell line Ras homolog gene family A

Abbreviations

RIG-I RLRs R-M systems RNA RNF43 RNI RNS ROC RocF ROCK ROS RPTP rRNA RSPO RTK RT-PCR RUNX1 RUNX3 s1/s2 SabA SBS SC SC-RNA-SEQ SDH SEER SeqA SFK SH2 SH3 SHH SHP1 SHP2 sIgA siRNA SIRT1 Siva1 SLB SLC1A2 sLex SLT SMC SMOX SNP SOD Soj

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Retinoic acid-inducible gene I RIG-I-like receptors Restriction-modification systems Ribonucleic acid Ring finger protein 43 Reactive nitrogen intermediate Reactive nitrogen species Receiver operating characteristic Urea-producing arginase Rho-Kinase Reactive oxygen species Receptor protein tyrosine phosphatase Ribosomal RNA R-spondin1 Receptor tyrosine kinase Reverse transcriptase-polymerase chain reaction Runt-related transcription factor 1 Runt-related transcription factor 3 2 allelic forms of the vacA gene Sialic acid binding adhesin Single base substitution Stem cell Single-cell RNA sequencing Succinate dehydrogenase Surveillance, Epidemiology, and End Results Sequestration protein A Src family kinase Src homology 2 Src homology 3 Sonic hedgehog SH2 domain-containing phosphatase-1 SH2 domain-containing phosphatase-2 Secretory IgA Small interfering RNA Sirtuin 1 SIVA1 apoptosis-inducing factor Single layer antiparallel β-sheet Solute carrier family 1 member 2 sialyl-Lewis x antigens Soluble lytic transglycoylase Structure maintenance of chromosomes Spermine oxidase Single-nucleotide polymorphism Superoxide dismutase Sporulation initiation inhibitor Soj

xxviii

SOX2 SOX9 SPEM SPF SPR Src SSB ssDNA STAMBPL1 STAT1 STAT3 STING STMN1 T3SS T4SS TAB2/3 TAK1 TAM TBK-1 TCA TCF/LEF TCGA TEER TetO TetR TFF2 TFF3 TGCT TGF-β TGF-βRII TH Thp1 TIFA TIMP3 TIR TIRAP TJ Tlp TlpB TLR TME TNF Tnfrsf19 TP53

Abbreviations

SRY (sex determining region Y)-box transcription factor 2 SRY (sex determining region Y)-box transcription factor 9 Spasmolytic polypeptide-expressing metaplasia Specific pathogen-free Surface plasmon resonance Sarcoma virus kinase Single-strand binding Single-stranded DNA STAM-binding protein like 1 Signal transducer and activator of transcription factor-1 Signal transducer and activator of transcription factor-3 Stimulator of interferon genes Stathmin 1 Type-III secretion system Type IV secretion system TAK1-binding proteins 2 and 3 TGF-β-activated kinase 1 Tumor Associated Macrophage Serine/Threonine protein kinase-1 Tricarboxylic acid T-cell-specific transcription factor/Lymphoid enhancer binding factor The Cancer Genome Atlas Transepithelial electrical resistance Tetracycline operator Tetracyclin repressor Trefoil factor 2 Trefoil factor 3 Tenosynovial giant cell tumor Transforming growth factor beta Transforming growth factor β receptor II Helper T cell Monocytic cell line TRAF-interacting protein with forkhead-associated domain Metalloproteinase inhibitor 3 Toll/IL-1 receptor domain TIR domain containing receptor protein Tight junction Transducer-like protein Transducer-like protein B Toll-like receptor Tumor microenvironment Tumor necrosis factor Tumor necrosis factor receptor super family 19 Tumor protein p53

Abbreviations

TRAF TRAIL TRD TRD1 TRD2 Treg TRM TROP2 TrxA UC ULF ULK1/2 UPRmt Ure UreB USP48 UV VacA Vav2 VCAM1 VEGFA VEGFR Vil1 WFDC2 WHO Wnt Wt WWOX XIAP XRCC Y2H YAP YY1 ZNRF3 ZO-1

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TNF receptor associated factor TNF-related apoptosis-inducing ligand Target recognition domain Target recognition domain 1 Target recognition domain 2 Regulatory T cell Tissue-resident memory T cells Trophoblast cell-surface antigen 2 Thioredoxin A Ulcerative colitis Ubiquitin ligase for ARF Unc-51-like kinase 1/2 Mitochondrial unfolded protein response Urease Urease B subunit Ubiquitin Specific Peptidase 48 Ultraviolet Vacuolating cytotoxin A Rac-specific nucleotide exchange factor Vascular adhesion molecule 1 Vascular endothelial growth factor A Vascular endothelial growth factor Receptor Villin-1 WAP four-disulfide core domain 2 World Health Organization Wingless and Int-1 Wild-type WW domain containing oxidoreductase X-linked inhibitor of apoptosis protein X-ray repair containing oxidoreductase Yeast two hybrid Yes-associated protein Ying Yang binding motif Zinc and ring finger 3 Zonula Occludens-1

Gastric Stem Cell Biology and Helicobacter pylori Infection Jonas Wizenty and Michael Sigal

Abstract Helicobacter pylori colonizes the human gastric mucosa and persists lifelong. An interactive network between the bacteria and host cells shapes a unique microbial niche within gastric glands that alters epithelial behavior, leading to pathologies such as chronic gastritis and eventually gastric cancer. Gland colonization by the bacterium initiates aberrant trajectories by inducing long-term inflammatory and regenerative gland responses, which involve various specialized epithelial and stromal cells. Recent studies using cell lineage tracing, organoids and scRNA-seq techniques have significantly advanced our knowledge of the molecular “identity” of epithelial and stromal cell subtypes during normal homeostasis and upon infection, and revealed the principles that underly stem cell (niche) behavior under homeostatic conditions as well as upon H. pylori infection. The activation of long-lived stem cells deep in the gastric glands has emerged as a key prerequisite of H. pyloriassociated gastric site-specific pathologies such as hyperplasia in the antrum, and atrophy or metaplasia in the corpus, that are considered premalignant lesions. In addition to altering the behaviour of bona fide stem cells, injury-driven de-differentiation and trans-differentation programs, such as “paligenosis”, subsequently allow highly specialized secretory cells to re-acquire stem cell functions, driving gland regeneration. This plastic regenerative capacity of gastric glands is required to maintain homeostasis and repair mucosal injuries. However, these processes are co-opted in the context of stepwise malignant transformation in chronic H. pylori infection, causing the emergence, selection and expansion of cancer-promoting stem cells.

J. Wizenty · M. Sigal (B) Division of Gastroenterology and Hepatology, Medical Department, Charité—Universitätsmedizin Berlin, Berlin, Germany e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 S. Backert (ed.), Helicobacter pylori and Gastric Cancer, Current Topics in Microbiology and Immunology 444, https://doi.org/10.1007/978-3-031-47331-9_1

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1 Introduction Macroscopically, the stomach can be subdivided into five sections: cardia, fundus, corpus, antrum, and pylorus. Its lumen is lined by a simple columnar epithelium, which is in close contact with nutrients and bacteria and is organized into gland units. These gastric glands can be subdivided into base, isthmus, neck, and surface compartments that harbor specific subsets of specialized cells. Gastric glands undergo constant and rapid turnover with stem cells fueling the replacement of short-lived differentiated cells (Post and Clevers 2019). Gland homeostasis is maintained by epithelial and non-epithelial signals that orchestrate cell proliferation, regeneration, and differentiation into different lineages. Microscopically, glands in the corpus (and fundus) and the antrum (and pylorus) differ (Fig. 1): Corpus glands are longer and contain pit cells, neck mucus cells, long-lived parietal cells located in the neck region that produce gastric acid, longlived chief cells at the base that secrete digestive enzymes, and tuft cells that secrete prostaglandins (Lee et al. 1982; Karam and Leblond 1993; Karam 1993). The shorter antrum glands lack parietal and chief cells (Lee et al. 1982; Lee and Leblond 1985). Although both sites also contain enteroendocrine cells, these differ functionally: While the corpus contains enterochromaffin-like (ECL) cells, the antrum contains gastrin-secreting G cells, and somatostatin-producing D cells. The gland isthmus contains highly proliferative cells in both sites, but turnover kinetics appear to be more rapid in the antrum than in the corpus (Kitsanta et al. 2005). Helicobacter pylori colonizes gastric glands at all sites and causes gastric pathology, which is reflected by an alteration of the gland structure. All infected individuals develop gastritis, with some patients going on to develop severe pathologies such as gastroduodenal ulcers or gastric cancer, particularly after long-term colonization (Bauer and Meyer 2011). Gastric adenocarcinoma is the most severe complication of infection and occurs in a step-wise manner, with specific micromorphological gland alterations that are known as the Correa cascade (Correa and Piazuelo 2012). H. pylori infection promotes epithelial cell proliferation leading to mucosal hyperplasia and hyperplastic gastritis. The next step in the cascade is chronic-atrophic gastritis, which is characterized by a loss of specific specialized cell types in the gland, in particular parietal cells. Atrophic gastritis can be accompanied by two types of metaplastic changes, the so called “spasmolytic polypeptideexpressing metaplasia” (SPEM) or intestinal metaplasia (IM), which can be subdivided into the complete and incomplete forms. These changes are linked to a high risk of further transformation towards dysplasia and formation of invasive adenocarcinoma. Since all these changes are linked to an altered cellular organization of gastric glands, it is likely that they are driven by changes in gastric epithelial stem cells. In fact, recent studies have now revealed that stem cells can be directly and indirectly reprogrammed in the context of H. pylori infection, which may represent key events for the onset and progression of gastric (pre)malignant lesions. Here, we discuss recent insights into gastric stem cells, their interplay with H. pylori and their role in the development of gastric pathology.

Gastric Stem Cell Biology and Helicobacter pylori Infection

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Fig. 1 Model of the glandular organization of the stomach. Antrum and corpus glands consist of simple columnar epithelium with specialized cells varying in their composition and location. The antrum has three zones, including the pit containing pit cells, the isthmus with rapidly cycling stem cells, and the base containing slowly cycling stem cells and gland base mucous cells. The corpus glands consist of four zones with a pit, an isthmus containing rapidly cycling stem cells, a neck zone with neck mucous cells and parietal cells, and a base harboring chief cells. Rare endocrine cells (G and D cells in antrum, ECL cells in corpus) and tuft cells are present in both gland types. Created with BioRender.com

2 Gastric Stem Cells A highly proliferative cell population with an undifferentiated granule-free appearance in the isthmus of antrum and corpus glands was already revealed decades ago. These cells were shown to give rise to both base and surface cells (Karam and Leblond 1993; Karam 1993; Lee and Leblond 1985). More recently, lineage tracing, mostly by use of Cre recombinase, revealed several label candidates that are able to repopulate entire glands (Mills and Shivdasani 2011). Organoid technology and single-cell multi-omic techniques have further facilitated an in-depth understanding of processes that orchestrate tissue homeostasis, allowing the identification of key signaling pathways. While in the intestine the Wnt target gene leucine-rich repeatcontaining G-protein coupled receptor 5 (Lgr5) is the established marker for active stem cells in the crypt base (Barker et al. 2007), the “identity”, location, and molecular biology of stem cells in the stomach is less defined. In fact, Lgr5 does mark a subset of stem cells in the antrum, but also secretory cells in the gland base express Lgr5, while in the corpus, Lgr5 is expressed mainly in chief cells. Further, the identity and function of gastric stem cells appear to be context-dependent, with high plasticity potential that is controlled by niche factors secreted by neighboring epithelial

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cells, stromal mesenchymal cells, immune cells, vascular cells, or neurons (Bartfeld and Koo 2017). Niche mesenchymal cells located adjacent to the glands were shown to be the major source of Wnt during gastric development and regeneration. Inhibition of Wnt secretion in these stromal cells leads to severe stem cell damage and developmental defects (Kim et al. 2020).

2.1 Antrum Stem Cell Biology As in the intestine, Lgr5 has been found as a marker of antral gland base stem cells that are enriched for several Wnt target genes and showed 29% co-expression of Ki67 (Barker et al. 2010). The major subpopulation of these Lgr5 + antral base cells is marked by aquaporin 5 (Aqp5) expression on their apical surface, and simultaneous expression of the base mucous cell marker gastric intrinsic factor (Gif) (Fig. 2). Ki67 labeling and lineage tracing revealed that these cells can repopulate entire glands within 5 days (Tan et al. 2020). The Wnt target gene Axin2 is expressed by these Lgr5 + cells, as well as by adjacent, highly proliferative Lgr5neg cells in the lower isthmus, which can repopulate entire glands, including the base, upon depletion of the Lgr5 + population (Sigal et al. 2017). These Axin2 + Lgr5neg have been shown to likely co-express cholecystokinin 2 receptor (Cckbr or Cckr2) and runt-related transcription factor 1 (Runx1 or the enhancer element eR1) (Hayakawa et al. 2015b; Matsuo et al. 2017). Mechanistically, LGR5 as well as its homologue LGR4 act as receptors by binding R-spondin (RSPO) molecules, which prevents turnover of the Wnt receptor FRIZZLED, thereby leading to fully active Wnt signaling (de Lau et al. 2011). RSPO3 is secreted by stromal myofibroblasts beneath the gland base and activates and leads to expansion of Axin2 + Lgr4 + Lgr5neg cells in the lower isthmus, while simultaneously inducing differentiation of basal Lgr5 + cells into secretory cells that express and secrete antimicrobial factors (Sigal et al. 2017, 2019; Wizenty et al. 2022). Recent data indicate that in fact, the majority of antral Lgr5 + cells are secretory cells, while Lgr4, but not Lgr5, is required for antral homeostasis and proliferation in response to RSPO3 (Sigal et al. 2019; Wizenty et al. 2022). The Rspo3-Lgr4 pathway is required for maintaining the Lgr5 + stem cell state (Wizenty et al. 2022) and this cell pool is stabilized by a positive feedback loop via RSPO-induced expression of Lgr5, its own receptor (Yan et al. 2017). Depletion of Lgr5 + cells is followed by rapid recovery in an Rspo3-Lgr4 dependent manner. Interestingly, the formation of Lgr5 + secretory cells requires not only Wnt but also NF-κB signaling activity, which is also induced by RSPO3 in Lgr4 + lower isthmus cells (Wizenty et al. 2022). Single-cell RNA sequencing (sc-RNAseq) is a single-cell resolution sequencing technology enabling researchers to quantify the gene expression of individual cells in complex tissues. Sc-RNAseq data have revealed that basal stromal cells secrete inhibitors of bone morphogenic protein (BMP), such as chordin-like 1 (CHRDL1) and gremlin 1 (GREM1) while pit stromal cells secrete BMP ligands. The resulting BMP gradient promotes the differentiation of epithelial cells in the pit where the

Gastric Stem Cell Biology and Helicobacter pylori Infection

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Fig. 2 Stomach glands, their niche, stem cell markers and main signaling gradients. Epithelial cells are surrounded by niche cells, e.g. mesenchymal cells, supporting epithelial cells with important factors such as Wnt ligands, Rspo, BMP, EGF and acetylcholine, with spatial distribution along the gland axis. Stem cell markers as well as gland base mucus cell markers in the antrum and chief cell markers in the corpus are highlighted in blue. Created with BioRender.com

BMP signal is high, while the low BMP signal in the base ensures that basal stem cells do not differentiate. Similarly, addition of BMP ligands to organoid cultures leads to decreased proliferation and reduced expression of stem cell markers. The lineage commitment towards terminally differentiated pit cells is further enforced by a positive BMP feedback loop, with BMP2 ligand inducing expression of Bmp2 in the epithelial cells themselves (Kapalczynska et al. 2022). Conversely, a complete loss of BMP signaling in Axin2 + cells results in hyperplasia. In the intestine, the effects of BMP signaling on epithelial homeostasis are Wnt-independent, and instead caused by BMP-driven Smad proteins directly interfering with the promoter regions of stem cell signature genes (Qi et al. 2017). In the stomach, however, we found that Rspo3 expression in myofibroblasts is reduced upon BMP2 treatment, which may explain the spatial restriction of RSPO3 to the lower gland (Kapalczynska et al. 2022). Further markers, such as B lymphoma Mo-MLV insertion region 1 homolog (Bmi1) (Yoshioka et al. 2019) and basic helix-loop-helix family member a15 (Bhlha15 or Mist1) (Sakitani et al. 2017; Nienhüser et al. 2021), have been proposed as antral isthmus stem cell markers. Taken together, the lower isthmus of antral glands contains highly proliferative cells, whereas the base contains differentiated slow-cycling cells, that are nonetheless capable of repopulating entire glands upon damage (Fig. 2). Notch signaling seems to be critical for maintaining antral stemness: inhibition of NOTCH1/2 leads to reduced proliferation of Lgr5 + stem cells and increased

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proliferation of secretory lineages, as demonstrated in organoids from both mouse and human antrum tissue (Gifford et al. 2017). Recently, the NOTCH ligand deltalike canonical Notch ligand 1 (Dll1) was found to be expressed by gland base mucus cells adjacent to Lgr5 + Notch1high cells, regulating epithelial proliferation via direct ligand-receptor interaction (Horita et al. 2022). Although the Wnt and Notch pathways have partially overlapping targets, the exact interplay between both pathways in the stomach is not fully understood. In contrast to Lgr5 + cells, which undergo symmetric cell division maintaining a stable level with a few dominant stem cells (Leushacke et al. 2017), a small subpopulation of lower isthmus cells marked by Cckbr and Dll1 undergoes asymmetric cell division, keeping a quiescent stem cell state driven by gastrin signals (originating from G cells residing near Cckbr + stem cells), and generating a stem cell and a daughter cell with different fates. Loss of gastrin signals, e.g. by chemical injury, leads to a switch to symmetric cell division with rapid tissue regeneration (Chang et al. 2020). Furthermore, Lgr5 + cells express high levels of muscarinic acetylcholine receptor, with stem cell expansion also driven by cholinergic neurons and choline acetyltransferase-expressing tuft cells (Hayakawa et al. 2017b).

2.2 Corpus Stem Cell Biology In the corpus, proliferating cells of the isthmus region in the upper-middle part of the gland are labeled by stem cell markers distinct from those of the antrum (Karam and Leblond 1993). Undifferentiated SRY (sex determining region Y)-box transcription factor 2 (Sox2) + cells can give rise to all cell lineages (Arnold et al. 2011b), while spasmolytic polypeptide, aka trefoil factor 2 (Tff2) + cells can give rise to mucus neck, parietal and chief cells, but not pit cells or enteroendocrine cells (Quante et al. 2010). Sox9 + progenitor cells overlap with Sox2 + cells and can differentiate to generate all gastric epithelial lineages (Chen et al. 2023). Mist1 has been detected in chief cells and in a few isthmus cells distinct from Sox2 + cells, that were characterized as quiescent non-canonical Wnt5a-dependent stem cells supported by a perivascular niche (Hayakawa et al. 2015a; Nienhüser et al. 2021). These Mist1 + isthmus stem cells, but not the basal chief cells, supply daughter cells towards the pit and the base, as demonstrated using lineage tracing with ablation of either chief cells (using Lgr5-DTR-GFP mice) or stem cells (using treatment with 5-fluorouracil). Thus, Mist1 + isthmus stem cells give rise to entire glands, including a major downstream migration with differentiation to mucous neck cells and later transdifferentiation to chief cells. Undifferentiated eR1 + cells were prominently found in the isthmus stem cell zone and occasionally also in the chief cell region and contributed to all lineages of the gastric gland (Matsuo et al. 2017). Further suggested stem cell markers in the corpus isthmus are leucine-rich repeats and immunoglobulinlike domains 1 (Lrig1) (Choi et al. 2018), Bmi1 (Yoshioka et al. 2019) and IQ motifcontaining GTPase-activating protein 3 (Iqgap3) (Matsuo et al. 2021). However, based on these studies, these markers are not region-specific, and it remains unknown

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if they are expressed by different cell types in the isthmus or if isthmus stem cells expressing these markers are active or quiescent (Fig. 2). Cckbr + cells have also been detected in the corpus isthmus, but in contrast to the antrum, they increase proliferation under hypergastrinemia (Sheng et al. 2020). Also, Notch activation can promote isthmus proliferation in the corpus (Demitrack et al. 2017). The BMP antagonist Grem1, expressed by stromal mesenchymal cells adjacent to isthmus cells, inhibits BMP signaling and supports stemness (Worthley et al. 2015; Ye et al. 2018), while the differential interplay of BMP and epidermal growth factor (EGF) signaling is important for controlling the specific differentiation into pit, chief and parietal cells, as shown in organoids (Wölffling et al. 2021). EGF signaling, concomitant with BMP, promotes foveolar differentiation, whereas BMP signaling in the absence of EGF induces parietal cell differentiation and suppresses chief cell differentiation (Wölffling et al. 2021). Importantly, canonical Wnt signaling does not seem to play a key role in the corpus isthmus turnover under normal conditions, since Wnt target genes are only highly expressed in the base. Under normal conditions, chief cells located in the base, but not proliferative isthmus stem cells, are activated by stromal RSPO3 (Fischer et al. 2022). However, upon chemical injury, regeneration of secretory lineages is driven by short-term upregulation of RSPO3 levels in stromal cells, upregulated LGR5 receptors in the isthmus and activation of Yes-associated protein (YAP) signaling (Fischer et al. 2022). The stem cell markers tumor necrosis factor receptor superfamily member 19 (Tnfrsf19 or Troy) (Stange et al. 2013) and Lgr5 (Leushacke et al. 2017) are expressed by chief cells and lineage tracing experiments of these two loci have revealed that chief cells repopulate entire glands in a slowly cycling manner. However, upon loss of proliferative isthmus cells, e.g. by chemical injury, Wnt signaling is activated, and their turnover is highly accelerated. This indicates that differentiated chief cells can acquire “reserve stem cell” functions if required. Recently, p57 Kip2 was identified as a key molecular switch that is expressed in chief cells during homeostasis, but is rapidly diminished upon injury, activating proliferation (Lee et al. 2022a). In addition, Wnt ligand profiling in the corpus revealed that stromal WNT2B and WNT7B are important Wnt ligands in the base during homeostasis (Teriyapirom et al. 2023). However, some concerns about the stem cell capacity of chief cells have been raised and alternative scenarios have been proposed suggesting rapid apoptotic death of fully differentiated chief cells upon injury and instead regeneration by adjacent “transitional” cells that share features of chief and mucous neck cells (Kinoshita et al. 2018; Hata et al. 2020). The two independent stem cells zones in the corpus were validated recently based on clonal data, single-cell profiling, and long-term lineage tracing experiments: with stathmin 1 (Stmn1) + /Ki67 + rapid cycling isthmus stem cells maintaining the pitisthmus-neck region and Troy + and Lgr5 + slow cycling base stem cells maintaining the base (Han et al. 2019; Burclaff et al. 2020). This confirmed that isthmus stem cells are the main drivers of gland turnover, but simultaneously the base cells remain stable without support from the isthmus, raising questions about the exact stem cell dynamics within the gland and the dogma of gland monoclonality in the stomach (Fig. 2).

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3 H. pylori Infection H. pylori is a spiral-shaped, microaerophilic, gram-negative bacterium colonizing the human stomach. About half of the world´s population is infected with H. pylori, which is the major risk factor for gastric cancer (Parsonnet et al. 1997) and has been classified as a class 1 carcinogen by the WHO since 1994. Infection usually occurs in childhood via oral transmission and persists lifelong due to highly organized bacteria-host interactions, bacterial survival strategies and host adaptive responses. While most bacteria are free-swimming, some colonize the gastric epithelium and induce manifold signaling processes that in some patients promote chronic active gastritis, characterized by mucosal infiltration of immune cells. The link between infection, inflammation, and epithelial pathology is not fully resolved. Currently, the use of organoids and genetically modified mice that allow modifications of stem cells and nearly every epithelial cell type fuel research on H. pylori-induced tissue pathology.

3.1 Gland-Invading H. pylori Interact with Stem Cells The biogeography of infection may play a role in carcinogenesis since infection of stem cells deep in the glands may have more detrimental effects than infection of pit cells, which are shed into the lumen within hours or days. H. pylori is equipped with a complex motility and chemotactic system, enabling it to penetrate the protective mucus layer and colonize the gastric epithelium. Four chemoreceptors—the transducer-like proteins (TlpA, TlpB, TlpC, and TlpD)—sense numerous signals such as urea, amino acids, and metals (Johnson and Ottemann 2018) to control bacterial movement via the flagellum (Howitt et al. 2011; Ottemann and Lowenthal 2002; Sigal et al. 2015; Collins et al. 2018). Multiple bacterial adhesins—blood group antigen-binding adhesin (BabA), sialic acid-binding adhesin (SabA), Helicobacter pylori outer membrane protein HopZ, Helicobacter pylori outer inflammatory protein A (OipA), and adherence-associated lipoprotein A/B (AlpA/B)—ensure attachment of H. pylori to the epithelium, allowing the bacteria to manipulate cell behavior via injection of virulence factors through the type-IV secretion system (T4SS) (Ilver et al. 1998; Mahdavi et al. 2002; Peck et al. 1999; Yamaoka et al. 2000; Odenbreit et al. 1999; Backert et al. 2011; Fischer et al. 2020). More recently, the H. pylori outer membrane protein Q (HopQ) was identified as a carcinoembryonic antigenrelated adhesion molecule (CEACAM) targeting adhesin that mediates adherence to the epithelium for translocation of CagA (Javaheri et al. 2016; Moonens et al. 2018; Zhao et al. 2018). In fact, bacteria appear to preferentially sense and colonize pit cells, which trigger only a low-level inflammatory response (Aguilar et al. 2022). However, although the glands are shielded from luminal stomach content, a subpopulation of H. pylori is able to penetrate deep into the gastric glands and reach the gland stem cells. These gland-associated microcolonies enable persistence

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and expansion of the infection (Fung et al. 2019). Infection triggers hyperplastic gastritis by expansion of the epithelial stem cell pool marked by Wnt target genes such as Lrig1 (Noto et al. 2013), Lgr5 (Uehara et al. 2013) or Axin2 (Sigal et al. 2015, 2017). H. pylori was found to induce DNA damage specifically in Lgr5 + stem cells (Uehara et al. 2013). Approximately half of the antral glands are colonized by H. pylori and a subpopulation of H. pylori forms extracellular microcolonies, preferentially in contact with the surface of proliferative isthmus cells and some basal stem cells (Sigal et al. 2015). These stem cell-associated microcolonies are formed in the very early phase of infection before chronic gastritis is established. Importantly, lineage tracing from Lgr5 + stem cells revealed a locally accelerated expansion of stem cells and hyperplasia in infected glands compared to uninfected glands in the same mice, indicating that it is specifically the gland-invading bacteria that lead to increased gland turnover. No hyperplasia was observed with bacteria that lack the capacity to colonize glands due to mutations in the chemotaxis machinery or the T4SS (Sigal et al. 2015). Importantly, stem cells that are hyper-proliferating are at risk of giving rise to metaplasia and dysplasia upon accumulation of genetic and epigenetic changes (Hayakawa et al. 2017a).

3.2 H. pylori Induces Gastritis via Activation of NF-kB H. pylori infection commonly leads to activation of the NF-κB pathway in epithelial cells, which causes the expression of pro-inflammatory cytokines such as IL-8 or other members of the CXC chemokine family and recruitment of innate and adaptive immune cells to the site of infection (Backert and Naumann 2010). Only recently, the PAMP ADP-β-d-manno-heptose (ADP-heptose), a soluble metabolite of the lipopolysaccharide (LPS) synthesis pathway, was identified as the bacterial factor activating NF-κB in a T4SS-dependent manner (Zhou et al. 2018; Pfannkuch et al. 2019) (Fig. 3). In gastric epithelial cells, ADP-heptose binds the novel cytosolic PRR alpha-kinase 1 (ALPK1), a serine/threonine kinase that phosphorylates TRAFinteracting protein with FHA domain (TIFA), which in turn binds to tumor necrosis factor receptor (TNFR)-associated factor (TRAF) molecules to activate classical and alternative NF-κB pathways (Maubach et al. 2021). Notably, H. pylori-dependent activation of the NF-κB pathway exclusively involves TIFA, but not TNF-α or IL-1β. Several molecules upstream and downstream of NF-κB are modified upon infection, altering the homeodynamics of gastric epithelial (stem) cells, and laying the ground for long-term colonization and malignant transformation (Maubach et al. 2022; for more details see chapter “Gastric Epithelial Barrier Disruption, Inflammation and Oncogenic Signal Transduction by Helicobacter Pylori” of this book). Chronic gastritis is at risk of stepwise transformation into gastric cancer, in a process called the Correa cascade (Correa and Piazuelo 2012), which progresses from atrophy to IM to cancer. Gastric cancer arises in the context of chronic H. pylori infection upon multiple mutagenic events, most probably in long-lived, selfrenewable stem cells, while mutations in short-lived, differentiated cells are unlikely

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Fig. 3 H. pylori induces NF-κB activity in stem cells. H. pylori adheres to and activates Axin2 + Lgr4 + stem cells. ADP-heptose, a secreted bacterial intermediate of LPS, binds to the intracellular receptor ALPK1 thereby inducing NF-κB activity. NF-κB drives inflammation via transcription of pro-inflammatory cytokines (e.g. Cxcl1 and Cxcl2, which recruit neutrophils), epithelial proliferation and potentially causes DNA damage specifically in regulatory R-loops, which are RNA– DNA triple-stranded structures. Infection also enhances Rspo-Lgr4 signaling, which potentiates proliferative and inflammatory effects in stem cells. Created with BioRender.com

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to achieve the mutational threshold for malignant transformation (Hayakawa et al. 2017a). The Cancer Genome Atlas (TCGA) provides a framework for the mutational signatures of 4 gastric cancer subtypes but lacks information on the cellular origin or a dominant mutational sequence in dependence on niche signals (Cancer Genome Atlas Research Network 2014). One important aspect of H. pylori-induced carcinogenesis is the observation that infected cells show DNA damage. The induction of double-strand breaks (DSBs) by nucleotide excision repair (NER) endonucleases depends on the T4SS. H. pylori enhances the interaction of NER with NF-κB, which amplifies NF-κB target gene transactivation, promoting host cell survival and potentially carcinogenesis, especially when stem cells are affected (Hartung et al. 2015). CagA-carrying strains can also directly induce DSBs in host cells via inhibition of partitioning-defective 1b (PAR1b), a kinase phosphorylating breast cancer gene 1 (BRCA1). Unphosphorylated BRCA1 cannot exert its function in the DNA-repair pathway of homologous recombination, which may lead to errors in the repaired DNA via non-homologous end-joining (Imai et al. 2021). This suggests that glandasscoiated H. pylori may drive genomic instability (Kidane 2018; for more details see chapter “Helicobacter Pylori-Induced Host Cell DNA Damage and Genetics of Gastric Cancer Development” of this book).

3.3 H. pylori Triggers Hyperplastic Gastritis in the Antrum Although the direct bacteria-stem cell contact suggests that epithelial pathology is induced by a bacterial factor that directly controls stem cell behavior, strong evidence points to the involvement of an indirect signaling circuit via the stem cell niche. Mechanistically, genetic experiments revealed that expansion of the stem cell compartment and hyperplasia in the antrum are mediated via increased expression of Rspo3 in Myh11 + myofibroblast in the stroma surrounding the gland base (Sigal et al. 2017). RSPO3 drives proliferation of antral isthmus cells via LGR4 as well as differentiation of Lgr5 + basal cells into secretory cells that are marked by pepsinogen c (PGC), GIF or Griffonia simplicifolia lectin GS-II (GSII) and secrete antimicrobial factors such as intelectin 1 (ITLN1) and regenerating family member 3 gamma (REG3γ). These factors counterbalance the infection but are unable to achieve total clearance of bacteria from the gland (Fig. 4) (Sigal et al. 2019; Wizenty et al. 2022). Accordingly, depletion of Lgr5 + cells was shown to result in higher colonization levels in antral glands. By analyzing the distinct roles of the RPSO receptors Lgr4 and Lgr5 upon H. pylori infection, loss of Lgr4 was found to abrogate the stem cell responses and formation of early premalignant gland alterations, while loss of Lgr5 does not impair homeostasis. The Rspo3-Lgr4 axis induces NF-κB activation in proliferative isthmus cells, driving a robust pro-inflammatory response. This leads to epithelial expression of cytokines, mucosal neutrophil infiltration, and expansion of the gland base—hallmarks of hyperplastic gastritis in gland-invading H. pylori infection (Fig. 4). Studies using gastric organoids or mucosoids in the presence of the stem cell niche factors WNT and

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Fig. 4 Gastric gland pathology upon H. pylori infection. Site-specific tissue responses and evolving pathologies have been identified in antrum and corpus. Antrum glands exhibit hyperplastic gastritis, which is caused by proliferative isthmus and base stem cells as well as expansion of gland base mucous cells. This is mainly driven by Rspo secreted by stromal myofibroblasts. Base mucous cells in turn secrete Itln1, which counterbalances infection. Corpus glands lose parietal cells and exhibit increased proliferation in the isthmus and base. In the base, chief cells alter their differentiation state leading to SPEM and IM, which may progress to dysplasia. SPEM, IM and dysplastic cell markers are highlighted in blue. Created with BioRender.com

RSPO showed that stem cells respond with an NF-κB-driven inflammatory response to infection or ADP-heptose, while differentiated lineages have a highly diminished inflammatory response (Bartfeld et al. 2015; Boccellato et al. 2018; Wizenty et al. 2022). The resulting expression of pro-inflammatory cytokines was similar to that induced by exposure to TNF-α, pointing to a universal stem cell-dependent response to multiple stress factors. This mechanism explains how the epithelium, which is in contact with numerous bacteria, toxins, and chemicals present in the luminal content, is able to distinguish between harmless and harmful contacts that require differential responses: It strongly responds and structurally adapts only if harmful factors manage to reach the stem cell compartment and therefore pose a threat to the genetic integrity of the glands. Interestingly, the above mentioned DNA damage induced by ALPK1-mediated NF-κB activity occurs specifically in proliferating cells via co-transcriptional RNA/ DNA hybrids (R-loops) (Fig. 3) (Bauer et al. 2020). In addition, NF-κB signals also seem to be crucial for gland base identity and the regenerative response upon infection (Wizenty et al. 2022). This may be explained by the recent identification

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of two molecules, RAS protein activator like 2 (RASAL2) and cyclin-dependent kinase 1 (CDK1), which mediate NF-κB-driven β-catenin expression in the context of H. pylori infection, indicating that infected host cells can acquire a stem cell state with expression of Lgr5 and Axin2 (Cao et al. 2022; Zhu et al. 2023). In mice, H. pylori infection also causes a loss of the stromal and epithelial BMP2 expression that normally maintains differentiated cell fates. At the same time, infection increases expression of BMP inhibitors, resulting in enhanced self-renewal of isthmus stem cells, proliferation, and accumulation of gland base mucous cells with expression of anti-microbial agents (Kapalczynska et al. 2022). These effects are dependent on the inflammatory T cell cytokine interferon-gamma (IFN-γ), which is induced in a T4SS-dependent manner, and blocks BMP2 signaling in epithelial and stromal cells. Mice that lack IFN-γ do not display hyperplasia upon infection, pointing to a direct effect of inflammation on gland pathology (Sayi et al. 2009). Nonetheless, even long-term infection with H. pylori in normal mice does not lead to the emergence of gastric epithelial malignancy. Future studies are needed to address how dysregulated epithelial stem cell niche signals and DNA damage of stem cells contribute to the initiation or fixation of oncogenic mutations that drive gastric cancer.

3.4 H. pylori Triggers Atrophic Gastritis and Metaplasia in the Corpus In chronically infected mice and humans, key premalignant lesions, such as oxyntic atrophy, SPEM, and IM are frequently observed in the corpus (Arnold et al. 2011a). Lineage tracing experiments have shown that loss or inflammatory injury of parietal cells results in “atrophic gastritis” caused by altered chief cell differentiation. This aberrant chief cell differentiation pattern is characterized by decreased expression of chief cell granules containing enzymes such as PGC, alongside re-expression of neck cell markers such as GIF, GSII, MUC6, and TFF2, also known as spasmolytic polypeptide. It thus resembles the organization of the antrum glands and is also called “antralization” (Fig. 4) (Nozaki et al. 2008; Mills and Sansom 2015; Fischer et al. 2022). Mechanistically, this regenerative response to parietal cell atrophy involves extracellular signal-regulated kinases (ERK)-CD44 signaling (Khurana et al. 2013). New scRNA-seq analyses have identified specific markers for SPEM such as AQP5 co-localizing with CD44v9, gastrokine-3 (GKN3), and WAP four-disulfide core domain 2 (WFDC2) (Lee et al. 2022b; Bockerstett et al. 2020; Jeong et al. 2021). Importantly, chief cell plasticity may also contribute to the proliferation of pit cells (foveolar hyperplasia) (Caldwell et al. 2022) and the regenerative program in the corpus also includes expansion of mucous neck cells and isthmus stem cells. A second type of metaplasia typically found in the corpus, IM, is characterized by the presence of intestinal goblet cells and may derive from SPEM (Choi et al. 2016). IM has been subclassified into complete versus incomplete types, which often occur simultaneously. Complete IM glands harbor mature TFF3 + goblet cells and

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enterocytes. If the goblet cells are immature, the pathology is defined as incomplete IM and is associated with a high risk of gastric cancer (Lee et al. 2022b; Shao et al. 2018). As described above, AQP5 marks antral stem cells and is absent in the normal corpus, but new scRNA-seq data in humans have identified a specific upregulation of Aqp5 also in incomplete IM (Fig. 4) (Lee et al. 2022b). The progression from SPEM to dysplasia involves a distinct subset of mesenchymal cells, as recently shown by using scRNA-seq and co-culture of metaplasia-derived or cancer-derived fibroblasts with metaplastic gastric organoids with loss of CD44v9 and AQP5 (marking metaplasia) and gain of trophoblast cellsurface antigen 2 (TROP2) and CEACAM5 (marking dysplasia) (Lee et al. 2023). The recently coined term “paligenosis” describes a multi-organ regenerative program of mature, fully differentiated cells that involves cell structure degradation, progenitorassociated gene activation, and re-entry into the cell cycle (Willet et al. 2018; Bockerstett et al. 2020; Miao et al. 2020; Adkins-Threats and Mills 2022). Chief cells likely undergo paligenosis, either progressing to SPEM or dysplasia in case of chronic injury such as H. pylori infection, or upon certain mutations, or returning to normal chief cells in case of acute injury that is repaired, such as acid-induced ulcers. Importantly, SPEM appears to be independent of bona fide stem cells, as demonstrated by blocking them via injection of 5-fluorouracil in mice (Radyk et al. 2018). Notably, upon infection with H. pylori, the stem cell factor RSPO3 triggers profound chronic glandular hyperplasia and regeneration in the corpus via activation of YAP (Fischer et al. 2022). YAP expression, which is dependent on CagA, promotes pro-survival and pro-proliferation genes and expression of metaplasia markers, in a mechanism that involves the Wnt pathway (Messina et al. 2023). Indeed, overexpression of Rspo3 in mice has been shown to strongly promote SPEM (Fischer et al. 2022). In concordance, another study found that depletion of BMP receptor specifically in Lgr5 + cells is sufficient to induce metaplasia (IM and SPEM), as well as dysplasia, in mice infected with other Helicobacter species (Ye et al. 2018). In addition, H. pylori infection leads to CagA-dependent secretion of sonic hedgehog (Shh), which in turn induces programmed death-ligand 1 (PD-L1) expression in SPEM cells, and may partly explain the persistence of infected cells via escape from cytotoxic T cells. Indeed, pharmacological PD-1 inhibition led to the death of SPEM cells (Holokai et al. 2019). The progression of SPEM and/or IM to gastric cancer likely involves the accumulation of mutations in the context of chronic gastritis (Tan and Yeoh 2015). However, the underlying molecular mechanisms remain unknown. A report of a patient with an activating Kras mutation in SPEM has now provided direct evidence that SPEM can give rise to gastric cancer (Kumagai et al. 2023). In mice, recent studies used combined genetic targeting of the Wnt, Ras, and Trp53 pathways, which led to advanced, metastatic gastric cancer (Seidlitz et al. 2019) and showed dependency on Lgr5 + stem cells (Fatehullah et al. 2021). Mutations in Rnf43 and Znrf3 (both negative regulators of the Wnt receptor FRIZZLED), or in Cdh1 and Tp53, led to tumor independence from RSPO (Koo et al. 2015; Nanki et al. 2018). However, these tumors were not completely independent of Wnt. Wnt niche independence in gastric carcinogenesis can be achieved via activation of the MAPK pathway (by

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Kras mutation or Her2 overexpression) or direct upregulation of the Wnt2 gene e.g. by copy number gains (Teriyapirom et al. 2023). Upon H. pylori infection, gastric cancer could be generated by deletion of the Cdh1 or Apc gene with simultaneous Kras mutation in Mist1 + isthmus cells (Hayakawa et al. 2015a). As gastric atrophy also occurs in the context of Kras mutation and is a driver of isthmus cell expansion, this atrophy may also promote the rise of cancers with isthmus cell origin (Hata et al. 2020). Further studies are needed to determine the differential impact of plastic, de-differentiated progenitors versus bona fide stem cells on gastric carcinogenesis. These data together show how the stem cell niche activating pathways Wnt, Rspo, NF-κB and YAP, as well as paligenosis, orchestrate a plastic, pro-regenerative epithelial state in H. pylori infection. On one hand, this is an important response to injury that prevents barrier failure and ulceration; on the other hand, in the context of a chronic infection that is not resolved, it represents a potential risk for carcinogenesis (Liabeuf et al. 2022; Messina et al. 2023; Traulsen et al. 2021).

4 Concluding Remarks Gastric stem cells located within the gland maintain gland homeostasis in high dependency on surrounding niche signals that may coordinate a plastic regenerative response to injury. Animal studies have revealed two stem cell populations in the base and isthmus of gastric glands, marked by often overlapping genes that are usually target genes of signaling pathways regulating proliferation and differentiation. Alterations of stem cells or the signals that regulate them, disrupt epithelial health to a greater extent than alterations of pit cells, which are rapidly replaced. Infection with gland-invading H. pylori activates stem cells, causing increased stem cell renewal, hyperproliferative glands, and expansion of anti-microbial gland base cells in the antrum. Specifically in the corpus, H. pylori infection activates trans- and de-differentiation programs such as “paligenosis” that may allow cells to acquire stem cell functions and to emerge to premalignant lesions such as SPEM and/or IM. Since H. pylori infection is chronic, it drives a sustained epithelial adaptive, anti-microbial response that is unable to eliminate the infection and is therefore at risk of malignancy due to the sustained activity of regenerative and proliferative programs. The interplay of H. pylori with stem cells, either bona fide or re-aquired, appears to be a hallmark of the pathobiology that predisposes to gastric cancer. Gland-invading bacteria trigger aberrant inflammatory signaling and DNA damage in stem cells, thereby strongly affecting gland homeostasis. First studies found direct evidence that H. pylori virulence factors provoke extensive pathology if stem cells are infected. A loss of genetic integrity in stem cells may fail to be resolved properly during the complex and sustained active interplay between chronic bacterial stress and epithelial host responses, and may be further fostered by defects in the DNA repair machinery or the presence of additional mutations. Whether and by what mechanisms proliferative and inflammatory signals stimulate damaged cells in the competition against healthy neighboring cells, and how mutant stem cell clones

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become fixed in the tissue upon infection, need to be addressed in future studies. Fully understanding these processes may enable us to develop interception strategies to better prevent or treat gastric cancer.

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Clinical Pathogenesis, Molecular Mechanisms of Gastric Cancer Development Lydia E. Wroblewski and Richard M. Peek Jr

Abstract The human pathogen Helicobacter pylori is the strongest known risk factor for gastric disease and cancer, and gastric cancer remains a leading cause of cancer-related death across the globe. Carcinogenic mechanisms associated with H. pylori are multifactorial and are driven by bacterial virulence constituents, host immune responses, environmental factors such as iron and salt, and the microbiota. Infection with strains that harbor the cytotoxin-associated genes (cag) pathogenicity island, which encodes a type IV secretion system (T4SS) confer increased risk for developing more severe gastric diseases. Other important H. pylori virulence factors that augment disease progression include vacuolating cytotoxin A (VacA), specifically type s1m1 vacA alleles, serine protease HtrA, and the outer-membrane adhesins HopQ, BabA, SabA and OipA. Additional risk factors for gastric cancer include dietary factors such as diets that are high in salt or low in iron, H. pylori-induced perturbations of the gastric microbiome, host genetic polymorphisms, and infection with Epstein-Barr virus. This chapter discusses in detail host factors and how H. pylori virulence factors augment the risk of developing gastric cancer in human patients as well as how the Mongolian gerbil model has been used to define mechanisms of H. pylori-induced inflammation and cancer.

1 Introduction In the majority of countries across the globe the overall incidence of gastric cancer (GC) is declining, however, it is still a serious condition and GC remains the fourth leading cause of cancer-related mortality worldwide behind lung, colorectal, and liver cancers (Sung et al. 2021). Adenocarcinoma is the most common type of cancer that L. E. Wroblewski · R. M. Peek Jr (B) Division of Gastroenterology, Hepatology, and Nutrition, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA e-mail: [email protected] L. E. Wroblewski e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 S. Backert (ed.), Helicobacter pylori and Gastric Cancer, Current Topics in Microbiology and Immunology 444, https://doi.org/10.1007/978-3-031-47331-9_2

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affects the stomach, but other types of cancer can also arise, including lymphoma and leiomyosarcoma. The Cancer Genome Atlas Network has identified four molecular subtypes of GC: (i) Chromosomal instability with intestinal-type histology, (ii) Microsatellite instability, (iii) Genomically stable, and (iv) Epstein-Barr virus (EBV)related (Cancer Genome Atlas Research 2014). However, in all anatomic regions of the stomach, chromosomal instability tumors with intestinal-type histology predominate. Intestinal-type adenocarcinoma is initiated by the transition from normal mucosa to chronic superficial gastritis; this is followed by the development of atrophic gastritis (loss of acid secreting parietal cells), SPEM (spasmolytic polypeptideexpressing metaplasia) and/or intestinal metaplasia, finally leading to dysplasia and adenocarcinoma (Fig. 1) (Correa 1996; Sipponen and Marshall 2000; Song et al. 2015). GC is a multifactorial disease, and the interplay between many different factors including, but not limited to, bacterial virulence factors, host genetics, diet, and the gastric microbiota can influence disease progression and outcome (Fig. 2). In this chapter, we discuss mechanisms driving the development of GC in humans and the use of the Mongolian gerbil as a model that recapitulates GC development in humans.

2 Epidemiology and Risk Factors for Gastric Cancer Development Each year, approximately 1 million people worldwide are diagnosed with GC, of whom approximately 800,000 will die (Pilleron et al. 2019). Although the overall incidence of GC has been decreasing in most parts of the world (Ferlay et al. 2021) the incidence and mortality rates of GC are increasing in certain populations. Overall, males are two to three times more likely to develop GC than females, and there are significantly more GC cases in less developed regions of the world compared to more developed regions. Approximately half of the total GC burden is found in East Asia, especially China and Japan. GC incidence and mortality rates are also higher in central and eastern Europe, South America, and Central America (Ang and Fock 2014). In the United States, GC rates are increasing specifically among young female and Hispanic male populations (Siegel et al. 2015; Anderson et al. 2018).

2.1 Helicobacter pylori Helicobacter pylori infects more than 4.4 billion individuals and is the strongest known risk for GC, prompting its designation in 1994 as a WHO Group I carcinogen and a high-priority pathogen for which new therapies are urgently needed (Savoldi et al. 2018; Hooi et al. 2017). H. pylori selectively colonizes the gastric epithelium and has colonized humans for more than 60,000 years. Infection is usually acquired

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Fig. 1 The sequential stages of gastric carcinoma modified from the Correa cascade. H. pyloriinduced inflammation is the strongest known risk factor for gastric carcinoma. H. pylori colonize the gastric mucosa of approximately 50% of the world’s population. Almost all infected individuals develop gastritis and 1–3% of infected individuals will develop gastric cancer. In the gerbil model, almost all colonized gerbils develop gastritis and the majority develop gastric carcinoma

in childhood and in the absence of combined antibiotic therapy, can persist for the lifetime of the host (Wroblewski et al. 2010). Most H. pylori-infected individuals remain asymptomatic, but approximately 1–3% will develop gastric adenocarcinoma, and 0.1% develop MALT lymphoma (Hooi et al. 2017). In addition to H. pylori, other components of the gastric microbiota may also influence gastric disease progression

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Fig. 2 Risk factors for developing gastric cancer in the context of chronic H. pylori infection. Host diet, the gastric microbiome, host cell factors and infection with Epstein-Barr virus may all influence disease outcome in the context of H. pylori infection

(see Sect. 2.6 Human microbiome and chapter “Gastric Cancer: The Microbiome Beyond Helicobacter pylori” of this book).

2.2 H. pylori Virulence Factors Colonization with H. pylori is the strongest known risk factor for developing GC, however, most infected individuals do not develop GC. H. pylori virulence factors including urease, adhesins, vacuolating cytotoxin A and the cag pathogenicity island (cagPAI), play key roles in determining disease outcome (Cover and Blanke 2005; Franco et al. 2008; Wroblewski et al. 2010; Koch et al. 2015; Pachathundikandi et al. 2015) (Fig. 3). The best-studied factor involved in pathogenicity is the cagPAI, which contains genes that encode for proteins that form a bacterial type IV secretion system (T4SS) and the oncoprotein CagA (Fischer et al. 2020; for more details see Chapter “Impact of the Helicobacter Pylori Oncoprotein CagA in Gastric Carcinogenesis” of this book). CagA is translocated through the cagT4SS by adherent H. pylori across the bacterial and epithelial membranes into host cells. Approximately 60% of H. pylori isolates from Western countries contain the cagPAI and almost all strains from East Asia harbor this locus (Odenbreit et al. 2000; Fischer et al. 2001; Kwok et al. 2007; Shaffer et al. 2011). Infection with cagA-positive H. pylori strains is associated with developing intestinal and diffuse gastric adenocarcinoma at 2–3 times the frequency compared to persons infected with H. pylori strains that are cagA-negative (Parsonnet et al. 1997; Huang et al. 2003).

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Fig. 3 H. pylori virulence factors involved in the development of gastric cancer. H. pylori is genetically highly variable, and some strains are more strongly associated with the development of gastric disease. This schematic summarizes some of the host-cell alterations that H. pylori virulence factors elicit

The carboxyl-terminal part of CagA contains repeat phosphorylation glutamateproline-isoleucine-tyrosine-alanine (EPIYA) motifs, which may also be used as indicators of pathologic outcome (Hatakeyama 2004; Higashi et al. 2005; Naito et al. 2006). Four different EPIYA motifs (EPIYA-A, -B, -C, or –D) have been identified (Hatakeyama 2004; Higashi et al. 2005; Naito et al. 2006; Lind et al. 2014, 2016). EPIYA-A and EPIYA-B motifs are found in most strains, while the EPIYA-C motif is predominately found in Western strains and the number of EPIYA-C sites is associated with an elevated risk of developing GC in multiple populations (Basso et al. 2008; Estaji et al. 2022; Rodriguez Gomez et al. 2020). Strains that contain the EPIYA-D motif are typically East Asian strains and are associated with increased pathogenesis compared to strains harboring C-type CagA motifs (Hatakeyama 2004; Argent et al. 2008). Following translocation, CagA becomes tyrosine-phosphorylated at EPIYA motifs and can induce cellular responses with carcinogenic potential. The primary phosphorylation site is the tyrosine of the EPIYA-C or -D motif and phosphorylation of these motifs is required for binding to the Src homology region 2 domaincontaining phosphatase (SHP2) and increasing pathogenicity through mechanisms such as cytoskeletal rearrangements, cell proliferation and inflammatory responses

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(Mueller et al. 2012; Backert and Tegtmeyer 2017; Hatakeyama 2014; Higashi et al. 2004, 2002; Li et al. 2018). CagA also exerts phosphorylation-independent effects within host cells that contribute to pathogenesis. Non-phosphorylated CagA targets many cellular effectors including apical-junctional components, the hepatocyte growth factor receptor c-Met, the phospholipase PLC-γ, the adaptor protein Grb2, and the kinase PAR1b/ MARK2, leading to pro-inflammatory and mitogenic responses, disruption of cell– cell junctions, and loss of cellular polarity (Mimuro et al. 2002; Saadat et al. 2007; Murata-Kamiya et al. 2007; Churin et al. 2003; Amieva et al. 2003; Franco et al. 2005; Bagnoli et al. 2005; Suzuki et al. 2005; Takahashi-Kanemitsu et al. 2020). Independent of CagA, H. pylori can also induce mislocalization of the tight junction proteins occludin and claudin-7 and alter barrier function (Wroblewski et al. 2009, 2015). The protease high temperature requirement A (HtrA) protein family are serine proteases (Tegtmeyer et al. 2016). HtrA from H. pylori is a secreted virulence factor involved in the disruption of cellular junctions by cleaving occludin, claudin-8 and Ecadherin (Hoy et al. 2010; Schmidt et al. 2016; Tegtmeyer et al. 2017), and permits H. pylori to colonize and persist under harsh conditions (Zarzecka et al. 2019). Recently, H. pylori isolated from humans was found to contain natural mutations in the htrA gene, which resulted in an amino acid change at position 171 that was associated with protein trimer stability (Zarzecka et al. 2023). Further analysis identified a single nucleotide polymorphism (SNP) in HtrA at serine/leucine 171 that significantly correlated with GC. In vitro, the 171S-to-171L mutation activated HtrA trimer formation, which resulted in disruption of the junctional proteins occludin and E-cadherin, increased translocation of CagA, increased accumulation of β-catenin in cell nuclei, and augmented host DNA double-strand breaks, all pre-malignant changes (Sharafutdinov et al. 2023) (Fig. 3). Another widely studied H. pylori virulence factor is the cytotoxin VacA, which elicits multiple effects on host cells including vacuolation, altered plasma and mitochondrial membrane permeability, autophagy, and apoptosis (Cover and Blanke 2005; Boquet and Ricci 2012; Caso et al. 2021) (Fig. 3). The vacA gene is found in all strains of H. pylori and contains a number of variable loci in the 5’ region termed s, i and m regions. This 5’ terminus encodes the signal sequence and amino-terminus of the secreted toxin (allele types s1a, s1b, s1c, or s2), an intermediate region (allele types i1 or i2), and a mid-region (allele types m1 or m2) (Atherton et al. 1995; Rhead et al. 2007). Strains containing type s1, i1, or m1 alleles are more strongly associated with GC (Atherton et al. 1995, 1997; Miehlke et al. 2000). Infection with H. pylori VacA s1m1 strains is associated with increased risk for developing peptic ulcer disease (Matos et al. 2013). Similarly, Latin American strains with s1 and m1 genotypes increase the risk for developing gastric cancer and peptic ulcers. African strains with the s1 or m1 genotypes also augment the risk of peptic ulcers and gastric cancer (Sugimoto and Yamaoka 2009). In middle eastern populations, the s1m1 genotypes significantly increased the risk of gastric cancer and peptic ulcers (Sugimoto et al. 2009b).

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In human gastric organoids, VacA m1 and m2 alleles have been shown to have similar vacuolating activity (Caston et al. 2020). VacA and CagA may also counterregulate each other’s actions to manipulate host cell responses (Backert and Tegtmeyer 2010; Barker et al. 2010; Tsugawa et al. 2012). VacA is secreted from H. pylori as an 88 kDa monomer (p88) and assembles into multiple water-soluble oligomeric structures, including hexamers, heptamers, dodecamers, and tetradecamers (Chambers et al. 2013; Lupetti et al. 1996; Adrian et al. 2002). Oligomerization appears to be essential for VacA activity (Ivie et al. 2008; Genisset et al. 2006), and recent studies using Cryo-EM have provided structural insights into the process of VacA oligomerization (Su et al. 2019). H. pylori genomes contain approximately 30 different hop genes, which encode outer membrane proteins. HopQ binds to the human Carcinoembryonic Antigenrelated Cell Adhesion Molecule (CEACAM) receptor on the host cell surface and the interaction between HopQ and CEACAM has been shown to facilitate CagA translocation via the T4SS (Javaheri et al. 2016; Zhao et al. 2018; Nguyen et al. 2023; Belogolova et al. 2013; Moonens et al. 2018). There are two different families of hopQ alleles: type I and type II. Type I hopQ alleles were found significantly more frequently in cag-positive/type s1-vacA allele strains from patients with peptic ulcer disease, whereas type II hopQ alleles were more frequently observed in cagnegative/type s2-vacA strains from patients without peptic ulcer disease (Cao and Cover 2002).

2.3 Dietary Factors (Salt/Iron) The risk of developing gastric adenocarcinoma is also influenced by environmental factors such as diet. Diets that are high in salt, pickled, smoked or poorly preserved foods, those with a high meat content, and those with low fruit and vegetable content are most commonly associated with an increased risk for developing GC. Conversely, diets plentiful in fruits and vegetables are associated with less risk for developing GC (Tsugane and Sasazuki 2007; Epplein et al. 2008; Gonzalez et al. 2006, 2012; Kim et al. 2010, 2004; Ren et al. 2012). In the context of H. pylori infection, high dietary salt intake and low iron levels are highly associated with an increased risk for developing GC (Lee et al. 2003; Shikata et al. 2006; Noto et al. 2013a, 2022, 2015; Noto and Peek 2015; Loh et al. 2023). Iron deficiency is the most common nutritional disorder in the world and can be a result of a diet deficient in iron, blood loss, or colonization by certain H. pylori strains, which have been associated with hemorrhagic gastritis (Yip et al. 1997). Chronic H. pylori infection can further exacerbate iron deficiency through the development of gastric atrophy which leads to decreased acid secretion, reduced ascorbic acid levels and decreased iron absorption (Yip et al. 1997). Iron deficiency in H. pylori-infected individuals accelerates the development of gastric adenocarcinoma by increasing the virulence potential of H. pylori, and this has been further studied in the Mongolian gerbil model (see Sect. 3 for further discussion) (Noto et al. 2013a, 2022) and

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mouse models. Further, iron deficiency has been associated with increased levels of secondary bile acids, such as deoxycholic acid, in H. pylori-infected animals. A retrospective cohort analysis of the association between the use of bile acid sequestrant medications and the incidence of GC in 416,885 patients suggests that bile acid sequestrants are protective against GC (Noto et al. 2022). Furthermore, expression of the bile acid receptor, TGR5, paralleled the severity of gastric disease in humans. Expression of TGR5 increased in the progression from atrophic gastritis to intestinal metaplasia, dysplasia, and finally GC (Noto et al. 2022). In addition to host iron levels, high salt diets have also been found to modulate the virulence potential of H. pylori. The association between high dietary salt intake and increased GC risk has been reported in both humans and Mongolian gerbil models (discussed in Sect. 3) of H. pylori infection (Gaddy et al. 2013; Bergin et al. 2003). A cross-sectional study performed in Japan identified a significant correlation between the amount of salt excreted in urine and GC mortality rates in both men and women (Tsugane et al. 1992). In animal models and in vitro models, high salt diets have been reported to increase the expression of the H. pylori virulence factors CagA, VacA and UreA, in addition to selecting fur mutations that confer a selective advantage to H. pylori in high-salt conditions (discussed further in Sect. 3.4) (Loh et al. 2007, 2012, 2023, 2018; Gancz et al. 2008; Voss et al. 2015; Caston et al. 2019).

2.4 Host Constituents H. pylori induces a robust inflammatory response in its host and specific host gene polymorphisms are associated with further increases in the risk of developing GC (Usui et al. 2023). H. pylori infection increases gastric mucosal expression of the T helper (Th) type 1 (Th1) pro-inflammatory cytokine and inhibitor of gastric acid secretion, IL-1ß (Noach et al. 1994). In the context of H. pylori infection, individuals who harbor polymorphisms in IL-1ß that result in high expression levels of IL-1ß are at significantly higher risk for developing distal gastric adenocarcinoma compared to those with genotypes that limit IL-1ß expression (El-Omar et al. 2000). IL-1β polymorphisms can promote GC by augmenting IL-1β production, decreasing gastric acid production and increasing circulating cytokine levels (Furuta et al. 2002; El-Omar et al. 2001; Fox and Wang 2007). Individuals with high-expressing IL-1ß polymorphisms in conjunction with H. pylori cagA+ or vacA s1-type strains have a 25-fold or 87-fold increase in risk, respectively, for developing GC compared to uninfected individuals (Figueiredo et al. 2002). Polymorphisms that increase expression of the pro-inflammatory cytokine TNF, or that decrease the production of anti-inflammatory cytokines such as Il-10 are also associated with an increase in risk of developing GC (El-Omar et al. 2003).

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2.5 Epstein-Barr Virus (EBV) EBV is a human herpes virus that is associated with GCs. EBV-positive GCs comprise almost 10% of GCs and represent a distinct subset of GC identified by The Cancer Genome Atlas (TCGA) (Cancer Genome Atlas Research 2014). Synergistic interactions between EBV and H. pylori in the gastric epithelium may promote progression towards GC. A case–control study has shown that the combination of EBV and H. pylori induces severe inflammation and, in this way, augments the risk of developing intestinal type GC (Cardenas-Mondragon et al. 2015). In a recent study, EBV was shown to methylate the host phosphatase SHP1 and thereby prevent SHP1 from dephosphorylating CagA. This perturbation increases the oncogenic activity of CagA and may augment the synergistic effect of EBV and H. pylori (Saju et al. 2016).

2.6 Human Microbiome The development of a new technology for analyzing microbial communities has expanded our understanding of the human gastric microbiome. The gastric microbiome is known to play a critical role in maintaining homeostasis, while perturbations in the microbiome can contribute to the development and progression of GC (Guo et al. 2020; Ai et al. 2023; Mannion et al. 2023). In the normal human stomach, Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, and Fusobacteria are the major phyla identified (Bik et al. 2006; Liu et al. 2019, 2022; Delgado et al. 2013). In contrast, in H. pylori-infected individuals, H. pylori is the predominant bacterium found in the stomach and microbial diversity is decreased (Yu et al. 2017; Ndegwa et al. 2020; Mannion et al. 2023). In a study using reverse transcribed 16S rRNA as the amplification template, the metabolically active bacteria found in the upper gastrointestinal tract in individuals with and without H. pylori infection was analyzed (Schulz et al. 2018). In the stomach, consistent with other studies, Helicobacter species were found to dominate the microbiota in H. pylori -infected individuals, and the relative abundance of Actinobacteria, Bacteroidetes, Firmicutes and Fusobacteria were decreased (Schulz et al. 2018). Infection with H. pylori was also shown to influence the microbiota of the duodenum and oral cavity (Schulz et al. 2018). In a prospective population-based study, H. pylori was identified as one of the main factors in gastric microbial dysbiosis and this dysbiotic microbiota was associated with chronic atrophic gastritis, intestinal metaplasia and dysplasia. Successful H. pylori eradication resulted in restoration of the gastric microbiota to similar status of uninfected individuals (Guo et al. 2020). A population-based study in China using deep sequencing identified that the microbiota in chronic atrophic gastritis or intestinal metaplasia exhibited lower abundances of Actinobacteria, Bacteroidetes, Firmicutes and Fusobacteria compared to normal or superficial gastritis. Actinobacteria, Bacteroidetes and Firmicutes were found in greater abundance in dysplasia/ GC compared to intestinal metaplasia (Kadeerhan et al. 2021). A meta-analysis

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of microbiota from 6 independent studies across the stages of GC development identified Veillonella, Dialister, Granulicatella, Herbaspirillum, Comamonas, Chryseobacterium, Shewanella and Helicobacter as biomarkers for discerning GC from superficial gastritis (Fischer et al. 2022). Further, gastric transplantation of stomach microbiota harvested from patients with premalignant and malignant lesions into germ-free (GF) mice induced intestinal metaplasia and dysplasia (Kwon et al. 2022). Other investigators have defined the gastric microbiota in adults residing in either a low-risk or a high-risk GC region in Colombia. Significant differences were present (Yang et al. 2016), and colonization with either of 2 such differentially abundant species modified the ability of H. pylori to induce gastric injury in GF mice (Shen et al. 2022). In a 15-year intervention study, antibiotic treatment targeting H. pylori significantly reduced the incidence of GC, even though fewer than half of treated individuals remained free of H. pylori infection (Ma et al. 2012), suggesting that antibiotics that modify the microbiota can attenuate the development of GC despite the presence of H. pylori. See chapter “Gastric Cancer: The Microbiome Beyond Helicobacter pylori” of this book for further details.

2.7 Yes-Associated Protein Yes-Associated Protein (YAP) is a key effector of the Hippo tumor suppressor pathway. Li et al. reported that YAP is significantly upregulated in human gastric carcinogenesis and H. pylori infection induced YAP and downstream effectors in gastric epithelial cells in a cag-dependent manner (Li et al. 2018). Recently, it has been demonstrated that the interaction between CagA and PAR1b prevents nuclear translocation of the tumor suppressor, BRCA1, by inhibiting PAR1b kinase-mediated phosphorylation of BRCA1 (Imai et al. 2021). Nuclear expression of BRCA1 (BRCAness) was found to lead to replication fork instability and subsequent DNA double-strand breaks; however, cells expressing CagA were found to evade apoptosis. Simultaneously, apoptosis was shown to be suppressed through activation of Hippo signaling via CagA-mediated PAR1b inhibition, which prevented formation of the YAP/p73 pro-apoptotic complex and allowed cells the ability to repair double-strand breaks through error-prone mechanisms. In the presence of functional p53, proliferation of CagA expressing cells is inhibited by p21. In the absence of p53, CagA-expressing cells displaying BRCAness proliferate. Since loss of cellular p53 usually occurs due to aging-associated somatic TP53 mutation this may explain why H. pylori-infection in young individuals results in mostly mild disease while in elderly individuals, GC is more prevalent (Imai et al. 2021).

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3 Mongolian Gerbils as a Model for Gastric Cancer Development Mice remain the most frequently used animal model for the investigation of H. pylori-induced gastric carcinogenesis; however, the Mongolian gerbil is also used for reasons outlined below. The Mongolian gerbil is a small rodent member of the Cricetidae family. The gerbil has been increasingly used in research focused on H. pylori pathogenesis as it represents an efficient and cost-effective rodent model that recapitulates many features of H. pylori-induced gastric inflammation and carcinogenesis in humans, allowing for targeted investigation of the bacterial determinants and environmental factors that lead to H. pylori-induced disease.

3.1 Gastric Cancer Development in Mongolian Gerbils In 1991, the first published description of the Mongolian gerbil model reported that following oral inoculation, H. pylori colonized the gastric-mucosal layer of gerbils and induced mild gastritis following a two-month infection (Yokota et al. 1991). Similar to the disease process in humans, subsequent studies demonstrated that gerbils develop gastric ulcers, duodenal ulcers and intestinal metaplasia following long-term infection with H. pylori (Hirayama et al. 1996; Matsumoto et al. 1997; Honda et al. 1998; Ikeno et al. 1999; Ohkusa et al. 2003; Franco et al. 2008). Carcinomas that developed in H. pylori-infected gerbils typically occurred in the distal stomach and the pyloric region and contained well-differentiated intestinaltype epithelium, reflecting many features of intestinal-type gastric adenocarcinoma in humans. Consistent with reports in humans, H. pylori eradication in the gerbil model significantly reduced the severity of gastritis, premalignant lesions, and incidence of gastric adenocarcinoma (Matsumoto et al. 1997; Keto et al. 2001; Nozaki et al. 2002; Nozaki et al. 2003)}. In humans, two types of metaplasia can develop following H. pylori colonization, inflammation, and gastric atrophy: intestinal metaplasia and SPEM (see Introduction and Fig. 1). The development of intestinal-type GC is associated with intestinal metaplasia and SPEM; however, investigations into the origin of intestinal metaplasia have been somewhat limited because mice do not develop intestinal metaplasia in response to H. pylori infection (Correa 1988; Hattori 1986; Hattori and Fujita 1979; Xia et al. 2000; Schmidt et al. 1999; Yamaguchi et al. 2002; Halldorsdottir et al. 2003). Mongolian gerbils infected with H. pylori, however, do develop intestinal metaplasia, dysplasia and cancer making them fundamentally different from mouse models and more similar to humans (Hirayama et al. 1996; Honda et al. 1998; Watanabe et al. 1998; Yoshizawa et al. 2007). Recent studies using Mongolian gerbils have demonstrated that H. pylori-infected gerbils developed SPEM early and within 9 weeks of

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infection. SPEM initially occurred in the intermediate zone along the lesser curvature and subsequently invaded the greater curvature, which is similar to the development of metaplasia in humans. In early stages of H. pylori infection in gerbils, SPEM is organized in straight glands; however, at later stages of infections, SPEM glands became distorted and expanded. Following 6 months of infection, intestinal metaplasia developed (Yoshizawa et al. 2007).

3.2 Host Constituents Mongolian gerbils were the first model used to identify the role of IL-1β in the development of GC. IL-1β is a Th1-type cytokine that is increased within the gastric mucosa of H. pylori-infected individuals (Noach et al. 1994; see also Sect. 2.4 Host constituents). In gerbils infected with H. pylori for 6 or 12 weeks, IL-1β levels increased, while gastric acid secretion decreased. Moreover, treatment of H. pyloriinfected gerbils with an IL-1β antagonist abolished the loss of acid secretion, thus implicating IL-1β in the development of achlorhydria in the stomach of H. pyloriinfected gerbils (Takashima et al. 2001). Experiments using gerbils have also highlighted altered expression of other inflammatory mediators including inducible nitric oxide synthase (iNOS) and COX2 following H. pylori infection (Matsubara et al. 2004; Sakai et al. 2003). Cyclooxygenase 2 (Cox-2) mRNA expression was significantly increased following a 1- and 3-month infection with H. pylori, and this was not seen in gerbils infected with H. pylori lacking the T4SS component CagE (Sakai et al. 2003). Expression levels of IL-1β, TNF and iNOS mRNA were shown to be increased following a 2-week H. pylori infection, while in the fundic region, protein expression levels of IL-1β, TNF and iNOS were increased following a 4- and 8-week infection in gerbils (Matsubara et al. 2004). The gerbil model has also been used to demonstrate the role of transcription factor NF-κB activation within the context of H. pylori-induced inflammation. In an independent study, quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ) was used to compare gastric cell scrapings from H. pylori-infected and uninfected gerbils. 2764 proteins were quantified and 166 were significantly altered in abundance by H. pylori infection. Pathway mapping identified significant changes in many signaling pathways including those involved in inflammation, proliferation, differentiation, apoptosis, and regulation of the cell cycle (Noto et al. 2019a). H. pylori infection of gerbils has also been shown to increase serum levels of gastrin, which can promote gastric epithelial cell proliferation (Peek et al. 2000; Konturek et al. 2003). The Mongolian gerbil model has also been a very useful model for investigating ‘The Colombian Enigma’. Almost 90% of the Colombian population is infected with H. pylori; however, the incidence rates of GC differ greatly in high versus low altitude regions. In the high altitude Andean region, there is a higher prevalence of precancerous lesions compared to the low risk coastal region (de Sablet et al. 2011;

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Correa et al. 1976). Mongolian gerbils that were infected with H. pylori strains originating from the high-risk region induced more spermine oxidase (SMOX), oxidative DNA damage, dysplasia and adenocarcinoma than H. pylori from the low-risk region suggesting that activation of polyamine-driven oxidative stress could be used as a marker of GC risk and a target for chemoprevention (Chaturvedi et al. 2015).

3.3 Crucial H. pylori Determinants in Mongolian Gerbils Many advances have been made utilizing the gerbil model to investigate cancerassociated microbial determinants. As discussed in Sect. 2.2, cagPAI is one of the most studied H. pylori virulence factors. One limitation of using murine models of H. pylori infection is that clinical cag+ strains often fail to colonize (Sozzi et al. 2001; Philpott et al. 2002). In contrast, clinical cag+ strains of H. pylori readily colonize gerbils and maintain a functional cagT4SS secretion system (Peek et al. 2000), which together with the development of more severe disease, allows for a robust investigation of the role of cagPAI in the context of H. pylori-induced inflammation and cancer. As with human infections, gerbils infected with cag+ strains develop significantly more severe gastritis than those infected with cag− strains, echoing the importance of the cag island in H. pylori-mediated inflammation and pathogenesis (Ogura et al. 2000; Saito et al. 2005; Ohnita et al. 2005; Shibata et al. 2006). In vivo adaptation of H. pylori strains has been demonstrated to increase the virulence potential of H. pylori strains in gerbils. In one study, a single gerbil was infected with a human H. pylori isolate, 3 weeks post-challenge a single colony output derivative was isolated and used to infect an independent population of gerbils. Following in vivo adaptation, gastric dysplasia and adenocarcinoma developed, phenotypes that were not observed with the input H. pylori strain (Franco et al. 2005). Serial infections of gerbils with H. pylori have provided insights into how the host modifies cagT4SS function through alterations in cagY, a structural component of the cag T4SS. Changes in the genetic composition of cagY were shown to parallel cagT4SS function, and the development of dysplasia or cancer selected for attenuated cagT4SS virulence phenotypes (Suarez et al. 2017). In terms of genetic analysis, using whole genome sequencing techniques, sequences of H. pylori strains isolated from experimentally infected gerbils were compared to the sequence of the input strain (Beckett et al. 2018). The mean annualized SNP rate per site was similar to rates reported in H. pylori-infected humans. Many of the mutations occurred within or upstream of genes that are associated with iron-related functions ( fur, tonB1, fecA2, fecA3, and frpB3) or which encoded outer membrane proteins (alpA, oipA, fecA2, fecA3, frpB3 and cagY ). One of the SNPs detected in the output strains, FurR88H, conferred a survival advantage when H. pylori was co-cultured with neutrophils (Beckett et al. 2018). In studies focused on the T4SS per se, a recent study utilized a tetracyclin repressor (tetR)/tetracycline operator (tetO) system to conditionally regulate cagT4SS activity in the gerbil model of H. pylori infection (Lin et al. 2020). In experiments, where

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gerbils were exposed to more than three months of continuous cagT4SS activity, higher rates of dysplasia and/or GC were observed than when cagT4SS activity was limited to early or late stages of infection. However, when the activity of cagT4SS was confined to just the initial 6 weeks of infection, gastric inflammation still developed, and GC was detected in a small fraction of gerbils (Bartpho et al. 2020). These data support a hit-and-run model of carcinogenesis whereby an infectious agent triggers carcinogenesis during the initial stages of infection and the ongoing presence of the infectious agent is not required for development of cancer. The Mongolian gerbil model has also been used to investigate other H. pylori virulence factors including the role of the OMP OipA in H. pylori-induced gastric pathogenesis (Franco et al. 2008; Sugimoto et al. 2009a). Mongolian gerbils infected with oipA-deficient mutants developed significantly less inflammation than gerbils infected with wild-type strains and did not develop gastric dysplasia or GC (Franco et al. 2008). These findings are consistent with human population data (see Sect. 2.2 H. pylori virulence factors).

3.4 Dietary Factors in the Gerbil (Salt/Iron) The gerbil model has been widely used to investigate potential relationships between diet and GC risk in the context of H. pylori infection. Epidemiologic studies of diet in humans are limited by reliance on accurate patient reporting and difficulty in ascertaining diets that were consumed decades prior to the development of GC. Increased salt consumption has been shown to increase the risk for GC in humans (Sect. 2.3 Dietary factors), and the effects of high-salt diets on H. pylori infection and GC have also been investigated using the gerbil model. One study showed that gerbils maintained on a high-salt diet that were H. pylori-infected had a significantly higher incidence of gastric adenocarcinoma than H. pylori-infected gerbils maintained on a normal-salt diet (Gaddy et al. 2013). In a follow up study, H. pylori strains were analyzed from experimentally infected gerbils, and compared to input strains; the output strains from gerbils maintained on a high-salt diet produced higher levels of proteins involved in iron acquisition, including a mutation in fur (encoding the ferric uptake regulator variant Fur-R88H) and resistance to oxidative stress (Loh et al. 2015). In a recently published work, it was reported that the fur-R88H mutation augments H. pylori fitness in vitro under high-salt conditions and exerts the opposite effect under normal-salt conditions. FecA is a known ferric citrate transporter and analysis of the transcription profiles revealed that fecA2 plays a role in H. pylori fitness under both high-salt environments and normal salt environments (Loh et al. 2023). The role of iron deficiency in influencing disease outcome in the context of H. pylori infection has also been studied in Mongolian gerbils. In a study where gerbils were maintained on iron-replete or iron-depleted diets and then challenged with H. pylori, more severe gastritis and increased frequency of gastric dysplasia and gastric adenocarcinoma were reported among gerbils maintained on iron-depleted

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diets compared to gerbils maintained on iron-replete diets (Noto et al. 2013b). These phenotypes were only present in animals infected with a cagA+ strain and infection with a cagA− isogenic mutant strain abrogated the response (Noto et al. 2013b). H. pylori output strains isolated from gerbils maintained on an iron-depleted diet exhibited an enhanced ability to translocate CagA and induced higher levels of IL-8 compared to output strains isolated from gerbils maintained on an iron-replete diet (Noto et al. 2013b). It has also been demonstrated that H. pylori infection causes iron deficiency anemia in the Mongolian gerbil model. In the presence of H. pylori infection, gerbils maintained on a high-salt/low-iron diet for 16 weeks exhibited a higher incidence and an increased severity of iron deficiency anemia compared to H. pylori-infected gerbils maintained on a regular diet (Beckett et al. 2016).

3.5 Gerbil Microbiome In comparison to the human and mouse microbiome, very little is known about the gerbil microbiome. There have been a limited number of studies to determine if H. pylori induces dysbiosis of the gastric mucosal microbiota similar to what occurs in humans. Using qualitative and quantitative DNA- and RNA-based taxonomic microbiota analyses, human, mouse, and gerbil stomach samples were demonstrated to exhibit similarities at higher taxonomic levels but differences at lower taxonomic levels (Wurm et al. 2018). Microbiota changes in H. pylori infected Mongolian gerbils have also been studied and—like in humans—the microbiota of Mongolian gerbils is modified by long-term infection with H. pylori (Yin et al. 2011; Osaki et al. 2012; Heimesaat et al. 2014). Lactobacillus, Bifidobacterium, Clostridia, and Enterococcus were abundantly expressed among both H. pylori-infected and uninfected gerbils; however, the abundance of Bifidobacterium and Clostridia were significantly lower among H. pylori-negative gerbils (Osaki et al. 2012). Recently, the gerbil gastric mucosal microbiota, within the context of H. pylori infection and low iron has been more definitively defined using 16S rRNA sequencing. Infection with H. pylori was found to significantly decrease α-diversity and alter microbial community structure in a cagA-dependent manner. Concordant with earlier reports, Lactobacillus was found in abundance, but other abundant operational taxonomic units were different and included Enterobacteriaceae and Porphyromonadaceae. When gerbils were infected with H. pylori and maintained on an iron-deplete diet there were no significant differences in α- or β-diversity, phyla, or operational taxonomic unit abundance compared to infected gerbils maintained on iron-replete diets, despite increased H. pylori-induced injury in the gerbils maintained on an iron-deplete diet. Interestingly, when microbial composition was stratified based only on the severity of gastric injury, significant differences in α- and β-diversity were present among gerbils harboring premalignant or malignant lesions compared to gerbils with gastritis alone (Noto et al. 2019b).

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4 Concluding Remarks Globally, GC leads to a high number of cancer-related deaths each year. Infection rates of H. pylori vary across the globe, with some areas approaching 100%, however, 97–99% of colonized persons will never develop GC. The risk of developing GC is dependent on numerous factors including H. pylori strain-specific virulence factors, the host genotype, environmental factors such as diet as well as the microbiome. The gerbil model has provided and will continue to provide critical information on the interactions among these factors and will aid in understanding the dynamic of host genetics, dietary factors, and the microbiome in the context of chronic H. pylori infection, with the goal of identify individuals who are at the highest risk of developing GC.

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Mitochondrial Function in Health and Disease: Responses to Helicobacter pylori Metabolism and Impact in Gastric Cancer Development Javier Torres and Eliette Touati

Abstract Mitochondria are major cellular organelles that play an essential role in metabolism, stress response, immunity, and cell fate. Mitochondria are organized in a network with other cellular compartments, functioning as a signaling hub to maintain cells’ health. Mitochondrial dysfunctions and genome alterations are associated with diseases including cancer. Mitochondria are a preferential target for pathogens, which have developed various mechanisms to hijack cellular functions for their benefit. Helicobacter pylori is recognized as the major risk factor for gastric cancer development. H. pylori induces oxidative stress and chronic gastric inflammation associated with mitochondrial dysfunction. Its pro-apoptotic cytotoxin VacA interacts with the mitochondrial inner membrane, leading to increased permeability and decreased ATP production. Furthermore, H. pylori induces mitochondrial DNA damage and mutation, concomitant with the development of gastric intraepithelial neoplasia as observed in infected mice. In this chapter, we present diverse aspects of the role of mitochondria as energy supplier and signaling hubs and their adaptation to stress conditions. The metabolic activity of mitochondria is directly linked to biosynthetic pathways. While H. pylori virulence factors and derived metabolites are essential for gastric colonization and niche adaptation, they may also impact mitochondrial function and metabolism, and may have consequences in gastric pathogenesis. Importantly, during its long way to reach the gastric epithelium, H. pylori faces various cellular types along the gastric mucosa. We discuss how the mitochondrial response of these different cells is affected by H. pylori and impacts the colonization and bacterium niche adaptation and point to areas that remain to be investigated.

J. Torres Unidad de Investigacion en Enfermedades Infecciosas, UMAE Pediatriıa, Instituto Mexicano del Seguro Social, Ciudad de Mexico, Mexico E. Touati (B) Equipe DMic01-Infection, Génotoxicité et Cancer, Département de Microbiologie, UMR CNRS 6047, Institut Pasteur, Université Paris Cité, F-75015 Paris, France e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 S. Backert (ed.), Helicobacter pylori and Gastric Cancer, Current Topics in Microbiology and Immunology 444, https://doi.org/10.1007/978-3-031-47331-9_3

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1 Introduction Mitochondria are central organelles playing essential roles for eukaryotic cells, not only energy production. Mitochondria function as a central signaling hub, coordinating stress responses, immunity, lipid, and amino acid metabolism, as well as cell fate decision, survival, or death (Shen et al. 2022). To ensure their crucial functions, mitochondria have established their own network with other organelles and cellular components. Mitochondria use this network to transport metabolites and small signaling molecules, and to transfer components through the formation of vesicles to other cell compartments. Mitochondria constantly receive information about cell status and answer by sending signals to maintain cell function and health through messenger molecules to regulate biosynthesis, metabolism, and to control protein folding and activity. Thus, the inter-organelle signaling and interconnection with subcellular compartments are essential for the correct function and health of the cell. Their dysfunction may be the origin of a plethora of diseases including cancer (Kopinski et al. 2021). As a key regulator of cell physiology, mitochondria also play a critical role in the defense against pathogens which have developed strategies to target this organelle and modify its functions to reprogram host cell physiology to their own benefit so that they can generate suitable niches for colonization, access to nutrients, persistence, and evasion of the immune response (Blanke 2005; Escoll et al. 2016). H. pylori colonizes the stomach of about 50% of the human population and can persist for decades in the harsh gastric environment, thanks to mechanisms developed to coexist with its host acquired after thousands of years of co-evolution. The gastric mucosa responds to the presence of H. pylori with a chronic inflammatory response that in most individuals becomes a state of homeostasis under very strict mechanisms of control. However, a fraction of the infected individuals (less than 2%), which after decades of H. pylori colonization, ends up in developing gastric cancer (GC) (for more details, see Chapter “ Clinical Pathogenesis and Molecular Mechanisms of Gastric Cancer Development”). As a result, close to 800,000 individuals die from GC annually (Correa 1992; Salama et al. 2013; Zhang et al. 2020). Clinical, epidemiological, and experimental evidence have confirmed that H. pylori is the main risk factor to develop GC; hence, it is the only bacterium recognized as a type 1 carcinogenic agent (IARC 1994, 2012). GC is the fifth and the fourth most common cancer and cause of cancer-related death, respectively. It is an insidious disease mostly due to its asymptomatic phenotype until advanced cancer stages resulting in a poor prognosis and high mortality. The H. pylori secreted-pore forming toxin VacA targets the mitochondria, then inserted in the mitochondrial inner membrane (MIM), leading to increased membrane permeability and cytochrome c release (Willhite and Blanke 2004; Galmiche et al. 2000) (see Sect. 3.2). VacA is a pleiotropic toxin which among others leads to disruption of mitochondrial function, modulation of autophagy, apoptosis as well as inhibition of T cell activation (Backert and Tegtmeyer 2010; Kim and Blanke 2012; Foegeding et al. 2016). It is up to now the only H. pylori factor known to be associated with mitochondrial dysfunction. However, we previously reported

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VacA-independent mechanisms modulating mitochondrial DNA (mtDNA) replication/transcription and import components, indicating that other H. pylori factors can be responsible for mitochondrial response (Chatre et al. 2017). During colonization of the gastric glands, H. pylori interacts with various cell types including gastric epithelial cells (GECs), parietal cells, chief cells, and immune and stem cells (SCs), each characterized by a specific function and metabolism. Thus, the consequences of the infection on mitochondria will depend on the mitochondrial activity and physiology of the cell type interacting with H. pylori. In this chapter, we present diverse aspects of the role of mitochondria as a signaling hub in cell metabolism and cell fitness. We also describe how and why mitochondria can be considered as a strategic target for pathogens and the consequences of the disruption of mitochondrial functions during host–pathogen interaction. The different mechanisms by which H. pylori infection affects mitochondrial metabolism and physiology, particularly the action of the VacA cytotoxin, are reviewed. We also discuss the mechanisms and strategies developed by H. pylori to successfully colonize the gastric mucosa and grow in specialized niches and how these affect the metabolic activity of mitochondria. MtDNA defects and mitochondria dysfunctions are associated with carcinogenesis and cancer progression (Kopinski et al. 2021). Their impact in the context of H. pylori-induced inflammation and GC will be discussed.

2 Mitochondria, Essential Organelle in Cell Physiology 2.1 Mitochondria Function with a Highly Dynamic Network Mitochondria are of bacterial origin, derived from a mitochondria-like αproteobacterium ancestor, engulfed about a billion years ago by anaerobic nucleated cells via phagocytosis to become endosymbiont for the benefit of both bacteria and host (Embley and Martin 2006). As bacteria, mitochondria have their own genome, a circular double-strand DNA molecule with CpG-rich motifs. In humans, mtDNA has a size of 16.6 kb present in multicopies (2–10/mitochondrion), with 37 genes coding for 13 components of the respiratory oxidative phosphorylation system (OXPHOS), 2 rRNAs and 22 tRNAs. All the other mitochondrial components are nuclear DNA (nDNA)-encoded, synthesized on cytoplasmic ribosomes and transferred to the organelle. The membrane composition of mitochondria is similar to that of prokaryotes. Within the membranes is the mitochondrial matrix which houses mtDNA, ribosomes, proteins, and metabolites (Boguszewska et al. 2020). Quality control mechanisms ensure mitochondria homeostasis and maintain mitochondria pool through a dynamic equilibrium between fusion and fission processes (Westermann 2010). Mitochondrial fusion joins two mitochondria at the mitochondrial inner membrane (MIM) and mitochondrial outer membrane (MOM) interfaces via the action of three membrane GTPases, mitofusin 1 (MFN1), mitofusin 2 (MFN2),

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and optic atrophy protein 1 (OPA1). Fusion allows the exchange of gene products and metabolites between the two fused mitochondria to complement defects. Mitochondrial fusion also results in extended mitochondrial networks, providing advantages to cells under high energy demand. In contrast, fission consists in the division of mitochondrion in two mitochondria. The main fission protein is a large dynaminlike GTPase, DRP1 (Liu et al. 2020). Fission is responsible for correct partitioning and cellular distribution of the organelles, and cytochrome c release during apoptosis. Both fusion and fission are crucial during mitosis to assure equal segregation of mitochondria between daughter cells. Their imbalance has been observed in several human diseases including cancer. Under stress conditions during the early phase of damage, the mitochondrial membrane potentially changes leading to membrane depolarization and activation of mitochondrial autophagy-related proteins. Damaged mitochondria are encapsulated by autophagosomes to form mitophagosomes, a process called mitophagy, critical to maintain proper cellular function (Onishi et al. 2021). After the fusion of mitophagosomes and lysosomes, mitolysosomes are formed leading to degradation of the mitochondria. The three known pathways involved in mitophagy include PINK1/Parkin, MOM, and MIM receptor-mediated pathways. Defects in mitophagy are associated with various human pathologies (Killackey et al. 2020). The best studied mitochondrial mechanism in response to cellular stress is apoptosis, a programmed cell death required for tissue homeostasis. Upon pro-apoptotic stress, BAX and BAK, two pro-apoptotic BCL2 homologs, induce mitochondrial outer membrane permeabilization (MOMP) and cytochrome c release (Fig. 1a). Then, cytochrome c binds to apoptosis protease activating factor 1 (APAF1) leading to the formation of apoptosome and activation of caspase-9, which then cleaves and activates caspase-3 and caspase-7 causing cell death (Bock and Tait 2020). MOMP also induces pro-inflammatory signaling (Bock and Tait 2020). The extrinsic pro-apoptotic pathway is initiated by tumor necrosis factor (TNF)-family cell death receptors that lead to caspase-8 activation and cell death through the activation of caspases-3 and -7. Under sublethal apoptotic stress conditions, caspase inhibition, and maintenance of metabolic activity, a small fraction of mitochondria undergoes MOMP, with restauration of mitochondria pool and cell survival (Ichim et al. 2015). This process called minority MOMP is a source of DNA damage, as observed in response to pathogens such as H. pylori. A recent study showed that an H. pylori sublethal induction of mitochondrial apoptosis pathway correlated with inflammation contributing to DNA damage and carcinogenesis (Dörflinger et al. 2022).

2.2 Mitochondria, a Central Biosynthetic and Signaling Hub In MIM, through OXPHOS ADP is phosphorylated to ATP by ATP synthase, the ultimate step of the mitochondrial respiratory chain complex. The electron transfer chain (ETC) consists in successive oxidation–reduction reactions and electron transfer from NADH and FADH2 to oxygen across five protein complexes (Fig. 1a). The Complex

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◄Fig. 1 Mitochondria respiratory and signaling functions. a Through the respiratory chain and TCA, mitochondria generate energy to cells in the form of ATP. Under stress conditions, mitochondria release cytochrome c to induce cell death. DAMPs released by mitochondria into the cytosol include ROS that activates transcription factors, N-FP corresponding to formylated peptides, and mtDNA which are essential in the activation of inflammation and innate immune response. DAMPs also include cardiolipin, a unique phospholipid component of MIM, required for optimal activity of OXPHOS and ATP production. Cardiolipin is also involved in immune signaling. The TCA generates NADH and FADH2 required to transfer electrons to ETC in MIM. A functional ETC generates mitochondrial membrane potential required to produce ATP. This process referred to as OXPHOS required the presence of O2 . Complexes I and II led to replenish NAD + and FAD, respectively, to the TCA. TCA metabolites such as fumarate, succinate, α-KG, and acetyl CoA play a signaling role with direct consequences on chromatin remodeling, resulting in deregulation of gene expression and impact on innate and adaptative immunity (fumarate, succinate, acetyl CoA), stem cells pluripotency and lymphangiogenesis (α-KG, acetyl CoA), and tumorigenesis (fumarate, succinate). Mitochondria also directly interact with other cellular compartments as illustrated with ER, constituting a site of transfer of cholesterol, Ca2+ , and lipids. b TCA is a source of metabolites as signaling molecules in main biosynthetic pathways including the synthesis of fatty acids, amino acids, and nucleotides (see text for details). ACLY: ATP-citrate lyase; ACSS1: acetyl-CoA synthetase short-chain family member 1; α-KG:α-ketoglutarate; CS: citrate synthase; CPS: carbamoyl phosphate synthase; ER: endoplasmic reticulum; FH: Fumarate hydratase; GDH: glutamate dehydrogenase; GLS: glutaminase; GOT2: glutamic-oxaloacetic transaminase 2; c, m LDH: cytosolic or mitochondrial lactate dehydrogenase; MPC: mitochondrial pyruvate carrier; MIM, MOM: mitochondrial inner or outer membrane; NFP: N-formylated peptide; PC: pyruvate carboxylase; SDH: succinate dehydrogenase

I, a NADH:coenzyme Q oxidoreductase of 46 subunits, catalyzes the oxidation of NADH by quinone coenzyme Q10 (CoQ10) to transfer two electrons to Complex III and export 4 protons (H+ ) from the mitochondrial matrix to the intermembrane space, creating a H+ gradient at the origin of a membrane potential across the MIM. Complex II includes 4 subunits, referred to as succinate dehydrogenase (SDH). It catalyzes the oxidation of succinate to fumarate, transferring electrons from succinate to CoQ. Complex III is constituted of 10 subunits, referred to as a cytochrome c reductase. It allows the transfer of two electrons to Complex IV, composed of 20 subunits with cytochrome c oxidase activity, which catalyzes the transport of two H+ from the matrix to the intermembrane space, leading to the reduction of O2 to H2 O. The ultimate step (OXPHOS) consists in the ATP synthase: complex V, and H+ transfer from the intermembrane space, leading to the synthesis of ATP from ADP and inorganic phosphate (iP). Approximately, 30–32 molecules of ATP/ glucose molecule are produced through the ETC, compared to 2 by glycolysis. ROS and RNS are byproducts of the respiration oxidative metabolism, responsible for oxidative stress when produced in excess (Goncalves et al. 2015). Under normal condition, the major source of ROS is the mitochondrial energy metabolism. Excessive production of ROS increases mitochondria fragmentation via deregulation of mitochondrial fusion and fission proteins (Wu et al. 2011). Importantly, high levels of ROS are produced via reverse electron transfer (RET) which mainly occurs when the pool of CoQ is over-reduced with electrons from Complex II (Chouchani et al. 2014). The inhibition of Complex III or IV promotes RET. Of note, a decrease in the level of Complex I and increased activity of Complex II have been reported in macrophages in response to bacterial infection (Garaude et al. 2016). Moreover,

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macrophages respond to lipopolysaccharide (LPS) by interrupting ATP production via OXPHOS and promote oxidation of succinate by Complex II causing an increase in mitochondrial membrane potential, thus triggering RET-ROS (Mills et al. 2016). This deregulation is associated with inflammation and production of IL-1ß and IL-10. The tricarboxylic acid cycle (TCA) or citric acid cycle is a central energyproducing pathway which participates in both anabolism and catabolism. It is essential that its activity is constantly maintained at a minimal level to answer quickly to cellular biosynthetic requirement. TCA is initiated by the condensation of acetylCoA with oxaloacetate to synthetize the citrate, subsequently converted into its isomer isocitrate (Fig. 1b). Importantly, acetyl-CoA acts as a metabolic intermediate and a precursor in the synthesis of fatty acids and amino acids (glutamate, proline, and arginine). Two oxidative decarboxylation steps convert isocitrate to αketoglutarate (α-KG) and subsequently to succinyl-CoA, with the release of two CO2 and two NADH. Succinyl-CoA is converted to succinate, oxidized to fumarate, a reaction catalyzed by the succinate dehydrogenase (SDH), which links TCA and ETC (Fig. 1a). Importantly, constant feedback between TCA and OXPHOS maintains a stable state in cells to ensure balance between nutrients supply and cellular requirements. Acetyl-CoA participates in multiple cellular processes and has a central role in cell homeostasis, maintaining equilibrium between anabolism and catabolism (Jankowska-Kulawy et al. 2022). The TCA requires a continuous supply of acetylCoA, provided from different sources as pyruvate via glycolysis or fatty acid oxidation. Pyruvate is imported into the mitochondrial matrix by the mitochondrial pyruvate carrier (MPC) (Bricker et al. 2012), and converted into acetyl-CoA via oxidative decarboxylation by the pyruvate dehydrogenase complex (PDHC), a potent sensor of the TCA activity (Behal et al. 1993). Branched-chain amino acids, Leu, Ile, Trp, are also a source of acetyl-CoA (Brosnan and Brosnan 2006). The cytosolic pool of acetyl-CoA is produced from citrate by ATP-citrate lyase (ACLY) (Zhao et al. 2016). Variation in the pool of acetyl-CoA has signaling impact, mostly related to its ability to acetylate histones with consequences on chromatin remodeling and gene expression, thus reprogramming various biological processes as the immune response, SC functions, and even carcinogenesis (Sivanand et al. 2018). LPSstimulated macrophages induce ACLY and increase cytosolic acetyl-CoA, necessary for the production of pro-inflammatory effectors ROS and nitric oxide (NO) (Infantino et al. 2013). ACLY level and activity are regulated by a Akt-mTORC1dependent mechanism, a link between metabolism and immunity (Covarrubias et al. 2016). Another component of TCA, α-KG, can be provided from glutaminolysis that converts glutamine to glutamate and ammonia by glutaminase (GLS). Glutamate is then oxidized to α-KG by glutamate dehydrogenase (GDH), providing replenishment of the TCA (Fig. 1b). α-KG is a substrate for chromatin-modifying enzymes such as lysine demethylases KDM2-7 and ten-eleven translocation hydrolases (TET1-3), impacting gene expression and cell fate decision. In IL4-activated macrophages, the increased production of α-KG impairs histone demethylase activity and promotes an

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anti-inflammatory response by the suppression of IKKß activation required for the NF-κB-mediated pro-infammatory response (Liu et al. 2017). Succinate is considered as an oncometabolite. It accumulates in SDH-deficient cells which show a DNA hypermethylation and promotion of malignancy (Hao et al. 2009). Succinate also participates in the regulation of innate immunity. As mentioned above, in LPS-activated macrophages, high levels of succinate induce IL-1ß and increase ROS through Complex I-dependent RET (Mills et al. 2016). Fumarate, also suggested as an oncometabolite, is associated with epigenetic reprogramming via DNA hypermethylation due to TET inhibition and triggers epithelial-mesenchymal transition (EMT). Fumarate is responsible for succination of proteins, increased ROS signaling by binding to glutathione (GSH) (Sullivan et al. 2013), and is accumulated through glutamine replenishment of the TCA, in response to LPS-activated macrophages (Arts et al. 2016). Mitochondria produce metabolites, which enter anabolic pathways for macromolecules biosynthesis. As an example, glutamine, the most abundant amino acid in plasma, is produced from the condensation of glutamate with ammonia by the glutamine synthetase (GS) (Fig. 1b). Glutamate is a source of nitrogen and contributes to the urea cycle through conversion to aspartate by the glutamic-oxaloacetic transaminase 2 (GOT2) and exported by the mitochondrial aspartate-glutamate carriers AGC1/2. While ammonia can be produced by amino acid lyases and nucleotide deaminases, the largest source in mammals comes from urease-positive bacteria of the microbiota, which catabolize 15–30% of urea (Fig. 1b). Another TCA compound is citrate, exported to the cytosol and converted into acetyl-CoA used for lipid biosynthesis. Citrate leads to oxaloacetate which can generate aspartate, used for purine and pyrimidine synthesis. As a signaling organelle, mitochondria have established communication routes with other organelles and subcellular compartments. As reported in a recent review (Shen et al. 2022), mitochondrial signaling includes the transport of metabolites or small messenger molecules as also the formation of vesicules as carriers for mitochondrial products to other cellular compartments. Mitochondria-ER contacts (MERCs) are important sites that coordinate mitochondrial morphology and biogenesis and are active sites to segregate and distribute mtDNA nucleoids during mitochondria division. MERCs also contribute to exchange of metabolites, such as lipids, cholesterol, and Ca2+ , between mitochondria and ER (Fig. 1a). Ca2+ transferred from ER to mitochondria controls ROS which in turn impacts Ca2+ levels, highlighting an important link between Ca2+ transport and oxidative stress. Proteins belonging to the peroxisome-proliferator activated receptor coactivator-1 family (PGC1) are master regulators of mitochondria biogenesis and response to oxidative stress (RiusPérez et al. 2020). PGC1 and deacetylase sirtuin 1 (SIRT1) regulate OXPHOS and mitochondrial fusion, promoting mitochondrial biogenesis (Wai and Langer 2016). PGC1-α is also activated by AMP-activated Protein kinase (AMPK), a major regulator of cell metabolism and mitochondria biogenesis, a pathway required for antimicrobial host defense (Yang et al. 2014). Stress adaptation also includes reprogramming of mitochondrial to nuclear transcriptional process. A strict coordination between mitochondrial and nuclear replication/transcription machinery, either

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anterograde (nucleus to mitochondria) or retrograde (mitochondria to nucleus) is essential to optimize mitochondrial function and to satisfy cells’ energy demand. The mitochondrial unfolded protein response (UPRmt ) is initiated by a mitochondriastress signaling cascade leading to nuclear transcriptional upregulation of genes encoding mitochondrial stress proteins (Zhao et al. 2002; Shen et al. 2022). UPRmt is activated by a variety of stress signals, including loss of mtDNA, defects in OXPHOS, and imbalance of fusion/fission process. The activation of a retrograde signaling pathway by mitochondrial stress has been previously reported to trigger ISR and involves ATF4, leading to communicate metabolic dysfunction to the nucleus (Quirós et al. 2016; Ryan et al. 2021). Dysregulation of this mitonuclear crosstalk plays an important role in disease progression including cancer.

2.3 Mitochondria and Immune Response: Implication in Host Antimicrobial Defense Mitochondria-related damage-associated molecular patterns (DAMPs) can be released upon infection, stress, or damage and initiate an immune response (Fig. 1a). DAMPs include mtDNA, cardiolipin, N-formyl peptides (NFPs), ROS, ATP, and succinate (Rodríguez-Nuevo and Zorzano 2019). They are recognized by cytoplasmic receptors such as NOD-like receptors, RIG-I-like receptor, and AIM-2-like receptor, which activate innate immunity. The presence of mtDNA in the cytosol may be a response to infection, corresponding to a warning signal leading to NFκB activation and inflammation. Because of its CpG composition similar to bacterial DNA, cytosolic mtDNA can be detected by TLR9 and the NLRP3 inflammasome (Pachathundikandi et al. 2015). In macrophages, in response to TLR activation during infection, mitochondria produce increased levels of ROS through the activation of the NADH oxidase (NOX) complex, leading to the induction of TNF and IFN-γ, which in turn stimulate ROS production and inflammation (Shekhova 2020). MtROS activate NLRP3 inflammasome and trigger pyroptosis, a macrophage-specific cell death pathway associated with the clearance of infection (Weinberg et al. 2015). Bacterial pathogens have developed highly sophisticated mechanisms to reprogram cellular physiology to their benefit. Bacteria secrete or directly deliver mitochondrial effectors into host cells that alter mitochondrial membrane integrity and activate mitochondria-dependent cell death pathways (Blanke 2005; Escoll et al. 2016). Most of these factors are pro-apoptotic toxins and induce mtROS. They can be translocated into the host cell through a type-III secretion system (T3SS) as Map and EspF from enteropathogenic E. coli or VopE from Vibrio cholerae. Both Map and EspF possess an N-terminal mitochondrial leading sequence (MLS), to be addressed to mitochondria. A well-studied example is Legionella pneumophila, which has a dozen of mitochondrial effectors that modulate cellular metabolism, mitochondria dynamic, cell death, and autophagy, mainly through the cag type IV secretion system (T4SS) (García-Rodríguez et al. 2023; Fischer et al. 2020). At an early time-point,

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L. pneumophila-infected macrophages show a T4SS-independent increased glycolysis and OXPHOS activity, whereas later during infection a second T4SS-dependent phase increases glycolysis, while OXPHOS and mitochondrial ATP production are reduced, mimicking a Warburg effect as in cancer cells (Escoll et al. 2017). The mitochondria dynamic is disrupted by infection, facilitating the elimination of infected cells. Like L. pneumophila, H. pylori also induces a DRP1-dependent mitochondrial fission caused by its pro-apoptotic toxin VacA (Jain et al. 2011). How H. pylori deals with mitochondria metabolism and signaling functions to get through the different steps required for its successful gastric colonization and its consequences in gastric pathologies are discussed in the following paragraphs.

3 H. pylori, Colonization of Gastric Glands, Metabolism, and Impact on Mitochondria 3.1 H. pylori, the Conquest of the Gastric Glands, Adaptation, and Dialogue with Mucosal Gastric Cells H. pylori has co-evolved with humans for thousands of years reaching an exquisite adaption to one of the most hostile niches in the human body, the gastric mucosa. The biogeography of the stomach is very complex with physiologically different regions, including the body, incisura, and the antrum and in each region an array of diverse cell types in each gland, from the specialized cells in the tip of the gland, progenitor cells in the neck, and SCs in the bottom, plus enterochromaffin-like (ECL), parietal, or other cells. A successful colonization relies on a number of strategies to survive drastic changes in pH, strong flagella to continuously swim from the mucous to the cell surface of cells in the epithelia, where H. pylori has to express adhesins specific to each cell type and secrete molecules that in most cases lead to a healthy dialogue between H. pylori and gastric mucosal cells (GMCs). Most probably, the nature of the dialogue differs from cell-to-cell types, although this is an area where very little is known. Biogeography of H. pylori colonization has been recently addressed and initial results show that the H. pylori-stomach relationship is a rather complex style of life. We have learned that in the early times of this relationship, during childhood, soon after H. pylori arrives in the stomach, the gastric mucosa responds by eliciting an inflammatory response to acknowledge the presence of H. pylori, and after a tuning period a chronic, balanced inflammatory state is reached, which is meant to remain under control for years and for the benefit of the two species (Blaser et al. 2019; Miller and Williams 2021). This is a process much like the one we see with gut microbiota and the intestinal mucosa. In fact, the induction by H. pylori of an inflammatory response in the gastric mucosa and the resulting recruitment and activation of diverse epithelial and immune cells train the gastric innate immune system and induce the differentiation of regulatory T cells, important in the prevention of autoimmune

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diseases as discussed elsewhere (for more details, see chapter “Immune Biology and Persistence of Helicobacter Pylori in Gastric Diseases” of this book). Thus, when studying the role of H. pylori in carcinogenesis we must keep in mind that in most cases the two species (human-H. pylori) share the same niche in peace with mutual benefits as expected from the components of our human microbiota. That would mean that acid tolerance, mobility, adherence, intimate contact with the T4SS, and injection of H. pylori molecules, like CagA and VacA to GMC, are part of the usual and healthy tools used in the H. pylori-GMC dialogue. When and why do some words of this dialogue go wrong increasing the risk for tissue damage? Why may H. pylori act as a pathobiont under yet unknown conditions? That is the challenge for science. If we could follow H. pylori from the time it approaches the gastric milieu to the point it establishes an intimate contact with cells of the epithelium (Fig. 2a), we may foresee H. pylori swimming in the gastric juice where it survives acid, thanks to the buffering activity of urease, a complex enzyme that produces NH4 + clouding the bacteria. Urease is a nickel-dependent enzyme that was acquired by gastric Helicobacter species probably since the beginning of the H. pylori-human relationship (Vinella et al. 2015), and is essential for colonization of the gastric mucosa. Then H. pylori must swim through the viscous mucus layer to the epithelial cells aided by strong flagella, by its helicoidal shape and by a stress response (Martínez et al. 2016). Swimming is not random, and H. pylori sense gradients of compounds with the Tlp chemoreceptor proteins, and the Che- family of proteins are responsible to transmit the message to H. pylori that lead the bacteria to the right place (Keilberg and Ottemann, 2016; Schreiber et al. 2004). ChePep controls chemotaxis in H. pylori by regulating rotation of the flagella and is necessary for the colonization of the gastric glands (Howitt et al. 2011). Once in the vicinity of epithelial cells, H. pylori localizes the intercellular junctions where it gets the right nutrients and environmental conditions for a successful colonization (Backert et al. 2017). With the help of adhesins like BabA, H. pylori anchors to ABO blood group antigens on the cell surface and starts sending messages to the guest cell (Bugaytsova et al. 2017). When in the right spot, H. pylori starts the interaction with GECs by injecting CagA to perturb the polarity of the cell in order to use the apical surface as permissive niche (Tan et al. 2009), where H. pylori can now access nutrients including iron (Fig. 2b). Previous studies suggested that H. pylori colonization was limited to the tip of the glands, but recent reports show that H. pylori extends its colonization to other regions of the gastric mucosa from the tip to the bottom, particularly the niches of the progenitor and the SCs (Sigal et al. 2015; Wizenty et al. 2020). This pattern of colonization (Fig. 3a–c) allows H. pylori to establish a chronic colonization, aimed to last all lifetime of its host without causing disease (Israel et al. 2001). This is of major importance because most reports up to date have studied the interaction of H. pylori with GECs, which in fact may have a limited role in the process of carcinogenesis. We need to look at cells in the other niches, particularly precursor and SCs, which because of their function and lifetime may have a more significant role in carcinogenesis. Precursor and SCs constantly divide and differentiate to replenish the differentiated cells all along the gland. Terminally differentiated cells ascend the gland to reach

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Fig. 2 Paths followed by H. pylori to colonize the gastric mucosa. a A human gastric tissue illustrating H. pylori (green) across the mucus layer covering the epithelia and bacteria in close contact with epithelial cells. The image illustrates where different components of H. pylori may be at work in the process. b An upside-down view of human gastric mucosa showing H. pylori (green) sitting mostly in intercellular spaces where conditions are favorable for an intimate contact H. pylori-epithelial cell. Known virulence factors expressed preferentially at this stage are indicated

the surface, where they are shed into the lumen every 2–3 days. If H. pylori growing at the surface of epithelial cells at the tip of the glands is turning over every 2–3 days, how does it survive for years in its host? We propose that the gastric glands provide additional niches for H. pylori to maintain a chronic reservoir that can replenish the transient populations along the superficial mucosa—analogous to a “bacterial SC” population (Markandey et al. 2021). Of note, and like the gastric glands, the colonic crypts also serve as a critical niche for commensal microbes to maintain stable colonization in the murine gut (Lee et al. 2013). Our findings in the stomach highlight the significance of the neck and crypt as microniches for H. pylori, probably allowing its persistence (Fig. 3c). Besides the differences in bio-localization, each cell type in the gastric glands performs different functions and has different metabolic activities, which in turn will affect the response of the subcellular targets including the mitochondria (Barker 2014; Markandey et al. 2021). SCs of the gastric mucosa are responsible to maintain tissue homeostasis by supplying progenitor cells engaged to become each of the different cell types in the glands. In SCs, mitochondria regulate cell activation, functions, and the fate of each cell, the mother of all mitochondria (Zhang et al. 2018). Besides functions, there are other differences: SCs present low mitochondrial number, which depend mainly on glycolysis for ATP production (Rafalski et al. 2012). The right fate of SCs is a tightly regulated process that might be affected by mtDNA integrity or mitochondrial ROS (mtROS). Very little is known about the interaction of H. pylori with gastric SCs, although a study with human gastric tissues revealed that H. pylori can reach the base of the glands and grow as microcolonies in the junction zone of SCs. The presence of H. pylori was associated with a significant increase

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Fig. 3 Images showing the in vivo H. pylori colonization of different regions of gastric glands. a Human gastric specimens showing antral glands colonized by H. pylori (green) across different regions of the glands. b Zoom of areas with H. pylori colonization, showing dense growth (biofilm) in the neck where progenitors cells are actively growing (mitosis) and region at the bottom of glands where stem cells (white) are in close contact with growing H. pylori (green). c A cartoon describing the sites susceptible to H. pylori colonization along the glands, also showing differences in H. pylori growth density. d Photo showing a longitudinal cut of the stomach of mice experimentally infected with H. pylori, showing differences in growth of H. pylori (green) in the antrum and corpus

of SCs proliferation, an effect that was stronger with cagPAI + than cagPAI- strains and supported by a significant increase of the expression of Lgr5, Axin2, and olfactomedin4 gene markers (Sigal et al. 2015). The role of mitochondria in the above H. pylori-associated changes in SCs has not been studied, although its importance in cell proliferation and commitment are well documented and most probably affected by the presence of H. pylori. Another poorly studied niche is the compartment of progenitor cells in the neck of the glands, of major importance to maintain the tightly regulated turnover of cells. A study by Sigal and co-workers (Sigal et al. 2015) strongly suggested that the compartment of progenitor cells is probably the region where growth of H. pylori is more abundant forming a dense biofilm (Fig. 3a, b), closely interacting with progenitor cells in mitosis, as if they mutually stimulate to favor proliferation. Again, the role of mitochondria in this probable symbiotic relationship has not been studied. Any reaction of progenitor cell´s mitochondria to the presence of H. pylori should impact the function of these cells as seeds for all differentiated cells in the glands. The region of the stomach where H. pylori colonization occurs is important. The site preferred by H. pylori is the antrum, whereas colonization in the corpus is scarce (Fig. 3d). The corpus contains a number of different cell types, including those involved in acid production, and parietal and ECL cells whose function is paramount in gastric carcinogenesis since long-lasting achlorhydria may lead to GC

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(Correa 1988). It has been suggested that infection of parietal cells by H. pylori may cause mitochondrial damage, release of ROS, apoptosis, and atrophy, as observed in patients with mutation in ATP4Ap.R703c (Benítez et al. 2020), a point that has to be confirmed. In summary, biogeography of the stomach mucosa is complex with different regions and each with particular types of cells forming the glands from top to bottom. H. pylori colonizes the mucosa and may interact with some but not all the cell types, although this has not been thoroughly studied. The response of each of these cell types to the presence of H. pylori will be different, according to the function of the infected cell and this includes damage to the mitochondria. We have learned that besides the surface mucous cells, H. pylori also interacts with stem and progenitor cells, although very little is known about the effects of this interaction. It is not known if H. pylori can interact with other cells in the gastric mucosa and the possible consequences of this interaction.

3.2 Mitochondria, a Prime Target for the VacA Cytotoxin The pro-apoptotic cytotoxin VacA is one of the major virulence factors of H. pylori that promotes gastric colonization and plays an essential role in the bacterial escape from host immune response (see also chapter “Immune Biology and Persistence of Helicobacter Pylori in Gastric Diseases” of this book). VacA is a secreted oligomeric autotransporter protein that binds to GEC surface via lipid rafts and forms anionselective membrane channel leading to plasma membrane permeability and depolarization (Cover and Blaser 1992). In vitro, VacA induces membrane-bound vacuoles associated with mitochondrial dysfunction and cell death (de Bernard et al 1997; Foegeding et al. 2016). Subsequent to its cellular internalization, VacA reaches the mitochondria through early and late endosomal trafficking, causing swelling of endosomes to form vacuoles (Fig. 4a) (Foegeding et al. 2016; Cover and Blanke 2005). This is associated with the activation of the pro-apoptotic factors BAX and BAK, causing cytochrome c release and caspase activation (Calore et al. 2010; Yamasaki et al. 2006). In late endosomal/lysosomal membranes, VacA-induced anion-selective channels allow influx of choride, which consequently stimulates the activity of the vacuolar ATPase and decrease luminal pH. Vacuolation is promoted by ammonium chloride (NH4 Cl), a weak base that diffuses into the endosomes where it is protonated. As previously reported, addition of NH4 Cl in cell culture inhibits the intracellular degradation of VacA with no effect on its trafficking (Foegeding et al. 2019). Once at mitochondria, VacA integrates as a MIM anion-channel and leads to dissipation of transmembrane potential (Δψm) with increase of membrane permeability and decrease of ATP production (Willhite and Blanke 2004). VacA-anion channels in MIM can compromise the mitochondrial ion equilibrium, impairing the proton motive force and the respiratory chain (Rassow and Meinecke 2012). Recently, 25 mitochondrial proteins were found able to bind VacA, allowing its translocation to MIM with the contribution of the phosphoglycerate mutase 5 (PGAM5) (Wang

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et al. 2022). This leads to impair mitochondrial membrane potential and to induce mitophagy via the action of PINK1, a mitochondrial serine/threonine-protein kinase, and Parkin, a cytosolic E3-ubiquitin ligase. A DRP1-dependent disruption of mitochondria dynamic has been reported in GECs treated with recombinant VacA comprising p33 and p55, associated with cellular death. Mitochondria fragmentation is induced within 60 min of incubation and progress to punctiform organelles after 150 min, indicating a disruption of the mitochondrial network (Jain et al. 2011). Disruption is followed by BAX activation with VacA-induced MOMP and cytochrome c release. The increase of gastric cell death during infection is also associated with the destruction of parietal cells responsible for gastric acid secretion, with drastic consequences for the host niche as promoting gastric atrophy and contributing to cancer. Mitochondrial fission precedes decrease of mitochondrial membrane potential and VacA-induced mitophagy (Terebiznik et al. 2009) that involved a VacA-mediated inhibition of mTORC1 signaling pathway (Kim et al. 2018). VacA has also drastic consequences on the cellular amino acids pool, as the inhibition of VacA-induced mitochondrial fragmentation prevents amino acid starvation (Kim et al. 2018). VacA-intoxicated cells showed increased levels of the serine/ threonine kinase, general control non-derepressible 2 (GCN2), which senses cellular amino acid deficiencies, indicating a depletion in the amino acids pool. VacA also modulates mitochondria biogenesis. As we previously reported, it participates in the induction of the mtDNA polymerase G (POLG) and the transcription factor A mitochondrial (TFAM), with direct consequences on cellular mtDNA content (Chatre et al. 2017). Two hour infection of AGS gastric epithelial cells with H. pylori induces an early and strong transient expression of POLG and TFAM, and concomitantly increases the mitochondrial translocases TOM22 and TIM23 and mtDNA content (Fig. 4b). These effects need the contact of VacA with the epithelial cell surface. The treatment of cells with the acid-activated purified VacA toxin showed that its N-terminal hydrophobic domain and vacuolating activity are not required for this early regulation. At later time-points, TOM22, TIM23, POLG, and TFAM were also induced by a ΔvacA mutant, but at a lower extent, compared to wild-type H. pylori, indicating that other bacterial components and/or derived-metabolites may be involved. The same effects were also observed in INS-GAS mice transgenic for the human gastrin that develop gastric intraepithelial neoplasia (GIN) after 6 and 12 months of H. pylori infection (Chatre et al. 2017). These findings highlight the participation of mitochondrial dysfunction and deregulation in the development of H. pylori-induced gastric pre/neoplasia.

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Fig. 4 Schematical overview of the consequences of H. pylori at mitochondria resulting from the action of its virulence factors and derived-metabolites, NH3 , H2 S, ROS, and RNS. a Action of VacA which interacts with lipid rafts and induces vacuoles in the cytosol. Presence of ammonium chloride activates vacuolation. VacA then reaches MIM through endosomal trafficking, leading to cytochrome c release and apoptosis. VacA also interacts with DRP1, promoting mitochondria fission, preceding mitophagy (see text for details). Urease activity that allows gastric acidity adaptation is a source of ammonia which in the presence of CO2 produces ammonium and bicarbonate. CagA translocated in cell through the T4SS is a source of ROS by the promotion of inflammation. CagA is also involved in the induction of the mitochondrial protease Lonp1 in response to UPRmt , as a result of mitonuclear communication to assure mitochondrial component quality. H. pylori LPS, NapA, and Hp(2–20) are also efficient inducers of ROS produced by immune cells. b Vac A is transferred in the MIM where it forms anion-channel, through interaction with the mitochondrial translocases TOM20, TOM40, and TOM70. H. pylori impacts mitochondria function through VacA-dependent and independent mechanisms. In H. pylori-infected GECs, the mtDNA polymerase POLG and the transcription factor TFAM are induced, concomitantly to the translocases TOM22 and TIM23, thus promoting mitochondria biogenesis. Production of ammonia is directly associated with amino acid biosynthesis through the urea cycle, connecting H. pylori-derived metabolites with mitochondria function. MtROS are essentially produced from OXPHOS, ETC activity. Production of H2 O2 can also result from the methionine pathway which leads to the synthesis of cysteine from cystathionine, catalyzed by CSE which leads to the production of H2 S and ATP. Another producer of H2 O2 is CagA, through the activation of SMO which catalyzes the conversion of spermidine to spermine. Under these conditions, H. pylori is an efficient inducer of mtDNA damage and mutations. MNC: mitonuclear communication; UPRmt : mitochondrial unfolded protein response; T4SS: type IV secretion system; PMN: polymorphonuclear cells; M: Macrophages; T: T cell; B: B cell; CSE: cystathionine γ lyase; ODC: ornithine decarboxylase; SMOX: spermine oxidase. Yellow symbols: mtDNA damage. Blue circles in mitochondrial matrix: ribosomes.

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3.3 H. pylori-Derived Metabolites Have an Important Role in Mitochondrial Damage The drastic effects of H. pylori on mitochondria may also result from its derivedmetabolites able to deregulate metabolic and cell signaling pathways and impact macromolecules biosynthesis. Ammonia produced by urease may have effects on both, H. pylori and its host. Urease is a nickel-dependent enzyme (Kumar et al. 2022), composed of UreA and UreB assembled into UreA/B which tetramerizes in [(UreA/B)3 ]4 (Farrugia et al. 2013). It hydrolyzes urea (CO(NH2 )2 ) to produce ammonia (NH3 ) and carbon dioxide (CO2 ) (Fig. 4a). Two other H. pylori ammonia-producing enzymes are the acidinduced amidase AmiE and the formamidase AmiF that hydrolyze the amide bond of short-chain aliphatic amides leading to NH4 + and OH− (Skouloubris et al. 2001). Ammonia crosses host cells and mitochondrial membranes, increasing the intracellular and intramitochondrial pH. The VacA-induced vacuolation inhibits mitochondria respiration (Tsujii et al. 1992) and could be related to the decrease of respiratory capacity and ETC Complex I defect (Machado et al. 2013). Ammonia also serves as nitrogen source for the synthesis of amidated amino acids as glutamine, involved in many biosynthetic pathways (proteins, nucleosides, amino acids, and cofactors biosynthesis), indicating that by modulating the level of ammonia, H. pylori impacts host cell biosynthesis (Fig. 1b). H. pylori GGT is also a source of ammonia resulting from the consumption of glutamine (Ricci et al. 2014), which provides the energy for gastric colonization and allows to refill the mitochondrial carbon pool. This should have direct consequences on immune response to infection as glutamine is used by immune cells as a source of energy. In mitochondria, GLS deaminates glutamine into glutamate, which is then converted to α-KG by GDH, replenishing the TCA (Fig. 1b). High levels of ammonia can accelerate the conversion from glutamate to glutamine by GS, which is highly produced by the stomach. As a consequence, ammonia promotes the depletion of ATP and reduces the viability of gastric mucosal cells (Kubota et al. 2004). Importantly, glutamine inhibits the DNA demethylase TET1 (Gong et al. 2017), suggesting that its variation during H. pylori infection can impact TCA activity and then affect energy supply with epigenetic consequences. Ammonia detoxification affects arginine levels through the conversion of arginine to urea and ornithine via the urea cycle (Fig. 1b). Thus, ammonia may impair the TCA, providing a direct link between H. pylori-derived metabolites and mitochondria respiratory dysfunction, and consequently macromolecules biosynthesis. Hydrogen sulfide (H2 S), a gaseous biological transmitter, is related to methionine metabolism. H2 S is able to S-sulfidrate proteins and modulate their activity, as the α subunit ATP5A1 of the MIM ATPase (Modis et al, 2016). H2 S remains a regulator of energy production in mammalian cells under stress condition, involved in the regulation of acid-induced HCO3 − secretion and mucosal protection (Ise et al. 2011). Its role in gastric cell proliferation has been documented (Sekiguchi et al. 2016). Metabolomic analysis using gas chromatography-tandem mass spectrometry (GC– MS/MS) of H. pylori-infected AGS cells showed that methionine metabolism was the

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most altered pathway with high H2 S production and increased level of cystathionine (Kawahara et al. 2020). In response to stress, cystathionine is converted to cysteine by the cystathionine γ lyase (CSE) which is translocated to mitochondria where it increases the production of H2 S and ATP (Fig. 4b). Because of its effective scavenger of ROS and RNS, production of H2 S by H. pylori has been proposed to limit inflammation and help to reduce tissue damage. This mechanism and its role in the energy demand of H. pylori-infected cells deserve further investigation. On the other hand, H. pylori infection has marked effects on the host metabolome. A recent study investigated the metabolomic variation between the antrum and corpus regions of the stomach in H. pylori-infected mice and in gastric organoids (Keilberg et al. 2021). Depletion of urea, glutamine, cholesterol, lactate, and fumarate was observed in both antrum and corpus-derived infected organoids. Depletion of glutamine and aspartate was also confirmed in the corpus of mice. In agreement, a previous NMR-based metabolic analysis in the gerbil model showed that H. pylori infection disturbs the carbohydrate and amino acids metabolism of its host gastric mucosa (Gao et al. 2008). Higher levels of glucose and cis-aconitate, a TCA intermediate, were observed with alteration of the energy metabolism and high levels of glutamine in urine. As mentioned above, reprogramming of mitochondrial to nuclear transcriptional regulation is an important stress response of mitochondria. As previously reported, H. pylori induces Lonp1 protease via HIF-1α regulation (Luo et al. 2016). Lonp1 is required to maintain a proper mitochondrial function. Its overexpression in H. pylori-infected GECs promotes a glycolytic switch and contributes to cell overproliferation, suggesting a role in gastric carcinogenesis.

3.4 H. pylori-Induced Oxidative Stress and Inflammation Mitochondria are a major source of ROS with O2 − as a by-product of O2 consumed by ETC and OXPHOS (Turrens 2003). ROS production is catalyzed by nicotinamide adenine dinucleotide phosphate oxidase (NADPH-oxidase; Nox) localized at the cell membrane. O2 − can be converted to H2 O2 by SOD. In macrophages and neutrophils, H2 O2 can react with Cl− to produce HOCl at the origin of OCl− , highly efficient against pathogens. In H. pylori-infected GECs, overproduction of O2 − is associated with mitochondrial membrane depolarization, impairment of ETC, and GSH depletion as antioxidant defense (Calvino-Fernández et al. 2008). Pro-inflammatory H. pylori virulence factors stimulate the production of intrinsic ROS (Han et al. 2022). CagA, referred to as an H. pylori oncogenic protein (Tegtmeyer et al. 2017; for more details, see chapter “Impact of the Helicobacter Pylori Oncoprotein CagA in Gastric Carcinogenesis”), promotes the recruitment of inflammatory cells in the gastric mucosa leading to increased level of H2 O2 and oxidative DNA damage. Similarly, VacA participates in the oxidative stress through its damaging effects to mitochondria (see Sect. 3.2) and NF-κB activation (Kim et al. 2007). Furthermore, GGT, BabA, LPS, and the miniferritin neutrophil activating factor A (NapA), which recruits neutrophils (Codolo et al. 2022), have also been mentioned for their

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pro-inflammatory properties. In addition, as mitochondria releases non-formylated peptides (NFPs) under stress conditions (Rodríguez-Nuevo and Zorzano 2019), H. pylori releases NFPs as the cecropin-like peptide Hp(2–20), which interacts with the formyl peptide receptor-like 1 (FPRL1), activating several signaling pathways and innate immune response (Cuomo et al. 2021). Hp(2–20) contributes to low-grade inflammation, oxidative stress, and gastric carcinogenesis. ROS are also connected to amino acid metabolism pathways through the production of polyamines from L-arginine leading to L-ornithine, converted into polyamine putrescine by the ornithine decarboxylase (ODC). Putrescine leads to spermidine and spermine. Inversely, spermine oxidase (SMOX) catalyzes the conversion of spermine to spermidine and generates H2 O2 . The presence of H. pylori is associated with the induction of ODC and increase of polyamines concentration in vivo, mainly in macrophages. SMOX is also overexpressed in H. pylori-infected macrophages and promotes ROS in a CagA-dependent manner (Chaturvedi et al. 2007). H. pylori also produces NO, as it induces the inducible nitric oxide synthase (iNOS) in macrophages. However, the H. pylori-mediated up-regulation of ODC induces spermine, which inhibits L-arginine levels, and then iNOS and NO levels in macrophages (Chaturvedi et al. 2010) (Fig. 4b). As the main producer of ROS, mitochondria are central players in the fragile equilibrium between bacteria and host, although their role is influenced by the metabolic status and the type of cells interacting with H. pylori. Gastric achlorhydria, which results from the destruction of parietal cells during H. pylori infection, is known to create favorable conditions for gastric carcinogenesis. A recent study (Benítez et al. 2020) established a link between mitochondria alteration and gastric achlorhydria. Using 1-year old mice with the ATP4Ap.R703C mutation that impairs gastric acidity produced by parietal cells, they demonstrated an association between mitochondria dysfunction and mitochondria biogenesis, the induction of ROS and apoptosis, concomitantly with the development of atrophic gastritis and metaplasia.

4 H. pylori-Induced MtDNA Damage and Gastric Carcinogenesis H. pylori is a source of genetic instabilities and epigenetic deregulation, with major consequences on the progression of gastric cancer (Touati et al. 2003; Machado et al. 2009; Toller et al. 2011) (for more details, see chapter “Helicobacter Pylori-Induced Host Cell DNA Damage and Genetics of Gastric Cancer Development” of this book). MtDNA is more vulnerable than nDNA to damage since it lacks histones and is located near the MIM, in the vicinity of ROS source. MtDNA mutation and variation in mtDNA content have been found in most of tumoral tissues (Kopinski et al. 2021). We previously reported an increase of mtDNA levels in circulating leucocytes from GC patients, compared to healthy subjects (Fernandes et al. 2014). Importantly,

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mtDNA mutations in gene coding for OXPHOS subunits can have direct consequences on the mitochondrial respiratory activity and ROS production, contributing to malignancy. Similarly, mutation in nuclear gene coding for mitochondria components can impair mitochondria functioning and metabolism with consequences on chromatin remodeling and gene expression, as reported for gene coding for TCA enzymes in certain tumors (Hao et al. 2009). Using AGS cells, we previously reported that H. pylori induces mutation in the mtDNA D-loop region, which controls mtDNA replication/transcription, as in the mitochondrial genes ND1 and CO1, as also confirmed in patients with chronic gastritis (Touati 2010) and in H. pylori-infected mice in the presence of GIN (Chatre et al. 2017). H. pylori-induced mtDNA instabilities are associated with decreased respiration coupled to ATP turnover caused by impairment of the ETC Complex I (Machado et al. 2013). Moreover, sequential and higher accumulation of mitochondrial microsatellite instabilities (MSI) has been reported in H. pylori-positive patients during the progression from chronic gastritis to GC, suggesting that mtDNA instabilities are early events in the gastric carcinogenesis cascade (Ling et al. 2016). Mitochondrial haplotypes are defined by combination of SNP in mtDNA and grouped as haplogroups specifically associated with some human diseases (Mitchell et al. 2014). A recent study reported a higher proportion of haplogroup B than haplogroup D, among H. pylori-positive subjects. Interestingly, H. pyloriinfected fibroblasts showed higher decreased mitochondrial respiration with mtDNA haplogroup B than D, indicating that H. pylori-mediated metabolic impairment can be modulated/impacted by mtDNA haplogroup (Lee et al. 2021). Finally, mitochondrial stress as induced by infection can promote the release of mtDNA into the cytosol since the early activation of inflammatory pathways. This cytosolic mtDNA can be transferred and integrated into nDNA as nuclear mtDNA segments (NUMTs). About 1000 NUMTs (about 400 kb of DNA sequence) from about 2000 to 10 000 mtDNA copies/cell are present in human nDNA and 3 to 4 times more, found in tumor cells as reported in colorectal cancer (Srinivasainagendra et al. 2017; Kopinski et al. 2021). However, due to the heteroplasmy of mitochondria, the consequences of NUMTs should result more from genetic instabilities related to their nDNA insertion rather than their loss from the mitochondrial genome. Insertion of NUMTs into nDNA occur during double-strand breaks (DSB) repair by nonhomologous end-joining (NHEJ) DNA repair (Ricchetti et al. 1999) and represent a potential source of genetic instabilities. As a DNA-DSB inducer, H. pylori could promote NUMTs. This point remains to be investigated to further understand the contribution of mtDNA damage to the GC process.

5 Concluding Remarks It is important to remember that H. pylori colonizes over 50% of our species and in most cases without causing disease. This means that most of the responses of the GECs to H. pylori do not alter homeostasis and may even be beneficial to the human

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host, as seeing with components of the gut microbiota. Inflammation is commonly observed in response to microbiota in mucosas and skin, and only when this gets out of control then risk for disease increases. The same can be said for most changes observed in cells interacting with H. pylori, like in cytoskeleton, tight junctions, or nucleus and of course the changes observed in mitochondria. The challenge to science is to understand when these changes get out of control threatening the health of its host. Mitochondria are key regulators of cellular metabolism and main energy suppliers, as also key components for proliferation, speciation, defense, and a healthy life maintenance. As such, pathogens often choose mitochondria as their main target because of its central regulatory role in the physiology and metabolism of the cell. Due to the broad spectrum of mitochondria function, their role in the host response to H. pylori infection surely goes beyond the known consequences of the VacAmediated mechanism and ROS production. According to the constant increase of the knowledge in the mitochondria field, what is presently known about the consequences of H. pylori at mitochondria and their impact in the promotion of GC is only the tip of the iceberg. Indeed, the direct consequences and the associated mechanisms of H. pylori-derived metabolites on macromolecules biosynthetic pathways, on cell metabolism and catabolism remains to be further investigated as also the impact of H. pylori infection on DAMPs release and their role in gastric tumorigenesis. DAMPs include mtDNA of which the relargage in the cytosolic compartment is associated with inflammation. Whether mtDNA can be translocated as NUMTs in the nucleus in infected cells, promoting genetic instabilities and gastric carcinogenesis, remains an open question. Another important aspect is the cellular response to the presence of H. pylori, depending on the type of cell, which is complex considering that H. pylori may colonize diverse cells in the gastric mucosa, each with a specialized function. It will be of particular interest to study the nature of interaction between H. pylori and SCs and the specific mitochondrial response, as SCs constitute a niche that we now know is susceptible to H. pylori colonization and might be of particular interest considering their predominant role in GC which usually develops after decades of interaction of H. pylori with the human gastric mucosa. For this, we probably should keep in mind that the target, mitochondria, is a bacterial ancestral symbiont of human cells and that H. pylori is also a bacterium that has co-evolved with humans since the very beginning of our species and in the end, this is a dialogue between two bacteria. Acknowledgements We are grateful to Dr. Manuel Amieva (Stanford University, CA, USA) for providing excellent photos of human and mouse gastric mucosa (Figs. 2 and 3). This work is supported by Odyssey Reinsurance and Institut Pasteur Transversal Research Programs to ET (PTRs 217 and 332-20). The figures were created with BioRender.com.

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Immune Biology and Persistence of Helicobacter pylori in Gastric Diseases Sonja Fuchs, Ruolan Gong, Markus Gerhard, and Raquel Mejías-Luque

Abstract Helicobacter pylori is a prevalent pathogen, which affects more than 40% of the global population. It colonizes the human stomach and persists in its host for several decades or even a lifetime, if left untreated. The persistent infection has been linked to various gastric diseases, including gastritis, peptic ulcers, and an increased risk for gastric cancer. H. pylori infection triggers a strong immune response directed against the bacterium associated with the infiltration of innate phagocytotic immune cells and the induction of a Th1/Th17 response. Even though certain immune cells seem to be capable of controlling the infection, the host is unable to eliminate the bacteria as H. pylori has developed remarkable immune evasion strategies. The bacterium avoids its killing through innate recognition mechanisms and manipulates gastric epithelial cells and immune cells to support its persistence. This chapter focuses on the innate and adaptive immune response induced by H. pylori infection, and immune evasion strategies employed by the bacterium to enable persistent infection.

S. Fuchs and R. Gong contributed equally to this work. S. Fuchs · R. Gong · M. Gerhard · R. Mejías-Luque (B) Institute for Medical Microbiology, Immunology and Hygiene, TUM School of Medicine and Health, Department Preclinical Medicine, Technical University of Munich (TUM), Trogerstraße 30, 81675 Munich, Germany e-mail: [email protected] S. Fuchs e-mail: [email protected] R. Gong e-mail: [email protected] M. Gerhard e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 S. Backert (ed.), Helicobacter pylori and Gastric Cancer, Current Topics in Microbiology and Immunology 444, https://doi.org/10.1007/978-3-031-47331-9_4

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1 Introduction Helicobacter pylori is a Gram-negative, micro-aerophilic, spiral-shaped, and flagellated bacterium, which infects the human stomach in childhood and persists lifelong. Several virulence factors enable H. pylori to colonize its host despite the hostile gastric environment and the presence of a strong immune response elicited by and directed against it. One of the most abundant H. pylori proteins is urease, which catalyzes the hydrolysis of urea to ammonia and carbon dioxide. This enzymatic activity enables the survival of H. pylori in acidic environments, facilitating bacterial colonization, but at the same time, it modulates the host’s immune response (reviewed by Baj et al. 2020). Other well-known virulence factors of H. pylori are the cytotoxin-associated gene A (cagA), the vacuolating cytotoxin gene A (vacA), gamma-glutamyl-transferase (GGT), and neutrophil-activating protein A (NapA). All of those factors are involved in the modulation of the immune response during H. pylori infection and in its pathogenesis. CagA is encoded by the cag pathogenicity island (cagPAI) along with a type IV secretion system (T4SS) allowing the translocation of CagA directly into host cells (Fischer et al. 2020). Once translocated, CagA becomes phosphorylated and affects signaling in host cells in a phosphorylationdependent and independent manner (reviewed by Baj et al. 2020; Suzuki et al. 2009; Tegtmeyer et al. 2017). VacA is a cytotoxin involved in the formation of pores in host cells and is considered to support the survival of H. pylori by generating a protective intracellular reservoir for the bacterium (Capurro et al. 2019). GGT converts glutamine and glutathione into glutamate and ammonia or glutamate and cysteinylglycine, respectively. GGT-mediated changes in metabolites inhibit the function of immune cells and of gastric epithelial cells (GECs) (reviewed by Ricci et al. 2014). NAP is a virulence factor that stimulates neutrophil infiltration and activation during H. pylori infection (reviewed by Baj et al. 2020). The action of these and other virulence factors enables the bacterium to persist for decades in its host. Although most H. pylori infections are asymptomatic, H. pylori-induced chronic gastritis can progress to more severe pathologies over time. Thus, having effective treatment options for the bacterium is of utmost importance. Understanding the inflammatory response against H. pylori and the mechanisms used by the bacterium to persist is important to identify vulnerabilities in order to develop targeted treatment strategies. In the next sections, we summarize the immune response elicited during H. pylori infection and discuss persistence strategies used by the bacterium to evade the host’s immune response.

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2 Immune Response to H. pylori 2.1 Innate Immune Response 2.1.1

Recognition of H. pylori

Pattern recognition receptors (PRRs) recognize conserved pathogen-associated molecular patterns (PAMPs). These receptors are classified into five groups: Toll-like receptors (TLRs), C-type lectin receptors (CLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), RIG-I-like receptors (RLRs) and absent in melanoma 2 (AIM2)-like receptors (ALRs) (Li and Wu 2021), and all of them have been reported to be involved in the recognition of H. pylori. In general, during H. pylori infection, PRRs expressed by innate immune cells and GECs, activate NF-κB or the inflammasome upon recognition of PAMPs, leading to the production of proinflammatory cytokines and type I interferons (Cheok et al. 2022). However, PRR expression differs between immune cell types, tumor cell lines, and primary cells. Thus, when interpreting results concerning the recognition of H. pylori by PRRs, the experimental setup always needs to be considered. TLRs Ten TLRs exist in humans, and all of them have been described to participate in the recognition of different H. pylori PAMPs (Pachathundikandi et al. 2015). TLR2 was suggested to be a primary sensor for H. pylori as it recognizes lipopolysaccharide (LPS) (Smith et al. 2003; Yokota et al. 2007; Cullen et al. 2011), and several H. pylori proteins including NAP (Amedei et al. 2006; Wen et al. 2021) and heat shock protein 60 (Zhao et al. 2007). In addition to TLR2, TLR4 was also reported to recognize LPS (Kawahara et al. 2001; Mandell et al. 2004). Moreover, TLR4 detects HP0175, a secreted peptidyl-prolyl cis–trans isomerase with various detrimental effects on host cells, including apoptosis and autophagy (Basu et al. 2008). The classical flagellin receptor TLR5 does not recognize H. pylori flagellin (AndersenNissen et al. 2005b) but is activated by two structural components of the T4SS, CagY (Tegtmeyer et al. 2020) and CagL (Pachathundikandi et al. 2019). Moreover, H. pylori DNA is translocated by the T4SS and is subsequently detected by TLR9 (Rad et al. 2009; Varga et al. 2016; Tegtmeyer et al. 2022). In addition, signaling of the RNA-sensors TLR7 and TLR8, which are expressed inside intracellular vesicles, was suggested to have an important role during H. pylori infection by eliciting the production of type I interferon (Lee et al. 2022). Once activated by their respective H. pylori ligands, TLRs are tightly involved in the regulation of the inflammatory response against the bacterium as they modulate signaling pathways important for cytokine secretion in different immune cell subsets (Pachathundikandi et al. 2023). For instance, TLR2-mediated recognition of H. pylori elicits interleukin 8 (IL-8) (Wen et al. 2021) production in human neutrophils and an IL-1β (Jang et al. 2020) response in murine neutrophils and in the human neutrophillike HL-60 cell line. In murine macrophages, TLR2 signaling was correlated with

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the production of IL-6 and IL-1β, while in human DCs, TLR2 signaling promotes the secretion of tumor necrosis factor (TNF) and of granulocyte–macrophage colonystimulating factor (GM-CSF). Activation of TLR4 signaling in human monocytes and DCs has also important regulatory effects on cytokine expression. In monocytes, TLR4 signaling was found to be crucial to induce an IL-12 and IL-10 response (Obonyo et al. 2007), while in DCs it induces the secretion of IL-12 and IL-18 (Neuper et al. 2020). DCs are also important for the release of IL-10, which affects subsequent T cell responses as IL-10 polarizes T cells to a regulatory phenotype. Conversely, TLR4-inhibition on human DCs decreased the levels of interferon gamma- (INFγ), IL-17A-, and Forkhead box protein P3 (FoxP3)-expressing T-cells in co-culture experiments (Kabisch et al. 2014). When activated in the gastric epithelial cell line MKN45, TLR4 signaling was found to up-regulate the expression of Natural killer group 2, member D ligand (NKG2DL), which mediates natural killer (NK) cell cytotoxicity (Hernandez et al. 2021). In human monocytes, TLR5-dependent signaling promotes the production of IL-8 and TNF (Kumar Pachathundikandi et al. 2011), while TLR8 signaling leads to the production of type I interferon (Lee et al. 2022). Infection of TLR5-deficient mice proved that recognition of H. pylori by this receptor efficiently controls the infection (Pachathundikandi et al. 2019). In contrast to that, infection of TLR9-deficient mice resulted in a higher expression of TNF and INF-γ and a higher infiltration of neutrophils at the beginning of the infection, indicating that TLR9 promotes antiinflammatory effects during H. pylori infection (Otani et al. 2012). Contrary to the findings in mice, TLR9-dependent signaling was also shown to be involved in the production of IL-8 in human neutrophils (Alvarez-Arellano et al. 2014). In summary, TLR-mediated recognition of H. pylori is a major driver for subsequent innate and adaptive immune responses (Fig. 1). CLRs CLRs, which are mainly expressed by dendritic cells (DCs) and macrophages, commonly recognize bacterial-derived carbohydrates. Relevant CLRs during H. pylori infection are DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and macrophage inducible calcium-dependent lectin receptor (Mincle). DC-SIGN detects Lewis antigens (Bergman et al. 2004), in particular fucosylated H. pylori ligands (Gringhuis et al. 2009). Upon LPS treatment, DCSIGN was found to be up-regulated in the human gastric epithelial cell line GES-1 and in murine primary stomach epithelial cells and it cooperates with TLR4 to activate the NLR family pyrin domain containing 3 (NLRP3) inflammasome (Chen et al. 2020b). As TLR2 typically mediates inflammasome activation in H. pylori (Kim et al. 2013), the mechanism behind this newly found interaction with TRL4 remains to be studied. This directs GECs to secrete IL-1β and IL-18, which triggered a Th1 response in co-culture experiments (Chen et al. 2020b). In contrast to that, recognition of Lewis antigen variants by DC-SIGN in DCs blocks Th1 development (Bergman et al. 2004). Moreover, DC-SIGN signaling in DCs favors the expression of Th2-attracting chemokines (Gringhuis et al. 2014). In addition, Mincle recognizes bacterial cholesterol-derived metabolites (Nagata et al. 2021) and Lewis antigens on

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Fig. 1 The innate and adaptive immune response to H. pylori. H. pylori is recognized by PRRs including TLRs, NOD1, DC-SIGN, and Mincle. Recognition of PAMPs and translocation of CagA induces cytokine secretion and AMP production by GECs. Innate immune cells including macrophages (M1 and M2), neutrophils (N1), and DCs are recruited in response to the bacterium. H. pylori-derived factors mediate this recruitment, activate these cells, and shape cytokine production. Neutrophil recruitment is dependent on bacterial NAP and on IL-8. Moreover, numbers of ILC2, NK cells, and eosinophils are increased upon H. pylori infection. DCs and neutrophils mediate the induction of a Th1/Th17 response and of Treg cells. Recently, TRM cells were found to contribute to the control of H. pylori. Moreover, specific IgA and IgG are produced by B cells upon H. pylori infection

H. pylori LPS (Devi et al. 2015). Mincle expressed by human macrophages induces the expression of IL-10, while suppressing the production of TNF upon recognition of H. pylori. Moreover, infection of Mincle-deficient mice revealed that Mincledependent recognition of cholesteryl acyl α-glucosides is an important mediator of T-cell priming during H. pylori infection (Nagata et al. 2021). NLRs The NOD1 receptor is well-known to be involved in H. pylori recognition as it senses peptidoglycan translocated by the H. pylori T4SS (Watanabe et al. 2010). NOD1-mediated signaling was shown to induce the production of the antimicrobial peptide (AMP) human-beta defensin 2 (hbD2) in the gastric epithelial cell line AGS, which efficiently killed H. pylori in vitro (Grubman et al. 2010). H. pylori infection of NOD1-deficient mice indicated that NOD1-signaling shapes the cytokine production

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by GECs (Tran et al. 2018; Suarez et al. 2019). In particular, expression of the cytokines IL-22, IL-1β, IL-33, and the chemokine 20 (CCL20) were reduced in the absence of NOD-1. This had important consequences on the T cell response as Th1, Th2, and Th17-related cytokines were elevated in infected NOD1 knockout mice (Suarez et al. 2019). Apart from NOD1, NOD2 was found to have a significant role in H. pylori infection, as NOD2 together with TLR2 was reported to be crucial for the production of IL-1β by H. pylori-infected DCs (Kim et al. 2013). RLRs and ALRs RLRs and ALRs recognize foreign intracellular RNA or DNA, respectively. During H. pylori infection, retinoic acid-inducible gene I (RIG-I) acts as a receptor for 5, triphosphorylated RNA (Rad et al. 2009). Moreover, recent studies revealed AIM2 to be increased in H. pylori-positive human biopsies (Dawson et al. 2023). Infections of AIM2 knockout mice with H. felis also indicated a role of AIM2 in Helicobacter recognition, although this role remains controversial (Dawson et al. 2023; El-Zaatari et al. 2020). Thus, Dawson and co-workers (2023) found that inflammation in infected AIM2−/− mice was reduced, reflected by less CD3+ T cell infiltration in the gastric tissue. In contrast, El-Zaatari and colleagues (2020) observed increased CD3+ and, particularly, CD8+ T cell infiltration in infected AIM2−/− mice. These contradictory findings can be explained by the use of two different genetic backgrounds of the mice as well as by the different duration of the infection. Therefore, further research, especially with H. pylori infection models, is needed to understand the role of ALRs in H. pylori recognition and subsequent inflammatory response.

2.1.2

Gastric Epithelial Cells

Several cytokines and chemokines, including TNF, IL-1β, IL-6, IL-12, IL-8, monocyte chemoattractant protein-1 (MCP-1) and CCL-5 (Jung et al. 1997; Tanahashi et al. 2000; Kudo et al. 2005; Al-Sammak et al. 2013) were detected after infection of gastric epithelial tumor cell lines (AGS, Snu-5, KatoIII and MKN45) with H. pylori. This cytokine profile triggers the migration and activation of innate and adaptive immune cells. Eosinophil migration towards infected MKN45 cells was shown to be mediated by GEC-derived GM-CSF, CCL2, and CCL5 (Nagy et al. 2011), while DCs are recruited upon release of CXCL1, CXCL16, CXCL17, and CCL20. Also, inflammasome activation in GECs, resulting in IL-18 maturation, was implicated in neutrophil recruitment in a mouse model (Semper et al. 2019). Moreover, T-cell recruitment is controlled by GEC-derived chemokines. Thus, CX3CL1, expressed by GECs, promoted CD4+ T cell recruitment in a mouse infection model (Sun et al. 2022), while GEC-derived CCL20 was correlated with the migration of Tregs to the gastric mucosa in human biopsy samples (Cook et al. 2014). Once T cells are recruited, GEC-derived MCP-1 can support T cell activation. The gastric epithelial cell line MKN45 was found to secrete MCP-1 in the medium after treatment with H. pylori lysate, which stimulated the expression of the activation marker cyclooxygenase 2 (COX2) and cytokine secretion when added to T cells in culture (Futagami

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et al. 2003). In addition, matrix metalloproteinase 10 (MMP-10) in H. pylori-infected MKN45 cells and primary human GECs promotes CXCL16 production and further recruits CD8+ T cells (Lv et al. 2019). Furthermore, analysis of human gastric specimens revealed that GECs express a number of AMPs, such as cathelicidin LL37 (Hase et al. 2003), hBD2 (Pero et al. 2019), and hBD3 (Kawauchi et al. 2006) in response to H. pylori, while hBD1 was found to be constitutively produced (Nuding et al. 2013). Defensins and cathelicidins are cationic peptides that kill bacteria by disrupting their membranes (De Smet and Contreras 2005). HBD2 (Pero et al. 2019), hBD3 (Nuding et al. 2013), and LL37 (Nuding et al. 2013; Hase et al. 2003) were all reported to have anti-H. pyloriactivity in vitro. Moreover, H. pylori colonization of cathelicidin knockout mice was strongly enhanced compared to wild-type mice, indicating that human LL37, which is closely related to murine cathelicidin, is able to control H. pylori infection in vivo (Zhang et al. 2013). It has been reported that CagA promotes the secretion of TNF and IL-1β, the expression of chemokines CCL7 and CXCL16, and the synthesis of AMP LL37 from AGS via the activation of the mechanistic target of rapamycin complex 1 (mTORC1) (Feng et al. 2020). In addition, H. pylori was shown to activate phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR signaling in a CagA-independent manner in AGS cells, resulting in an increased protein synthesis (Sokolova et al. 2014).

2.1.3

Macrophages

In response to H. pylori infection, macrophages are recruited within two days to the site of the infection (Algood et al. 2007). They are activated by cytokines and the presence of H. pylori-derived molecules, including LPS (Innocenti et al. 2001), Hsp60 (Zhao et al. 2007), NAP (Amedei et al. 2006), and urease (Gobert et al. 2002). Moreover, the T4SS was also recently implicated in the activation of macrophages by the translocation of H. pylori-derived heptose metabolites through the T4SS. This leads to the activation of NF-κB and is crucial for the production of IL-8 by macrophages (Faass et al. 2021). Macrophages have several roles during infection including phagocytosis, antigen presentation, and cytokine production. However, transient elimination of macrophages from a mouse infection model did not influence H. pylori colonization, but reduced gastric pathology indicating that macrophages are rather gastritis mediators than effective immune cells during H. pylori infection (Kaparakis et al. 2008). Nevertheless, macrophages shape the immune response through their cytokine production. They secrete the cytokines IL-1β, IL-6, IL-10, IL-12, and IL-23 (Fehlings et al. 2012). Moreover, macrophages producing the chemoattractants IL-8 and growth-regulated protein alpha (Gro-α) were correlated with neutrophil infiltration in human biopsies (Eck et al. 2000). H. pylori induces the polarization of both macrophage subtypes (M1 and M2) in humans (Fehlings et al. 2012), while in the mouse model, M1 macrophages are the dominant subtype (Quiding-Jarbrink et al. 2010). This polarization depends on

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the virulence factor CagA, which induces heme-oxygenase 1 favoring a M2 phenotype (Gobert et al. 2014), and on the number of bacteria present in the vicinity of macrophages. Thus, in vitro, infection at a very high multiplicity of infection (MOI) suppressed the M2-phenotype, whereas the presence of less bacteria led to the polarization of M1 and M2 macrophages (Lu et al. 2020). In vivo, as shown by immunohistochemistry staining of the M2 marker CD206 and the M1 marker CD86 in human biopsies, the presence of M1 macrophages also depends on the infection level. In contrast to M1 macrophages, the numbers of M2 macrophages in the same study were not significantly increased by high H. pylori numbers but were highly correlated with the progression of H. pylori-induced gastritis to gastric cancer (Lu et al. 2020). In summary, macrophages shape further immune responses during H. pylori infection but are not able to control the infection efficiently and, especially M2 macrophages, contribute to disease progression.

2.1.4

Neutrophils

Neutrophils are among the first immune cells to be recruited to the site of infection (Algood et al. 2007). The H. pylori virulence factor NAP (Satin et al. 2000), the chemokine IL-8, and the hepatoma-derived growth factor (HDGF) (Chu et al. 2019) are known promotors of neutrophil infiltration. IL-8 is produced in response to H. pylori infection namely by GECs (Jung et al. 1997), neutrophils (Alvarez-Arellano et al. 2007), macrophages, and DCs (Fehlings et al. 2012). HDGF expression was found to be upregulated in GECs upon H. pylori infection of mice and HDGF levels were strongly correlated with neutrophil infiltration both in the murine and human context (Chu et al. 2019). In addition, H. pylori GGT was also shown to be important for neutrophil infiltration in a mouse infection model (Wustner et al. 2017). The main function of neutrophils is to kill invading pathogens by phagocytosis or by the release of toxic mediators such as reactive oxygen species (ROS), proteolytic enzymes, AMPs, and calprotectin. Indeed, the depletion of neutrophils in an IL10deficient mouse gastritis model infected with H. felis proved that neutrophils not only contribute to the clearance of Helicobacter but also increase gastritis at the same time (Ismail et al. 2003). Similarly, neutrophil depletion delayed clearance of H. felis from T cell receptor-deficient mice that were transferred with CD4+ CD25− T effector cells before infection. However, at least in part, clearance in this model was due to the neutrophil-dependent recruitment of T cells to the gastric mucosa and not only due to the toxic actions of neutrophils (Sayi et al. 2009). In addition to controlling the infection, neutrophils shape further immune responses directed against H. pylori by releasing cytokines like IL-8, IL-1β, and TNF (Alvarez-Arellano et al. 2007), however, the cytokine profile depends on the presence of certain virulence factors. Infection with cagPAI proficient strains is related to a pro-inflammatory cytokine response, whereas cagPAI− strains induced a rather antiinflammatory response in H. pylori-infected human neutrophils (Sanchez-Zauco et al. 2014). Flagellin A (FlaA), CagL, and NAP are also important bacterial proteins influencing the cytokine release by neutrophils. FlaA seems crucial for the induction of an

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IL-1β response (Jang et al. 2020), and the interaction of NAP with TLR2 is important for IL-8 secretion by neutrophils (Wen et al. 2021). Another mechanism important for neutrophil function during H. pylori infection is the interaction between the adhesin HopQ and CEACAM receptors. This interaction on neutrophils controls the production of macrophage inflammatory protein alpha (MIPα) and triggers phagocytosis (Behrens et al. 2020). As neutrophils fail to clear the infection, their ongoing oxidative action is discussed to promote gastric pathologies. In the context of tumor development, neutrophils were recently classified into two subgroups (N1 and N2). N1 cells produce high levels of ROS and cytokines, while N2 produces fewer cytokines, but more pro-tumoral factors (Mihaila et al. 2021). Considering these characteristics, it is accepted that H. pylori infection induces N1-like human neutrophils as indicated by a high expression of surface markers like CD11b, CD16, CD66b, and CD63 in combination with a low expression of CD62L and by the secretion of cytokines including IL-1β, IL-6, IL-8, CXCL1, MIP-1 by infected neutrophils (Whitmore et al. 2017). Although neutrophils are successfully recruited and activated in the presence of H. pylori, they fail to clear the infection due to diverse mechanisms (described below) employed by the bacterium to dampen their function. Over time, the continuous oxidative action of neutrophils might rather damage the tissue and drive gastric pathology.

2.1.5

Dendritic Cells

Chemokines secreted by GECs are crucial for the recruitment of DCs to the stomach upon H. pylori infection (Sebrell et al. 2019), and once in the epithelium they sense invading bacteria (Necchi et al. 2009) and are major determinants of the adaptive immune responses mounted against H. pylori. At the site of infection, H. pylori induces the activation and maturation of DCs leading to the production of several cytokines, including IL1-β, IL-6, IL-8, IL-10, IL-12, and IL-23 (Kranzer et al. 2004; Fehlings et al. 2012). Cytokine secretion and antigen presentation by DCs induce Th1 (Bimczok et al. 2010) and Th17 responses (Khamri et al. 2010). These general observations have led to recent studies in mouse models in order to address in detail the specific function of several DC subsets during H. pylori infection. Infection of mice deficient in the basic leucine zipper ATF-like transcription factor 3 (BATF3) showed that DCs expressing this transcription factor produce the chemokines CXCL9, 10, and 11 important to recruit Tregs (Arnold et al. 2019). In contrast, expression of the transcription factors interferon regulatory factor 4 (IRF4) and neurogenic locus notch homolog protein 2 (NOTCH2) rather impair DC-mediated Th1 responses as DC-specific deletion of IRF4 or NOTCH2 increased CD4+ T cell infiltration in infected mice (Zhang et al. 2020). In conclusion, DCs are central mediators of the H. pylori-directed Th1/Th17 response, but are also important for the induction of Tregs . How different DC subsets contribute to T cell responses is a topic of current research.

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Innate Lymphoid Cells, Natural Killer Cells, and Eosinophils

Innate lymphoid cells (ILCs) are a population of lymphocytes that lack antigenspecific receptors but respond to infection or to stress signals by producing cytokines (Panda and Colonna 2019). ILCs are categorized into three groups based on their cytokine production, transcription factors, and functions. ILC2s are the predominant subset in the gastric mucosa (Satoh-Takayama et al. 2020) and were observed to be increased in H. pylori-infected patients (Li et al. 2017) and mice (Satoh-Takayama et al. 2020). The immune function of ILC2s during H. pylori infection is not yet fully understood. However, Satoh-Takayama et al. (2020) showed that ILC2s secrete IL-5 leading to the production of immunoglobulin A (IgA) that coats H. pylori. This suggests that ILC2s are involved in B-cell responses, probably because IL-5 is important to enhance B-cell maturation. NK cells are an innate lymphocyte population that directly kill target cells. Upon H. pylori infection, DCs release IL-12 to activate NK cells to produce IFN-γ and TNF (Hafsi et al. 2004). Moreover, the cytotoxic response of NK cells was found to be activated by the TLR4-dependent induction of NKG2DL on H. pylori-infected MKN45 (Hernandez et al. 2021). Activated NK cells killed infected GECs efficiently in vitro (Hernandez et al. 2021). In conclusion, NK cells seem to be functionally important during H. pylori infection. However, further research in mouse models is needed to pinpoint their function in the H. pylori-directed immune response. Eosinophils are known to infiltrate the gastric mucosa during H. pylori infection (McGovern et al. 1991) and were also found close to GECs after an effective H. pylori vaccination of mice (Akhiani et al. 2004b). They have been reported to restrict effector T cell responses during the infection (Arnold et al. 2018). Arnold et al. found IFN-γ and IL-17 to be overexpressed in eosinophil-deficient mice during H. pylori infection. Moreover, they proved that the eosinophil-related control of the Th1 response depends on IFN-γ-mediated upregulation of PD-L1 (Arnold et al. 2018). In addition, decreased IL-10 secretion from CD4+ T cells in eosinophil-depleted mice undergoing vaccination suggests that eosinophils maintain a rather anti-inflammatory environment during H. pylori infection (Vaillant et al. 2021).

2.2 Adaptive Immune Responses 2.2.1

Cellular Immune Response

During H. pylori infection, the combined response of the adaptive and innate immune system is crucial. Antigen-presenting cells (APCs) play a significant role by capturing, processing, and presenting antigens to naïve T cells in the Peyer’s Patches (PPs) and mesenteric lymph nodes (MLNs). T cells are then activated by recognizing specific antigenic epitopes bound to MHC class I and II molecules on the surface of APCs and efficiently control the infection (as discussed below). As H. pylori is an extracellular bacterium, the concept that T cells combat the bacterium seems to be

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counterintuitive. However, this concept has just recently been corroborated in a mouse model of urinary tract infections. Here, Rousseau et al. (2023) showed that tissueresident memory T cells (TRM ) that develop during antigen persistence, mediate protection against recurring urinary tract infections with extracellular Escherichia coli, while experienced circulation-derived T cells did not contribute to protection. In line with this, independent reports using different models point to a central role of T cells during H. pylori infection. Several mechanisms might be involved in this process. In the first place, effector T cells produce an array of cytokines activating phagocytic activity of innate immune cells. In addition, specific subsets of T cells might eliminate host cells that have H. pylori tightly bound to them. Considering recent data showing that T cells recruited to the stomach express typical TRM markers (Koch et al. 2022), it is plausible that this T cell subset also participates in the protection from H. pylori infection. However, regulatory subsets of T and B cells suppress the pro-inflammatory response by secreting anti-inflammatory cytokines that inhibit the effector T cell response, resulting in immune tolerance during infection. CD4+ T Cells CD4+ T cells, including Th1, Th17, and Tregs are important for the immune response to H. pylori (Kabir 2011; Enarsson et al. 2006; Della Bella et al. 2023). A lower gastritis and higher bacterial colonization have been observed in H. pylori-infected CD4-deficient and IFN-γ-deficient mice compared to wild-type mice (Eaton et al. 2001). These results indicated that H. pylori infection induces a Th1-polarized response accompanied by the production of IFN-γ, which is necessary for the control of infection, but on the other hand, causes chronic gastritis, and eventually even gastric cancer (Karttunen et al. 1995; Bagheri et al. 2018). The robust Th1-biased environment is promoted by H. pylori NAP-dependent IL-12 and IL-23 secretion by neutrophils and monocytes. Moreover, the Th1 response is related to up-regulated Notch1 expression (Amedei et al. 2006; Xie et al. 2020; Rahimian et al. 2022). Th17 cells, producing IL-17A, were also found to contribute to bacterial control by increasing IL-8 secretion from a murine transformed gastric epithelial cell line (GSM06), and thereby recruiting neutrophils (Kabir 2011; DeLyria et al. 2009). Moreover, Th17 cells also amplify the Th1 response and later contribute to pathology, which has been proven by increased gastric inflammation and bacterial load in IL17 overexpressing mice, and reduced inflammation and IFN-γ secretion in IL-17 knockout mice during H. pylori infection (Shi et al. 2010). In addition, IL-23 also facilitates the Th17 response as IL-17 secretion was lower and inflammation was reduced in infected IL-23 mutant mice (Horvath et al. 2012). However, IL-17 also promotes gastric carcinogenesis after H. pylori infection through up-regulating the IL-17RC/NF-κB/NOX1 pathway (Kang et al. 2023). Additionally, H. pylori CagY predominantly drives IFN-γ and IL-17 secretion by gastric CD4+ T cells, which further promotes B cell proliferation and eventually may lead to the development of gastric MALT lymphoma (Della Bella et al. 2021). However, not only effector T cells but also high levels of Tregs are found in the H. pylori-infected gastric tissue (Cheng et al. 2012). These cells impair CD4+ and CD8+ T cell proliferation and cytokine secretion to suppress excessive immune responses

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(Sakaguchi et al. 2001). The increased gastric inflammatory response and reduced H. pylori colonization in CD25-depleted mice, which lowered gastric Tregs , indicate that Tregs are indeed highly immunosuppressive during H. pylori colonization and could thus contribute to local immune tolerance (Rad et al. 2006). Children and mice infected at a young age show a high amount of Treg cells and lower gastric pathology compared to adults (Harris et al. 2008; Arnold et al. 2011), which implies a protective effect of Tregs against gastric pathologies. Importantly, H. pylori directly supports the development of Tregs through various of its virulence factors (see below). CD8+ T Cells In the recent years, several studies have revealed a crucial function of CD8+ T lymphocytes in the immune response to H. pylori infection, and shown that a considerable proportion of T cells that infiltrate the stomach of H. pylori-infected mice are CD8+ and express high levels of IFN-γ (Ruiz et al. 2012; Wustner et al. 2017). Moreover, clinical studies conducted in children and adults revealed a link between a high infiltration of CD8+ T cells during H. pylori infection and the onset of gastric ulcers (Tan et al. 2008; Graham et al. 2004; Helmin-Basa et al. 2011; Booth et al. 2015). Likewise, findings from mouse studies indicated that in the absence of CD4+ T cells, mice infected by H. pylori or H. felis showed increased levels of gastric CD8+ T cells, which further accelerated the development of gastric ulcers (Tan et al. 2008; Fukui et al. 2007). However, CD8+ T cells not only mediate gastric pathologies but were also recently shown to effectively control H. pylori infection in the early infection phase (Koch et al. 2022). Aziz et al. reported that the metabolic profile of CD8+ T cells was altered in IL-10-deficient mice after H. pylori infection, which stimulated the release of IL-1β by CD8+ cells and promoted H. pylori colonization and tumorigenesis (Aziz et al. 2022). Thus, this result indicated that infection control mediated by CD8+ T cells might depend on IL-10. Tissue Resident Memory T Cells A localized infection occluding in a specific tissue or organ usually elicits a systemic and local immune response. Several studies observed that TRM cells expressing CD69 and CD103 could stay in the skin, lung, salivary glands, small intestine, and stomach after various infections without recirculating through the blood (Hombrink et al. 2016; Gebhardt et al. 2011; Watanabe et al. 2015; Sheridan et al. 2014; Hofmann and Pircher 2011; Koch et al. 2022). In the context of H. pylori infection, a protective function of gastric TRM cells was first observed in a vaccine study (Liu et al. 2019a). The authors reported that after vaccination of mice, more IFN-γ and IL-17 were secreted by CD4+ CD69+ TRM cells upon antigen stimulation, which significantly controlled H. pylori colonization (Liu et al. 2019a). Following studies revealed that vaccine-induced CD4+ CD69+ TRM cells strongly proliferated and stayed in the gastric tissue for long term without recirculating (Xu et al. 2019; Hu et al. 2020). In addition, increased neutrophil recruitment related to CD4+ CD69+ TRM cells induction was found to be involved in the protection against H. pylori infection (Xu et al. 2019; Hu et al. 2020). High numbers of CD4+ CD69+ CD103+ TRM cells were also

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found in stomachs of H. pylori-positive patients and exhibited a memory-like phenotype and high expression of chemokine receptors CXCR3 and CCR9 (Chen et al. 2020a). CD103 expression enhanced IFN-γ, TNF, and IL-17 production by gastric CD4+ CD69+ TRM cells from H. pylori-positive patients (Chen et al. 2020a). Furthermore, CagA-specific CD8+ CD69+ CD103+ TRM cells were recently found to participate in the control of H. pylori infection in mice (Koch et al. 2022). However, the specific function of TRM cells in the gastric tissue during H. pylori infection is still unclear, especially in the context of vaccine efficacy.

2.2.2

Humoral Immune Response

H. pylori infection not only triggers a potent T cell response with an essential role in mediating protection but also a specific antibody response. Strong local and systemic H. pylori-specific IgA and IgG antibodies are observed after infection, and, in fact, elevated serum Ig titers against H. pylori have been widely used for diagnosis of the infection (Godbole et al. 2020). However, there are contradictory results regarding the role of the humoral response in controlling H. pylori colonization. Studies using mice deficient in B-cells and antibodies (μMT mice) demonstrated comparable levels of H. pylori colonization in both μMT and C57BL/6 mice upon infection (Ermak et al. 1998). In addition, B cell-deficient mice were able to control H. pylori colonization to a similar extent as wild-type mice after immunization (Akhiani et al. 2004a). More recently, another study found no difference in H. pylori colonization between μMT and C57BL/6 mice by using siblings with the same microbiome (Arshad et al. 2020). Additionally, the prevalence of H. pylori infection in IgA-deficient patients showed no significant difference in the absence of IgA antibodies (Bogstedt et al. 1996). In summary, these results seem to indicate that H. pylori-directed antibodies do not contribute to H. pylori protection. However, results from studies involving μMT mice need to be interpreted cautiously because these mice can still produce secretory IgA (sIgA) that binds to luminal bacteria (Macpherson and Uhr 2004). Early studies reported that children who were breastfed by mothers with high titers of sIgA were protected from infection longer than others (Thomas et al. 1993). Later, screening of gastric samples, saliva, and breast milk from H. pylori-infected individuals revealed a specific sIgA response (Tummala et al. 2004). Interestingly, in IgA mutant mice after H. pylori infection, lower IFN-γ production and higher bacterial colonization were detected, suggesting that IgA responses facilitated by B cells even could potentially control H. pylori infection (Akhiani et al. 2005). A new subset of immunosuppressive B cells was recently identified. These cells found in normal human peripheral blood known as regulatory B cells (Bregs ) produce high amounts of IL-10 and are able to inhibit the proliferation of CD4+ T cells, decreasing the number of Th17 cells and Tregs in vitro (Hong et al. 2019). After H. pylori infection, the number of Bregs has been observed to increase in the human as well as in the mouse stomach during the early stage of infection before the induction of Tregs and provided a potential target for gastritis and colitis treatment (Li et al. 2019;

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Nahid-Samiei et al. 2020). Although B cells have been associated with gastric clinical symptoms such as gastritis in H. pylori-infected patients, the role of the humoral response, and in particular mucosal IgA, in H. pylori clearance is still controversial. Therefore, their functional immune modulation and subsequent pathogen clearance function need to be further studied.

3 Immune Evasion As mentioned in the previous sections, H. pylori infection induces a strong immune response orchestrated by the recognition of bacterial determinants by specific receptors expressed on immune cells and the subsequent secretion of a plethora of immunomodulatory molecules. However, the bacterium has developed efficient strategies to evade host immunity and mechanisms to manipulate the immune response with the aid of several virulence factors (Fig. 2). This results in the chronic persistence of H. pylori over decades and can lead to the development of gastric lesions and eventually gastric cancer, if not treated.

Fig. 2 H. pylori effectively evades the immune response by several mechanisms. The bacterium evades immune recognition by the modification of PAMPs and by manipulation of PRR-dependent signaling. Moreover, H. pylori is rather resistant to AMPs and inhibits AMP synthesis by GECs. The bacterium inhibits phagocytosis and limits the number of phagocytes. In addition to that, H. pylori is well-equipped to detoxify ROS and RNI and inhibits phagosome maturation. DCs are manipulated by H. pylori to favor Treg induction and to suppress the Th1/Th17 response. Moreover, T-cell proliferation and cytotoxicity are also inhibited by the bacterium

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3.1 Avoidance of Recognition and Manipulation of PRR Signaling As described above, immunity against H. pylori starts by its recognition through a number of receptors expressed on immune cells and on GECs. However, by modifying PAMPs and by manipulating the antigen presentation ability of phagocytes, H. pylori can avoid recognition. LPS is composed of three parts: Lipid A, O-antigen, and core oligosaccharide (Bertani and Ruiz 2018). H. pylori lipid A has a low biological activity because of its unique acylation (Moran et al. 1997) and phosphorylation pattern (Cullen et al. 2011). H. pylori phosphatases remove phosphate groups from lipid A, which reduces recognition by TLR4. However, phosphatases, involved in lipid A modification, might be downregulated during infection to increase fitness to calprotectin, which is an antimicrobial factor released by neutrophils (Gaddy et al. 2015). In this context, the binding of annexin V to phosphorylated lipid A can effectively shield LPS to interfere with TLR4 signaling (Schmidinger et al. 2022). Moreover, the LPS O-antigen is also modified by fucosyltransferases to resemble human blood group antigens to avoid recognition (Appelmelk et al. 2000). TLR5 recognition is evaded by H. pylori by modification of the TLR5-recognition domain of flagellin (Andersen-Nissen et al. 2005a). Moreover, Pachathundikandi et al. (2019) proposed that H. pylori hides or exposes CagL to avoid permanent TLR5 activation or to induce its activation when advantageous for the bacterium. In addition to the modification of PAMPs, H. pylori suppresses the antigen presentation activity of macrophages. The presence of H. pylori triggers the up-regulation of microRNAs that downregulate the class II major histocompatibility complex transactivator (CIITA) (Codolo et al. 2019) and upregulate the expression of CD300E, affecting the ability of macrophages to express the human leukocyte antigen II (HLAII) (Pagliari et al. 2017). The downregulation of CIITA depends on the presence of H. pylori-derived ADP-heptose, an important metabolite in LPS biosynthesis (Coletta et al. 2021). In addition to evading recognition by PRRs, H. pylori also manipulates their downstream signaling. Activation of TLR2 and TLR9 triggers rather anti-inflammatory responses as shown by the infection of TLR-2 (Sun et al. 2013) and TLR-9 (Otani et al. 2012) knockout mice. TLR-2 negative DCs produced less IL-12, TNF, IL-6, and IL23 during infection of TLR2-deficient mice, triggering an enhanced Th1 response. In contrast, less Tregs were induced, resulting in a reduced colonization of the knockout mice. Infection of TLR9-deficient mice leads to an increased neutrophil infiltration and production of cytokines TNF-α and INF-γ in the early phase of H. pyloriinduced gastritis. Moreover, TLR10 activation was found to attenuate the release of pro-inflammatory cytokines like IL-1β, INF-γ, and IL-12 by H. pylori-infected DCs (Neuper et al. 2020). Mechanistically, some strategies are known to enable the bacterium to manipulate PRR signaling. H. pylori was found to actively suppress RIG-I, a stimulator of interferon genes (STING) dampening the signaling of nucleic acid recognizing PRRs in

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GECs of H. pylori infected mice (Dooyema et al. 2022). Moreover, and in contrast to other pathogenic bacteria, H. pylori DC-SIGN ligands are fucosylated, which disassociates the downstream signaling complex and suppresses pro-inflammatory responses (Gringhuis et al. 2009). In summary, H. pylori actively avoids innate recognition and manipulates PRR-dependent signaling to avoid infiltration of immune cells and thereby ensure its long-term persistence in the stomach.

3.2 Resistance Against AMPs Although AMPs kill H. pylori efficiently in vitro (Nuding et al. 2013), the bacterium is able to persist in vivo despite the induction of AMP production in GECs upon infection. H. pylori evades AMPs by expressing factors to increase its resistance against them. Thus, H. pylori-derived outer membrane vesicles (OMVs) were recently shown to protect the bacterium from LL-37. This protective effect is either due to bacterial factors that are enclosed in OMVs or due to sequestration of LL-37 in OMVs as it was already shown for other Gram-negative species (Murray et al. 2020). Moreover, lipid A modification by phosphatases not only helps the bacterium to avoid recognition but also increases AMP resistance (Cullen et al. 2011). As cationic AMPs like LL-37 or hbD2 bind to negatively charged structures on the bacterial surface, the removal of phosphate groups by phosphatases prevents binding and thus function of AMPs. Apart from resisting the toxic action of AMPs, H. pylori also manipulates signaling cascades regulating the expression of AMPs. The virulence factors CagA and cholesterol-α-glucosyltransferase (CGT) enable the bacterium to downregulate AMP synthesis in GECs. H. pylori was shown to be able to inhibit the synthesis of hBD3 in the gastric epithelial cell line AGS by CagA-dependent activation of tyrosine phosphatase SHP-2, which subsequently inactivates the epidermal growth factor receptor (EGFR) (Bauer et al. 2012). Moreover, CGT depletes cholesterol from epithelial cell membranes, which was shown to reduce JAK/STAT signaling in infected MKN45, AGS, and primary human GECs and results in impaired expression of hBD3 (Morey et al. 2018). As revealed by the infection of NLRC4 knockout mice, H. pylori infection also leads to an NLRC4 inflammasome-dependent maturation of IL-18 in GECs, which activates NF-κB-signaling and downregulates expression of the constitutively expressed AMP hbD1 (Semper et al. 2019). This mechanism was shown to be of utmost importance for H. pylori persistence, as the colonization of NLRC4 knockout mice was enhanced compared to wild-type mice (Semper et al. 2019). Moreover, MMP-10 dependent CXCL16 production from H. pylori– infected AGS inhibits Reg3a together with E-cadherin, and zonula occludens-1 (ZO1), leading to impaired host defenses and contributing to inflammation in murine infection models (Lv et al. 2019).

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3.3 Inhibition and Survival of Phagocytosis To persist despite the presence of numerous phagocytes, H. pylori developed mechanisms to limit the number of active phagocytes in the vicinity of the bacterium and to avoid and survive phagocytosis. To reduce the number of phagocytes, H. pylori manipulates both macrophages and neutrophils. H. pylori inhibits the proliferation of macrophages by disrupting cell-cycle associated genes (Tan et al. 2015), and induces apoptosis of macrophages (Asim et al. 2010; Menaker et al. 2004) by altering the mitochondrial pathway and by extracellular signal-regulated kinase (ERK)-dependent formation of an activator complex that causes apoptosis. Inhibition of ERK during H. pylori infection in mice indeed attenuated apoptosis of gastric macrophages (Asim et al. 2010). Moreover, H. pylori was found to impair neutrophil activation CagA-dependently by inhibiting the expression of neutrophil protease activator cathepsin C (CtsC) in AGS and human primary GECs (Liu et al. 2019b). CtsC injected to infected mice cleared H. pylori efficiently, implicating that CtsC-downregulation is important for persistence. Moreover, H. pylori disturbs the polarity of neutrophils, which results in impaired chemotaxis towards IL-8 and a lower migration speed (Prichard et al. 2022). Another persistence mechanism involves the inhibition and survival of phagocytosis. Thus, H. pylori was shown to inhibit phagocytosis in a T4SS- (Ramarao et al. 2000) and alpha-glycosylation of bacterial-derived cholesterol-dependent manner (Wunder et al. 2006). If despite all these defense mechanisms H. pylori gets phagocytosed, the bacterium interferes with the killing machinery of phagocytes and is well-equipped to survive the release of toxic ROS and nitric oxide (NO). H. pylori disrupts NADPH-oxidase in neutrophils, releasing superoxide that can no longer accumulate in the phagosomes (Allen et al. 2005). In addition, phagosome maturation is inhibited by the virulence factor VacA (Zheng and Jones 2003) and by cholesteryl glucosides on the bacterial cell membrane (Lai et al. 2018). Despite HopQ-CEACAM3/6 interaction on neutrophils was found to trigger a strong oxidative burst and to enhance phagocytosis, this interaction was also shown to increase survival of H. pylori in a recently published study, indicating that H. pylori might use this interaction in a yet unknown way to manipulate neutrophils (Behrens et al. 2020). Moreover, H. pylori hampers NO production in macrophages by limiting host arginine, as H. pylori arginase (Gobert et al. 2002) and induction of host arginase II (Lewis et al. 2010) in response to the bacterium hydrolyze the amino acid. In addition, H. pylori expresses enzymes that detoxify ROS, including alkyl hydro-peroxidase (Wang et al. 2005), superoxide dismutase (Seyler et al. 2001), and catalase (Basu et al. 2004). Similarly, the H. pylori enzymes NO-reductase NorH (Justino et al. 2012) and GSNO-reductase FrxA (Justino et al. 2014) degrade toxic NO and the reactive nitrogen intermediate (RNI) S-nitrosoglutathione (GSNO). Both enzymes were shown to attenuate the NO-dependent killing of H. pylori by macrophages and were found to support H. pylori colonization in the mouse model (Justino et al. 2012, 2014). Moreover, urease-derived carbon dioxide reacts with the RNI nitrosoperoxycarbonate (ONOOCO2 − ) to the non-toxic metabolite NO3 − (Kuwahara et al. 2000).

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In addition, protein damage to the NO target arginase can be reverted by H. pylori thioredoxin A (TrxA) (McGee et al. 2006).

3.4 Manipulation of DCs and Modulation of Adaptive Immune Responses To evade T cell immunity, H. pylori can either manipulate DCs, which are central mediators of the T cell response elicited upon H. pylori infection, or interact with T cells directly. H. pylori induces a strong Treg response that favors bacterial persistence by several mechanisms. H. pylori infection of a mouse model with TGF-βdeficient DCs led to an increased production of pro-inflammatory cytokines and a more severe gastritis, indicating that TGFβ-production by DCs is important to mediate Treg responses (Owyang et al. 2020). Similarly, IL-18 knockout mice showed impaired DC-dependent Treg induction, which indicates that IL-18 is crucial for the conversion of naïve T cells to Tregs (Oertli et al. 2012). Moreover, there is cumulative evidence indicating that increased IL-1β and IL-18 expression mediated by NLRP3 inflammasome and caspase-1 activation, and up-regulated indoleamine 2, 3-dioxygenase (IDO) expression upon H. pylori infection also function to induce Tregs (Azadegan-Dehkordi et al. 2021; Engler et al. 2015; Kim et al. 2013; Fallegger et al. 2022). Several H. pylori factors including CagA, VacA, and GGT are important to skew DCs to a more tolerogenic phenotype favoring the induction of Tregs . In murine DCs, CagA interferes with downstream signaling blocking nuclear translocation of IRF3 and subsequent interferon production (Tanaka et al. 2010). In human DCs, CagA impairs IL-12 secretion and favors IL-10 production resulting in Treg induction (Kaebisch et al. 2014). VacA promotes immune tolerance by restoring transcription factor E2F2, which represses DC maturation (Kim et al. 2011). In addition, VacA was shown to have an immunomodulatory function targeting myeloid cells and skewing the T-cell response to Tregs by IL-23 suppression in DCs and by IL-10 induction in macrophages (Altobelli et al. 2019). GGT-derived glutamate interacts with glutamate receptors on DCs leading to cAMP-dependent signaling that inhibits IL-6 expression and favors the induction of Tregs . Moreover, the expression of miRNA375 is downregulated in the presence of H. pylori, hampering DCs maturation and promoting IL-6 and IL-10 secretion, which results in decreased numbers of CD4+ and CD8+ T cells (Zhang et al. 2021). H. pylori virulence factors have also been reported to interfere directly with T cells during infection. By interrupting IL-2 signaling and affecting the mitochondrial membrane potential of CD4+ T cells, VacA inhibits T cell proliferation further suppressing the Th1 response (Gebert et al. 2003; Torres et al. 2007; Zheng and Jones 2003). Moreover, VacA-mediated apoptosis in T cells is regulated by the activation of the mitochondrial pathway (Ganten et al. 2007) and is another evasion mechanism to limit T cell responses. However, how VacA would directly interact with T cells

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in the gastric mucosa has never been shown in vivo. GGT also inhibits T cell proliferation by decreasing extracellular glutamine, resulting in cell cycle arrest during the G1 phase (Schmees et al. 2007; Wustner et al. 2015). Moreover, GGT-dependent glutamine hydrolysis lowers mucosal glutamine levels and thereby hampers T cell activation and effector cytokine secretion. The outer membrane protein HopQ is another H. pylori virulence factor that directly inhibits T cell function. Thus, HopQ was found to inhibit IFN-γ secretion in CD4+ T cells and to limit the cytotoxic ability of CD8+ T cells and NK cells through its interaction with CEACAM1 (Gur et al. 2019). Finally, another virulence factor favoring persistent colonization is the urease B subunit (UreB). UreB can bind to myosin heavy chain 9 (Myh9), which increases mTORC1 signaling in macrophages and induces PD-L1 accumulation on H. pyloriinfected macrophages. Subsequently, PD-L1 on macrophages inhibits the activity of cytotoxic CD8+ T cells during H. pylori infection (Wu et al. 2021). This represents another indirect mechanism by which H. pylori escapes T-cell responses. In summary, H. pylori creates a tolerogenic environment through its virulence factors, which favors H. pylori persistence. This environment may prevent effector T-cell responses against potentially transformed host cells, thus eventually favoring tumor development.

4 Concluding Remarks While inducing a strong pro-inflammatory immune response, H. pylori uses various strategies to evade the immune response. The bacterium avoids recognition by modifying PAMPs and by manipulating PRR-dependent signaling. Nevertheless, AMPs are still produced and immune cells infiltrate the gastric mucosa upon H. pylori infection. Subsequently, H. pylori downregulates AMP synthesis via the virulence factors CagA and CGT. Moreover, action of phagocytes like macrophages and neutrophils is actively inhibited by H. pylori and the bacterium is well-equipped to survive oxidative and nitrosative stress. Additionally, H. pylori induces a strong Treg response by manipulating DCs limiting effector T cell response and pro-inflammatory cytokine section. Using the virulence factors CagA, VacA, and GGT, H. pylori also directly limits T cell responses. These immune evasion mechanisms enable the bacterium to persist for decades in its host, despite the presence of a robust innate and adaptive immune responses. As on-going activity of immune cells and the damaging effect on the gastric epithelium strongly contribute to H. pylori-associated gastric pathologies, it is of utmost importance to have effective therapies against this pathogen. Considering the growing number of antibiotic-resistant H. pylori infections, the development of alternative therapies or preventive measures becomes more and more important. Emphasis of vaccine studies should be put on inducing persistent protection without gastric cell damage such as TRM cells and neutralizing antibodies of virulence factors. Therefore, further research to understand the action and interplay of different immune cells and how bacterial virulence factors affect them is crucial to develop successful therapies and effective vaccines. Eventually, given the complex and multilayered

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immune response and immune evasion elicited by H. pylori, future vaccines may also require to address several of these aspects by counteracting immune evasion and at the same time eliciting broad immune responses comprising (neutralizing) antibodies, AMPs, and effector T cells in concert to be efficacious.

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Pathogenomics of Helicobacter pylori Yoshio Yamaoka, Batsaikhan Saruuljavkhlan, Ricky Indra Alfaray, and Bodo Linz

Abstract The human stomach bacterium Helicobacter pylori, the causative agent of gastritis, ulcers and adenocarcinoma, possesses very high genetic diversity. H. pylori has been associated with anatomically modern humans since their origins over 100,000 years ago and has co-evolved with its human host ever since. Predominantly intrafamilial and local transmission, along with genetic isolation, genetic drift, and selection have facilitated the development of distinct bacterial populations that are characteristic for large geographical areas. H. pylori utilizes a large arsenal of virulence and colonization factors to mediate the interaction with its host. Those include various adhesins, the vacuolating cytotoxin VacA, urease, serine protease HtrA, the cytotoxin-associated genes pathogenicity island (cagPAI)-encoded typeIV secretion system and its effector protein CagA, all of which contribute to disease development. While many pathogenicity-related factors are present in all strains, some belong to the auxiliary genome and are associated with specific phylogeographic populations. H. pylori is naturally competent for DNA uptake and recombination, and its genome evolution is driven by extraordinarily high recombination and mutation rates that are by far exceeding those in other bacteria. Comparative genome analyses revealed that adaptation of H. pylori to individual hosts is associated with

Y. Yamaoka · B. Saruuljavkhlan · R. I. Alfaray Department of Environmental and Preventive Medicine, Oita University Faculty of Medicine, 1-1, Idaigaoka, Hasama-machi, Yufu Oita 879-5593, Japan e-mail: [email protected] Y. Yamaoka Department of Medicine, Gastroenterology and Hepatology Section, Baylor College of Medicine, Houston, TX 77030, USA B. Linz (B) Division of Microbiology, Department Biology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstr. 5, 91058 Erlangen, Germany e-mail: [email protected] R. I. Alfaray Helicobacter pylori and Microbiota Study Group, Universitas Airlangga, Surabaya 60286, East Java, Indonesia © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 S. Backert (ed.), Helicobacter pylori and Gastric Cancer, Current Topics in Microbiology and Immunology 444, https://doi.org/10.1007/978-3-031-47331-9_5

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strong selection for particular protein variants that facilitate immune evasion, especially in surface-exposed and in secreted virulence factors. Recent studies identified single-nucleotide polymorphisms (SNPs) in H. pylori that are associated with the development of severe gastric disease, including gastric cancer. Here, we review the current knowledge about the pathogenomics of H. pylori.

1 Introduction The first genome of a Helicobacter pylori strain was published in the year 1997, the genome of strain 26695 that was isolated in the UK from a patient with gastritis (Tomb et al. 1997). This great achievement in the field of H. pylori research was soon followed by the genome sequence of strain J99 from a patient with duodenal ulcer in the US (Alm et al. 1999), which allowed a genome comparison of gastritis and peptic ulcer disease (PUD) strains. Despite the relatively small H. pylori genome size of approximately 1.6 Mbp, it took a long time, namely, 7 years, until the publication of a third H. pylori genome, the genome of strain HPAG1 from a patient with chronic atrophic gastritis (Oh et al. 2006). All three genomes contained the cag pathogenicity island (cagPAI), but HPAG1 was the only strain that contained a plasmid, though other H. pylori strains can possess one or several plasmids (Fernandez-Gonzalez and Backert 2014). Multilocus sequence analysis (MLSA)-based phylogeographic studies assigned isolates 26695 and HPAG1 to the H. pylori population hpEurope and J99 to the hpAfrica1 sub-population hspWAfrica (Falush et al. 2003b; Linz et al. 2007). Even though the pubmlst.org database (Jolley and Maiden 2010) contained over 800 H. pylori entries at this time, and genomes of multiple isolates from many other bacterial species were sequenced, there were only three published H. pylori genomes (Linz and Schuster 2007). However, in addition to H. pylori, genomes from other Helicobacter species were sequenced, namely of the mouse pathogen Helicobacter hepaticus (Suerbaum et al. 2003) and of H. pylori’s closest relative, H. acinonychis from big cats (Eppinger et al. 2006). The genome analysis showed that H. acinonychis arose from a host jump of H. pylori from humans to large felines (Eppinger et al. 2006) that was estimated to having occurred in Southern Africa ca. 50,000 years ago (50 kya) (Moodley et al. 2012). While the latter two genomes were cagPAI-negative, they contained other genomic islands (Linz and Schuster 2007). Given the importance of the cagPAI as a major H. pylori virulence factor, its sequence was determined from selected strains of world-wide origin (Azuma et al. 2004; Blomstergren et al. 2004; Censini et al. 1996; Olbermann et al. 2010). The observed lack of multiple H. pylori genomes in the early 2000s was partially associated with the lack of sophisticated sequencing technologies. All of the above genomes as well as the cagPAI sequences were generated by Sanger sequencing, which is very laborious for the generation of genome sequences. Next generation sequencing (NGS) methods at this time such as 454 pyrosequencing and Illumina sequencing represented milestones and leaps in sequencing technology, but both were associated with problems. While 454 pyrosequencing resulted in read lengths

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of over 500 bp, this technology was prone to sequencing mistakes, particularly in homopolymeric DNA stretches. Illumina reads on the other hand were very accurate but were very short with only 36 bp in length. Thus, H. pylori genome sequencing remained scarce, with only seven new genomes in 2008, three in 2009, and eight genomes added in 2010. Thanks to advanced read lengths in Illumina sequencing, this situation changed in 2011 with 27 new H. pylori genome entries in Genbank, followed by 116 new genomes in 2012, and then hundreds of new genomes annually. Since then, genome sequences were determined from a huge number of isolates in many different projects, including isolates from patients with chronic infection (Kennemann et al. 2011), transmission studies (Krebes et al. 2014; Linz et al. 2013), infection of human volunteers in vaccine trials (Estibariz et al. 2020), analysis of H. pylori pathogenicity in animal models (Ansari and Yamaoka 2022), phylogeographic analyses (Moodley et al. 2021; Thorell et al. 2017), sequencing of laboratory strains (Fischer et al. 2010), and genome-wide association studies (GWAS) to identify disease-specific genes (Berthenet et al. 2018; Tuan et al. 2021). Today (August 2023), the database at NCBI contains a total of 3,380 H. pylori genome entries. In addition, the genomes of numerous non-pylori Helicobacter species have been sequenced (Bauwens et al. 2018; Smet et al. 2018), and hundreds of H. pylori genomes are currently being worked on (https://dceg.cancer.gov/research/ how-we-study/genomic-studies/h-pylori-genome-project). The vast amount of H. pylori genomes, in combination with laboratory experiments, facilitated important progress in deciphering the pathogenicity of H. pylori. This included the specific presence of various virulence factors in individual phylogeographic populations, the correlation of particular genes or alleles to the geographical frequencies of gastric disease, and H. pylori pathogenomics in general. Also, H. pylori genomes enabled the reconstruction of human migrations in better resolution than from MLSA gene fragments alone. Recent progress even allowed the sequencing and reconstruction of the 5,300-year-old H. pylori genome of Ötzi the Iceman (Maixner et al. 2016, 2019). In the present chapter, we attempt to give an overview over the pathogenomics of H. pylori.

2 H. pylori Biogeography 2.1 Population Structure of H. pylori Initial single-gene sequence-based analyses noted considerable differences between H. pylori isolates of different geographic origin and showed that this bacterium exhibits a high degree of genetic diversity (Suerbaum et al. 1998; van der Ende et al. 1998; van Doorn et al. 1998a). The development of a MLSA scheme that was based on sequences of fragments from seven housekeeping genes (atpA, efp, mutY, ppa, trpC, ureI, yphC) (Achtman et al. 1999) and Bayesian algorithms implemented in the program Structure (Falush et al. 2003a) enabled a detailed analysis of the H. pylori population structure. Intra-familial and local transmission along with genetic

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isolation and genetic drift have facilitated the development of distinct bacterial populations. The no-admixture model in Structure, which assumes that each individual has derived all of its ancestry from only one population, allows to infer the number of distinct populations in the dataset and assigns the individual haplotypes to the inferred populations. Analyses of >2,500 H. pylori isolates from worldwide geographic and ethnic sources identified eight modern populations that were designated hpAfrica1, hpAfrica2, hpNEAfrica, hpEurope, hpEastAsia, hpNorthAsia, hpAsia2, and hpSahul based on their geographic distributions (Figs. 1 and 2) (Moodley and Linz 2009; Moodley et al. 2021). Additionally, current studies based on whole genome sequences and analysis of H. pylori genomes using FineStructure and Chromosome painting revealed the evolution of H. pylori subpopulations (hsp) in many countries (Moodley et al. 2021; Munoz-Ramirez et al. 2017, 2021; Suzuki et al. 2022). HpAfrica2 is the most divergent H. pylori population (Fig. 1) and is associated with the click language-speaking hunter-gatherers in southern Africa known as the San (Fig. 2, Table 1). Analyses showed that hpAfrica2 split from the other H. pylori lineages around 100 kya, and gave rise to H. acinonychis after a host jump from humans to large felines that occurred around 50 kya. The two subpopulations, hspNorthSan and hspSouthSan, split approximately 32–47 kya (Moodley et al. 2012). H. pylori of the hpAfrica1 population are found in Sub-Saharan Africa from West and Central Africa to southeastern and southern Africa, including Madagascar, but also frequently in the Americas, particularly among the Black population. Three distinct subpopulations have been identified: hspAfrica1SAfrica from southern Africa, hspAfrica1WAfrica from West Africa, and hspAfrica1CAfrica from central African countries (Fig. 2, Table 1) (Falush et al. 2003b; Linz et al. 2007, 2014a; Moodley et al. 2012; Nell et al. 2013). Isolates of the population hpNEAfrica were found in Ethiopia, Somalia, Sudan, among Nilo-Saharan language speakers in northern Nigeria (Linz et al. 2007), and among Baka pygmies and Bantu people from Cameroon (Nell et al. 2013). HpNEAfrica and hpAfrica1 separated 36–52 kya (Moodley et al. 2012). Two distinct hpNEAfrica subpopulations have been identified: hspENEAfrica and hspCNEAfrica (Nell et al. 2013; Thorpe et al. 2022). The hpAsia2 isolates were primarily found in Southeast Asian countries such as India, Bangladesh, Thailand, Malaysia, and the Philippines (Devi et al. 2007; Linz et al. 2007; Tay et al. 2009; Wirth et al. 2004) but also in northwestern Siberia among the Uralic speaker peoples Khanty and Nenet (Moodley et al. 2021). Three subpopulations have been identified within hpAsia2: hspIndia, hspLadakh, and hspUral (Figs. 1 and 2, Table 1). The hpSahul population is confined to indigenous peoples in Australia and Papua New Guinea and reflects the historical movement of humans into the Sahul continent. The two subpopulations, hspAustralia and hspNGuinea, split 23–25 kya (Moodley et al. 2009). The population hpEastAsia contains two subpopulations, hspEAsia and hspMaori. Isolates of hspEAsia are primarily present in East Asian countries, including China, Japan, Korea, Taiwan, Cambodia, Vietnam, and Malaysia (Breurec et al. 2011a; Falush et al. 2003b; Linz et al. 2007; Moodley et al. 2009; Tay et al. 2009). HspMaori isolates were originally found among Samoans, Tongans, and Maoris from New Zealand, hence the designation (Falush et al. 2003b), and subsequently among native Taiwanese, Melanesians in New Caledonia, and other Polynesian islanders

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Fig. 1 Distribution of the cag pathogenicity island in extant biogeographic H. pylori populations. Neighbor-joining (NJ) tree of concatenated sequences of seven housekeeping genes (length 3406 bp) of 876 H. pylori strains, including information about the presence or absence of the cagPAI. The sequences were extracted from the pubmlst.org database or extracted from genome sequences (Breurec et al. 2011a; Falush et al. 2003b; Kersulyte et al. 2010; Linz et al. 2007, 2014a; Moodley et al. 2009, 2012, 2021; Wirth et al. 2004). Information for the presence (filled triangles) or absence (empty circles) of the cagPAI is based on published data on the results of PCR reactions that span the ends of the cagPAI (Breurec et al. 2011b; Olbermann et al. 2010) or is based on genome sequences. Population assignments are indicated by the color coding of symbols that correspond to the labels next to the tree

(Moodley et al. 2009). The population hpNorthAsia accounted for 44% of the isolates from indigenous Siberians. While isolates assigned to the subpopulation hspAltai were restricted to central Siberia, the subpopulation hspIndigenousAmericas (former hspAmerind) was found in Northern Siberia, in Eastern Siberia, and in Beringia. hspIndigenousAmericas further contained isolates from Native North and South Americans (Linz et al. 2007; Moodley et al. 2021). Isolates of the population hpEurope are distributed throughout Europe, the Middle East, Iran, India, and Southeast Asia, but also among people with European ancestry in many other countries and continents (Fig. 2, Table 1) (Breurec et al. 2011a; Devi et al. 2007; Falush et al. 2003b; Latifi-Navid et al. 2010; Linz et al. 2007; Tay et al. 2009).

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Fig. 2 Geographical distribution of H. pylori populations and subpopulations. Each color represents a different population. Color code as in Fig. 1, except for pink that denotes admixed hpAfrica1 and hpEurope populations in the Americas

Sequence analyses indicated that hpEurope is a hybrid population that arose from the admixture of an Asian (ancestral Europe 1, AE1) with an African (ancestral Europe 2, AE2) ancestral population. AE1, that gave rise to the modern hpAsia2 bacteria, is believed to having originated in Central Asia after the out-of-Africa migration around 60 kya. The extant descendants of AE2 are hpNEAfrica bacteria from northeast Africa (Falush et al. 2003b; Linz et al. 2007; Moodley et al. 2012). As a result of this admixture, the genetic diversity among hpEurope isolates is higher than among other populations. Whole genome analyses identified several subpopulations within hpEurope, including hspEuropeNEurope, hspEuropeCEurope, hspEuropeSWEurope, and hspEuropeMiddleEast (Thorpe et al. 2022), as well as subpopulations hspSWEuropeColombia, hspSWEuropeHonduras, hspSWEuropeMexico, and hspEuropePeru that are specific for regions in Latin America (Gutierrez-Escobar et al. 2020; Munoz-Ramirez et al. 2017, 2021). In addition, several admixed subpopulations were identified in Siberia, such as hspSiberia1 and hspSiberia2 that are thought to be hybrids between hpAsia2 and hpNorthAsia and hpAsia2 and hpEastAsia, respectively, and hspKet that probably arose as a hybrid between hspSiberia2 and hpNorthAsia (Moodley et al. 2021). Furthermore, hspOkinawa from the Okinawa island in Japan likely diverged from hpNorthAsia strains about 32 kya, and hpRyukyu from Okinawa is assumed to having diverged from a Central Asian ancestor 45 kya (Suzuki et al. 2022). The distribution of H. pylori extends beyond the general classification provided here, and it is plausible that additional subpopulations or regional variations may exist that may be discovered in the future.

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Table 1 The populations and subpopulations of H. pylori and their predominant geographic distribution H. pylori populations

H. pylori subpopulations

Countries of isolation

References

hpAfrica2

hspNorthSan

Namibia, Angola

hspSouthSan

South Africa

Falush et al. (2003b); Moodley et al. (2012)

hspAfrica1WAfrica

Senegal, Gambia, Burkina Faso, Morocco, Algeria, Nigeria, Cameroon, South Africa

hspAfrica1SAfrica

Namibia, Angola, South Africa, Madagascar

hspAfrica1CAfrica

Cameroon, Namibia

hspAfrica1NAmerica

North, Central, and South American countries

hspAfrica1Nicaragua

Central American countries

hspMiscAmerica

Central and South American countries

hspCNEAfrica

Sudan, Cameroon, Nigeria, Algeria

hspENEAfrica

Sudan, Ethiopia, Somalia, Algeria

hspIndia

India, Bangladesh, Malaysia, Thailand, Philippines, Nepal

hspLadakh

India (Himalaya region)

hspUral

Russia (Ural Mountain regions)

hspEuropeNEurope

Europe as far east as Southeast Asian countries: UK, Sweden, Switzerland, Norway, Russia

hspEuropeCEurope

Central European countries: Greece, Belgium, Germany

hspEuropeSWEurope

Southwestern European countries: France, Portugal

hspEuropeMiddleEast

Middle East countries: Israel, Jordan, Iran

hpAfrica1

Admixed between hpAfrica1 and hpEurope

hpNEAfrica

hpAsia2

hpEurope

Falush et al. (2003b); Linz et al. (2014a); Nell et al. (2013)

Munoz-Ramirez et al. (2017, 2021); Thorell et al. (2017)

Linz et al. (2007); Nell et al. (2013); Thorpe et al. (2022) Linz et al. (2007); Moodley et al. (2021); Tay et al. (2009); Wirth et al. (2004)

Falush et al. (2003b); Gutierrez-Escobar et al. (2020); Linz et al. (2007); Thorell et al. (2017); Yamaoka et al. (2008)

(continued)

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Table 1 (continued) H. pylori populations

hpSahul

hpEastAsia

hpNorthAsia

H. pylori subpopulations

Countries of isolation

hspSWEuropeColombia

South America: Colombia

hspSWEuropeHonduras

Central America: Honduras

hspSWEuropeMexico

North America: Mexico

hspEuropePeru

South America: Peru

hspAustralia

Australian Aborigines

hspNGuinea

New Guinea highlanders

hspEAsia

High prevalence in China, Japan, Korea, Taiwan, Vietnam. Also in Malaysia, Singapore, Thailand, Cambodia

hspMaori

Indigenous Taiwanese, Melanesia (New Caledonia), Polynesian islanders (Wallis and Futuna, Samoa, Tonga), New Zealand, rare in Indonesia, Philippines, Japan

hspIndigenousNAmerica

Indigenous Americans in USA, Canada; Indigenous Siberians (Russia)

hspIndigenousSAmerica

Indigenous Americans in Peru, Venezuela, Colombia

hspAltai

Indigenous Siberians Altai region (Russia) Japan: Okinawa island

Suzuki et al. (2022)

hspSiberia1

Indigenous Siberians, Mongolians

Moodley et al. (2021)

hspSiberia2

Indigenous Siberians, Mongolians

hspKet

Indigenous Siberians, Ket

hspOkinawa Admixed sub-populations

hpRyukyu

Japan: Okinawa island

References

Moodley et al. (2009)

Breurec et al. (2011a); Falush et al. (2003b); Linz et al. (2007); Moodley et al. (2009); You et al. (2022)

Dominguez-Bello et al. (2008); Falush et al. (2003b); Moodley et al. (2021)

Suzuki et al. (2022)

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2.2 H. pylori and Human Migrations The H. pylori population structure is affected by several factors, most notably the high mutation rate, the extremely frequent genetic exchange between isolates, and the transmission route. H. pylori is primarily acquired during childhood through personto-person contact, often within families or communities (Alfaray et al. 2022; Schwarz et al. 2008; Tindberg et al. 2001). This transmission pattern leads to the formation of distinct bacterial populations within geographic areas and/or ethnicities. Localized admixture between isolates in close geographical vicinity distorts the difference between initially different local populations by homogenizing the genetic signals, which on a global scale results in clines of genetic isolation-by-distance (IBD) in H. pylori that are similar to those observed in its human host (Linz et al. 2007, 2014a; Serre and Paabo 2004). In addition, also similar to humans, the genetic diversity of H. pylori decreases with geographic distance from east Africa, the supposed origin of mankind. Computer simulations revealed that H. pylori accompanied anatomically modern humans during the migration out-of-Africa around 60 kya (Linz et al. 2007). Subsequent analyses displayed that modern humans were associated with H. pylori since their origin over 100 kya (Moodley et al. 2012). Thus, H. pylori has been diversified with human populations ever since. Due to its long evolutionary association with humans, the predominantly intra-familial transmission route and its high genetic diversity, the geographic distribution of H. pylori sequences reflects ancient and historic human migrations. Those include an ancient migration from India to Southeast Asia that brought hpEurope bacteria to Thailand, Cambodia, and Malaysia, and extensive migrations of traders of Chinese origin across Southeast Asia that are reflected in the distribution of hspEAsia (Breurec et al. 2011a). The population hpSahul among Australian Aborigines and New Guineans reflects the original settling of the ancient continent Sahul comprised of New Guinea, Australia, and Tasmania that were connected as a large landmass during the last ice age about 45 kya, when the sea level was much lower than today. The geographic separation of Sahul from Sundaland by several deep sea trenches resulted in genetic isolation and development of a distinct H. pylori population by selection and genetic drift (Moodley et al. 2009). The European colonial expansion in the sixteenth to nineteenth centuries spread hpEurope bacteria all over the world (Falush et al. 2003b). The distribution of hpAfrica1 bacteria reflects the expansion of Bantu societies that distributed hpAfrica1 bacteria from their homeland in tropical West Africa into the summer-rainfall regions of central, east, and southern Africa that were climatically suitable for the agricultural crops (Moodley and Linz 2009). The slave trade, which resulted in the forced migration of Africans, brought hpAfrica1 bacteria to the Americas (Falush et al. 2003b). The finding of hspIndigenousAmerica bacteria in both indigenous Siberians and Native Americans mirrors the peopling of the Americas via the Bering Strait (Moodley et al. 2021). An analysis of the geographic distribution and genetic diversity of hspMaori isolates solved a decades-long debate about the origin of the Polynesians, in which linguistics and archaeology provided evidence for a source on Taiwan, whereas human genetics favored insular Southeast Asia.

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The H. pylori data supported the start of the Austronesian expansion on Taiwan by showing the diaspora of one of several subgroups of the Austronesian language family along with one of several hspMaori clades from Taiwan via Melanesia into Polynesia (Moodley et al. 2009). The mummified corpse of the Iceman, due to its discovery site near the Ötz valley in the Tyrolean Alps in 1991 commonly known as “Ötzi”, is the oldest intact and naturally preserved mummy of a European from the Neolithic Copper Age (Zink and Maixner 2019). Ötzi was infected with H. pylori. Reconstruction and analysis of this 5,300-year-old H. pylori genome revealed that the Ötzi H. pylori represented a nearly pure specimen of the hpAsia2 population that existed in Europe before the hybridization with the bacteria of hpNEAfrica origin (Maixner et al. 2016). This suggested that the African population arrived in Europe only within the past few thousand years, much later than the originally estimated 10–52 kya (Moodley et al. 2012). Ötzi the Iceman carried a cagA-positive vacA s1a/i1/m1 type strain that in modern strains causes inflammation of the gastric mucosa. Indeed, proteins associated with inflammatory host responses were identified in gastric mucosal samples from the corpse, albeit in low numbers. However, the poor preservation of the stomach mucosa did not allow to elucidate whether Ötzi suffered from gastric disease (Maixner et al. 2016).

3 Pathogenomics of H. pylori 3.1 Genetics of H. pylori Virulence Factors and Pathogenicity The virulence of H. pylori is tightly linked to its capacity to colonize the human stomach and to the development of gastric disease (Yamaoka 2010). H. pylori virulence factors comprise a diverse range of classes, including toxins, adhesins, secretion systems, and other secreted factors (Fig. 3). These factors exert pivotal functions in H. pylori’s capacity to adhere to and colonize the gastric mucosa, to evade host immune surveillance, to trigger inflammatory responses, and to inflict tissue damage (Table 2). The cytotoxin-associated gene A (cagA, locus tag HP0547 in the genome of strain 26695), which is located at the cagPAI, plays a crucial role in the H. pylori pathogenicity and is considered one of the main virulence factors associated with gastric cancer (GC) development (Cover and Blaser 2009; Oliveira et al. 2021; Reyes 2023; Takahashi-Kanemitsu et al. 2020). The cagPAI encodes a type IV secretion system (T4SS) for the delivery of CagA into host gastric epithelial cells (Backert and Tegtmeyer 2017). Within the host cell, host kinases phosphorylate CagA at the tyrosine residues of the so-called EPIYA motifs, which initiates a series of intracellular signaling events that result in alterations to cellular morphology and the disruption of regular cellular functions (Hatakeyama 2014; Mueller et al. 2012). CagA usually contain three repeats of the EPIYA motifs with specific flanking sequences,

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Fig. 3 Key virulence factors involved in H. pylori pathogenesis. These virulence factors are implicated in disease development and in bacterial colonization and persistence

called EPIYA-A, EPIYA-B and EPIYA-C or EPIYA-D. While East Asian strains (hspEAsia) contain EPIYA-A, -B, and -D, European and African strains contain EPIYA-A, -B, and -C, with EPIYA-C sometimes present more than once (Olbermann et al. 2010). East Asian CagA were shown to elicit stronger induction of pro-inflammatory cytokines compared to CagA of Western-type strains. The function of CagA is reviewed in more detail in chapter “Impact of the Helicobacter pylori Oncoprotein CagA in Gastric Carcinogenesis” of this book. In addition, besides the injection of CagA, the structural T4SS components CagL and CagY can interact with host cell receptors such as integrin-α5 β1 (Koelblen et al. 2017; Kwok et al. 2007) and toll-like receptor 5 (TLR5) to trigger signaling (Pachathundikandi et al. 2015, 2019; Tegtmeyer et al. 2020). The CagY protein contains a large region composed of multiple direct sequence repeats. Recombination between individual repeat segments facilitates shuffling as well as in-frame deletion and duplication of repeat units, which provides considerable structural variability to the protein (Barrozo et al. 2013; Delahay et al. 2008). Moreover, these structural changes enable immune escape during infection, as they were shown to sufficiently affect CagY expression to enable or inhibit T4SS functions (Barrozo et al. 2013). It is important to note that the cagPAI genes belong to the auxiliary genes as they are not present in all H. pylori strains (Gressmann et al. 2005; Olbermann et al. 2010). All strains of the populations hpAfrica1, hpAsia2, and hpEastAsia possess the cagPAI (Fig. 1). In contrast, the cagPAI is missing in the hpAfrica2 population and thus also in H. acinonychis, and it is only variably present in strains from the populations hpSahul, hpNorthAsia, hpEurope, and hpNEAfrica (Fig. 1). A second T4SS, the comB DNA uptake system that is employed for DNA import during natural

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Table 2 An overview of several key virulence factors in H. pylori Virulence factor

Gene(s)

General information (e.g., function)

Reference(s)

Cytotoxin-associated cagA gene A

Translocated via the T4SS, undergoes phosphorylation and triggers various cellular responses leading to gastric diseases including gastric cancer

Backert and Tegtmeyer (2017); Cover and Blaser (2009); Hatakeyama (2014); Miftahussurur et al. (2023); Oliveira et al. (2021); Takahashi-Kanemitsu et al. (2020)

Vacuolating cytotoxin A

vacA

Secreted toxin that forms pores in the host cell membrane, leading to cellular damage

Foegeding et al. (2016); Palframan et al. (2012); Reyes (2023); Terebiznik et al. (2006)

Serine protease HtrA htrA

Periplasmic chaperone and protease. Inflicts epithelial damage by disruption of host cell junctions. Manipulation of host immune responses

Hoy et al. (2010); Sharafutdinov et al. (2022, 2023); Zarzecka et al. (2019)

Induced by contact with epithelium

iceA

IceA1 is linked to the development of peptic ulcer disease

Feliciano et al. (2015); Shiota et al. (2012); van Doorn et al. (1998b)

Outer inflammatory protein A

oipA

Associated with increased IL-8 production in gastric epithelial cells

Dossumbekova et al. (2006); Reyes (2023); Sallas et al. (2019); Thai et al. (2023); Yamaoka et al. (2000, 2002a) (continued)

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Table 2 (continued) Virulence factor

Gene(s)

General information (e.g., function)

Reference(s)

Sialic acid-binding adherence

sabA

Adhesin that mediates bacterial adherence to sialyl-Lewis x antigens at the epithelial cell surface. SabA expression is associated with severe gastric atrophy, intestinal metaplasia, and gastric cancer

Doohan et al. (2021); Mahdavi et al. (2002); Yamaoka (2008)

Blood group binding babA, babB proteins

Bind to the mucosal ABO/ Leb blood group antigens, facilitating H. pylori adhesion to the gastric epithelium

Aspholm-Hurtig et al. (2004); Doohan et al. (2021); Moonens et al. (2016)

Adhesin-like proteins

Facilitate adherence to host cells

Senkovich et al. (2011)

Neutrophil-activating napA / dps protein

Promotes H. pylori survival in iron-limited conditions, protects from DNA damage

Fu (2014); Fu and Lai (2022); Tsuda et al. (1994); Uzzau and Fasano (2000)

Gamma-glutamyl transpeptidase

ggt

Plays a role in nutrient acquisition and evasion of the host immune response

Schmees et al. (2007); Wang et al. (2022)

Urease

ureA, ureB, among others

Facilitates pH regulation and survival in the acidic gastric environment

Graham and Miftahussurur (2018); Olivera-Severo et al. (2017)

Flagella

flaA-B, flhA-B, fliF-S, flgM, among Facilitate others motility and chemotaxis; contribute to biofilm formation

alpA, alpB

Cheok et al. (2021); Gu (2017)

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transformation, is ubiquitously present among strains (Hofreuter et al. 2001). Similar to the cagPAI, two additional H. pylori T4SSs called TFS3 and TFS4 that supposedly represent conjugational DNA transfer systems are part of the auxiliary gene set and thus are variably present among individual strains (Kersulyte et al. 2003, 2009; Fischer et al. 2020; Tegtmeyer et al. 2022). In many strains, the genes that encode TFS3 and TFS4 are located at the co-called plasticity zones, large genomic regions that were found to contain a considerable part of genome-specific genes, including in the genomes of strains 26695 and J99. The average G + C percentage of the cagPAI and of the plasticity zones is lower (35%) than in the rest of the genome (39%), suggesting original acquisition from (a) currently unknown source(s) (Alm et al. 1999; Gressmann et al. 2005). The vacuolating cytotoxin A (VacA, HP0887) displays considerable genetic variability (Atherton et al. 1995; Foegeding et al. 2016). VacA is a toxin that induces the formation of pores in host cell membranes, which results in the development of cellular vacuoles from lysosomes and subsequent damage to the cellular structure (Palframan et al. 2012). Additionally, VacA alters various cellular signaling pathways, triggers mitochondrial membrane disruption and apoptosis, and interferes with immune cell functions (Foegeding et al. 2016; McClain et al. 2017). While the vacA gene is part of the core genome and is present in all strains, distinct allelic variations impact its expression, secretion, and cytotoxic activity. The VacA cytotoxicity is determined by three heterogenic regions of the protein. The signal peptide region (alleles s1a-c, s2) determines the vacuolating activity, the intermediate region (i1, i2, i3) contains the pore forming domain, which is involved in the development of GC, and the middle region (m1, m2) is associated with cell binding (Foegeding et al. 2016). The vacA s1/m1 and to a lesser degree s1/m2 strains were shown to be cytotoxic. In contrast, s2/m2 strains do not exhibit cytotoxicity (Atherton et al. 1995) due to a hydrophilic 12-amino-acid extension in the s2 region that prevents the formation of anion channels across the lipid bilayers (McClain et al. 2017). Interestingly, the vacA alleles are associated with the presence of the cagPAI. While cagPAI-negative strains contain the non-cytotoxic vacA s2/m2, cagPAI-possessing isolates contain s1/ m1 or s1/m2 vacA variants, which may be related to the antagonistic effects of CagA and VacA on scattering, elongation and apoptosis of infected host cells (Oldani et al. 2009; Tegtmeyer et al. 2009). While the oipA gene (HP0638) that encodes the outer (membrane) inflammatory protein A (OipA, also known as HopH) belongs to the H. pylori core genome (Gressmann et al. 2005), it is not expressed in all strains as this gene is subject to phase variation due to multiple CT dinucleotide repeats at the 5, -end of the gene. OipA is believed to play a role in the initiation and progression of inflammation and subsequent tissue damage (Dossumbekova et al. 2006; Yamaoka 2010). H. pylori strains that express OipA display increased colonization density, induce elevated pro-inflammatory cytokine production, and elicit more prominent neutrophil infiltration. Furthermore, strains that express oipA are significantly associated with the development of GC and peptic ulcer disease (Reyes 2023; Sallas et al. 2019; Thai et al. 2023).

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Another phase variable outer membrane protein is the sialic acid-binding adherence protein SabA (Baj et al. 2020), which binds human sialyl-Lewis x antigens in the gastroduodenal tract and enables H. pylori to tightly adhere to the gastric mucosa (Doohan et al. 2021; Mahdavi et al. 2002). SabA expression correlated with the activation of host pro-inflammatory responses, including activation of human neutrophils (Unemo et al. 2005), and has been associated with severe intestinal metaplasia, gastric atrophy, and the development of GC (Su et al. 2016; Yamaoka 2008). In general, H. pylori adhesins play a major role during the colonization of the gastric mucosa, including the blood group antigen binding proteins BabA and BabB (Ansari and Yamaoka 2017; Aspholm-Hurtig et al. 2004; Doohan et al. 2021). While the babB gene (HP0896, HopT or OMP19) appears to belong to the core genome, babA (HP1243, also called HopS or OMP28) was found to be strain specific and only variably present (Gressmann et al. 2005; Sharafutdinov et al. 2023). BabA is an important virulence factor: Two GWAS of H. pylori from Europe (Berthenet et al. 2018) and from East Asia (Tuan et al. 2021), as well as another study of over 1,000 H. pylori from worldwide sources (Sharafutdinov et al. 2023) identified the presence of babA to be strongly correlated to the development of severe disease, particularly GC. BabA binds mucosal ABO/Leb blood group carbohydrates on the gastric mucosa. The vast majority of BabA-expressing strains were shown to bind equally well to all three fucosylated blood group antigens A, B, and O, whereas a small subset of strains bound blood group O antigens with high affinity, but not A or B (Aspholm-Hurtig et al. 2004). The different binding affinities were caused by amino acid polymorphisms in the diversity loop 1 of the carbohydrate binding domain (CBD) that affected the size of the antigen binding pocket. An aspartic acid, an asparagine or a leucine at position 198 created a small binding pocket that only fit the Lewis b antigen characteristic for blood group O. In contrast, a serine at this position created a larger CBD that also facilitated binding of the larger antigens of blood groups A and B (Moonens et al. 2016). Other outer membrane proteins (OMPs) described as adhesins are AlpA (Omp20, HopC, HP0912), AlpB (Omp21, HopB, HP0913), and HopQ (Omp27, HP1177), all of which are considered part of the core genome present in all strains (Gressmann et al. 2005; Odenbreit et al. 2009). While AlpA and AlpB were shown to bind laminin (Senkovich et al. 2011), HopQ was identified as the surface-exposed H. pylori adhesin that specifically binds the human carcinoembryonic antigen-related cell adhesion molecule (CEACAM), in particular the members CEACAM-1, -3, -5, and -6 (Javaheri et al. 2016; Moonens et al. 2018; Tegtmeyer et al. 2019). Importantly, HopQ attachment to the CEACAM receptors enables T4SS-mediated delivery of CagA into host cells and enhances the release of pro-inflammatory cytokines such as interleukin-8 (Javaheri et al. 2016; Moonens et al. 2018; Tegtmeyer et al. 2019). Two families of hopQ alleles were described, type I and type II. While type I hopQ alleles are frequently associated with the more virulent cagPAI-positive strains, type II hopQ alleles are often found in cagPAI-negative strains (Cao and Cover 2002; Cao et al. 2005). Upon infection of the host, the H. pylori neutrophil-activating protein (NapA, HP0243), a protein that is highly conserved and expressed by all H. pylori isolates,

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stimulates toll-like receptor 2 (TLR2), which induces activation and transendothelial migration of neutrophils. NapA-induced neutrophil activation leads to the production of pro-inflammatory cytokines and the release of reactive oxygen species (ROS), which result in active chronic gastritis that can eventually develop into duodenal ulcer disease or gastric adenocarcinoma (Amedei et al. 2006; Brisslert et al. 2005; Fu 2014; Satin et al. 2000). NapA is an iron-binding protein (Tonello et al. 1999) and appears to support the survival of H. pylori under oxidative stress conditions and to protect the bacteria from DNA damage. The napA gene is also known as the dps gene encoding the DNA protection during starvation protein. In addition, T cells recovered from antral biopsies of H. pylori-infected patients showed significant T cell proliferation to NapA, indicating that NapA is involved in the activation of T cell-dependent immune responses (Amedei et al. 2006; Fu 2014). The gamma-glutamyl transpeptidase (GGT, HP1118), another ubiquitously present protein, plays a crucial role in the evasion of the host’s immune response. GGT inhibits T cell proliferation and function by induction of a cell cycle arrest in the G(1) phase, which contributes to immune evasion (Schmees et al. 2007; Wustner et al. 2015, 2017). GGT promotes the initial colonization by H. pylori, possibly through the acquisition of nutrients such as glutamine, but is not essential for this process. H. pylori infection of mesenchymal stem cells resulted in GGT-dependent histone methylation and promoted the proliferation, migration, self-renewal, and pluripotency of mesenchymal stem cells (Wang et al. 2022). Other colonization-associated factors are urease and flagella. Urease, which is encoded by the urease gene cluster (HP0067–HP0073), plays a crucial role in buffering of the pH in the immediate surrounding by hydrolysis of urea into ammonium and carbon dioxide. Partial neutralization of the pH by the released ammonium aids in the survival of H. pylori within the acidic milieu of the stomach (Athmann et al. 2000; Debowski et al. 2017) and can activate the inflammasome via TLR2 (Koch et al. 2015). Flagella are involved in motility, chemotaxis, and biofilm formation and affect the ability of H. pylori to colonize and persist in the gastric mucosa (Graham and Miftahussurur 2018; Olivera-Severo et al. 2017). The flagella are encoded by a set of flagellar genes that are organized in a hierarchical regulatory system. These genes include flaA, flaB, flhA, fliF through fliS, and flgM, among others (Gu 2017). The serine protease gene htrA belongs to the core genome of H. pylori (Gressmann et al. 2005; Tegtmeyer et al. 2016). HtrA is an essential periplasmic enzyme involved in folding and processing of periplasmic, outer membrane-bound and secreted proteins. During infection, HtrA released from H. pylori and other gastrointestinal pathogens compromises the functional integrity of the epithelial layer by cleavage of E-cadherin, which is the major component of the adherens junctions, and the tight junction proteins occludin and claudin-8 (Schmidt et al. 2016; Harrer et al. 2017; Hoy et al. 2010, 2012; Linz et al. 2023). Disruption of the epithelial cell junctions enables paracellular migration of the bacteria and their effector proteins to the basolateral membranes, where CagA is injected into the epithelial cells (Backert et al. 2018; Tegtmeyer et al. 2017). In addition, HtrA affects host cell transcription of various inflammation, apoptosis, and carcinogenesis-related genes (Sharafutdinov et al. 2022). A recent analysis identified three SNPs in the htrA gene that significantly

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correlate with H. pylori-induced GC (Sharafutdinov et al. 2023). Functional studies on one of those SNPs unraveled the underlying mechanisms (see below).

3.2 GWAS to Identify Genes and/or SNPs Associated with Disease Outcome Genome-wide association studies (GWAS) have significantly transformed our understanding of genes affecting the risk of disease development. GWAS have played a crucial role in identifying genetic variants and SNPs associated with disease outcome in H. pylori-infected individuals. SNPs and germline mutations associated with an increased GC risk were identified in a variety of human genes increased disease risk (Castano-Rodriguez et al. 2014; McLean and El-Omar 2014; Rudnicka et al. 2019; Usui et al. 2023; Wu et al. 2021). While these studies examined the occurrence of disease-related SNPs in human genes, only two GWAS reports focused on correlating SNPs in H. pylori genes to gastric disease occurrence (Berthenet et al. 2018; Tuan et al. 2021). The first GWAS conducted on 173 H. pylori strains from Europe (population hpEurope) identified 32 genes that differed significantly in frequency between gastritis isolates and isolates from patients with GC. GC risk was associated with the presence of eight cagPAI genes (cagβ, cagY, cagX, cagU, cagT, cagI, cagH, and cagE), three OMP genes including babA (HP1243) and hopQ (HP1177), urease gene ureG, and with variations and SNPs in less-well studied genes (Berthenet et al. 2018). A second study performed on 240 H. pylori isolates of the subpopulation hspEAsia identified 11 SNPs and three DNA motifs associated with GC (Tuan et al. 2021). The genetic variations associated with GC were found in or immediately upstream of two OMP genes (HP0807, HP1467), chemotaxis sensor tlpC (2 SNPs), and several housekeeping genes. Furthermore, multiple SNPs were found to be positively selected during H. pylori colonization in the Mongolian gerbil model, including R88H in the iron regulator Fur, which may potentially increase bacterial resistance against oxidative stress (Beckett et al. 2018). In addition, a synonymous SNP was identified in cagA, although the biological relevance may be questionable (Tuan et al. 2021). While the current GWAS reports have provided valuable insights into the genetic variations associated with peptic ulcer and GC, it is essential to conduct further investigations using larger datasets. Moreover, the role of the identified SNPs in pathogen-host interactions is unknown as no functional analyses were performed to determine their biological relevance, an issue that needs to be studied in the future.

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3.3 Cancer-Associated Mutations in H. pylori Serine Protease HtrA An alternative to GWAS was recently shown by the association of SNPs in a single H. pylori gene to the frequency of gastric disease. An analysis of the htrA gene in 1,043 H. pylori genomes from worldwide sources revealed three htrA SNPs that significantly correlated with the frequency of GC (Sharafutdinov et al. 2023). Using this genome dataset, significant disease association was further demonstrated for the presence of the cagPAI, presence of the babA gene, of the virulent hopQ type I, and the cytotoxic vacA s1/m1 and s1/m2 alleles. This approach demonstrated the feasibility of analyzing the presence of SNPs within single genes from the same data set in correlation to disease. Importantly, this study unraveled the molecular mechanisms by which one of the identified HtrA SNPs, a serine (S) to leucine (L) change that is located at amino position 171 in the protease domain of the protein, promotes GC development. Allele 171L was more frequently found in H. pylori isolated from patients with malignant changes, i.e. with metaplasia or adenocarcinoma, than in patients with mild or no disease (gastritis and asymptomatic) (Fisher’s exact test, p = 0.0003). The functional analyses revealed that the 171S-to-171L mutation amplified HtrA proteolytic activity through structural stabilization of HtrA trimers (Sharafutdinov et al. 2023), which is the proteolytically active form of the enzyme (Zarzecka et al. 2023), and enhanced the disruption of the gastric epithelial layer by cleavage of epithelial cell junction proteins, including tight junction protein occludin and tumor-suppressor protein E-cadherin in the adherens junctions (Sharafutdinov et al. 2023). Cleavage of junctional components at the apical surface generated gaps between neighboring epithelial cells, compromising the epithelial barrier function and enabling the invasion of bacteria. 171L HtrA-type, but not 171S HtrA possessing H. pylori strains, triggered increased cell proliferation through release of β-catenin from the E-cadherin/β-catenin complex and subsequent translocation from the membrane and accumulation of β-catenin in the nucleus. Furthermore, 171L HtrA-type strains increased injection of H. pylori oncoprotein CagA into epithelial cells and enhanced inflammation through strong elevation of transcription factor NF-κB activity and elevated secretion of pro-inflammatory cytokines such as IL8 (Sharafutdinov et al. 2023). Based on studies indicating that H. pylori infection induces double strand breaks (DSBs) in chromosomal DNA of the host, which elicit the acquisition of mutations and genome instability (Bauer et al. 2020; Hartung et al. 2015; Koeppel et al. 2015), the authors further checked host chromosomal DNA damage following H. pylori infection. Again, 171L HtrA possessing H. pylori induced a higher rate of DSBs in host chromosomes than their 171S HtrA counterparts. Thus, the 171L HtrA variants are more efficient at cleavage of cell junctions and tumor suppressor E-cadherin, at delivery of CagA into epithelial cells, at promoting cell proliferation by nuclear accumulation of β-catenin, at causing NF-κB-mediated and IL-8-mediated inflammation, and at causing DSBs in host chromosomes, all of which collectively promote GC development. Thus, this SNP is a remarkable example of a bacterial mutation that promotes gastric disease development during

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infection and may turn out as a useful biomarker for risk predictions in H. pylori infections (Sharafutdinov et al. 2023).

4 Genome Evolution: Mutation, Recombination, and Genome Shuffling 4.1 Accumulation of H. pylori Mutations and Recombination During Infection Thanks to extraordinarily high mutation and recombination rates that are among the highest in the bacterial kingdom, H. pylori accumulates a large amount of genomic changes during infection. Genomic changes occur due to mutation as well as due to frequent uptake of DNA from external sources, which requires at least temporary co-colonization with an unrelated strain. Over the past 15 years, several studies were dedicated to analyze the in vivo evolution of H. pylori in the human host. In all these studies, single polymorphisms flanked by at least 200 bp identical sequence on both sides were regarded as mutations and thus used for the calculation of the mutation rate. Groups of two or more polymorphisms that were separated by less than 200 bp and flanked by >200 bp identical sequence on both sides, termed clusters of nucleotide polymorphisms (CNPs), were considered as imported DNA. First analyses of H. pylori evolution during infection, that were undertaken before substantial advances in sequencing technology made whole genome sequencing of multiple strains affordable, were based on sequences of several gene fragments. An analysis of the seven gene fragments used in MLSA plus three virulence-associated genes ( flaA, flaB, vacA) from isolates taken sequentially at intervals of 3–36 months from patients with chronic infection revealed a synonymous H. pylori mutation rate of 4.1 × 10–5 (Falush et al. 2001). A follow-up study of the same plus multiple other paired isolates that spanned periods between sampling points of up to 10 years and analyzed sequences from 78 gene fragments (covering ~39 kb) determined a mean mutation rate of 1.36 × 10–6 changes per site per year (Morelli et al. 2010). Subsequent whole genome-based studies that allowed a more comprehensive analysis of bacterial evolution during infection and transmission adjusted the average mutation rate to 2.5 × 10–5 changes per site per year (Kennemann et al. 2011; Linz et al. 2014b). In contrast, isolates recovered from infection of human volunteers during vaccine studies and genome comparisons of family isolates from Japan indicated an about five times lower mutation rate of ~5 × 10–6 (Estibariz et al. 2020; Furuta et al. 2015; Nell et al. 2018). Overall, the estimated mutation rates were quite similar between studies indicating a relatively uniform mutation rate among worldwide isolates. The average mutation rate of 1.38 × 10–5 (range 9.14 × 10–6 to 1.85 × 10–5 ) predicted from genomes of 40 antrum and corpus isolate pairs from the same hosts (Didelot et al. 2013) now appears to the accepted standard in the field.

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The degree by which H. pylori sequence changes accumulate differed widely between infected individuals. While the mutation rate was relatively uniform, the effect of recombination varied significantly from one host to another. Among the 40 H. pylori genome pairs from antrum and corpus of the same host, at least four did not contain any recombinant genes. In contrast, other genome pairs displayed very high recombination rates and possessed imported DNA in up to 95 genes (Didelot et al. 2013). Equally wide ranges of DNA import were also detected in genomes of multiple H. pylori clones grown from biopsies taken from different stomach compartments (antrum, corpus, fundus) of the same hosts (Ailloud et al. 2019). Genomes from some patients showed no sign of DNA import of unrelated strains, as did H. pylori isolates after experimental challenge of human volunteers in two vaccine studies (Estibariz et al. 2020; Nell et al. 2018). H. pylori genomes of other patients contained very few and only short CNPs that were probably the result of intra-chromosomal translocation (Ailloud et al. 2019), similar to H. pylori from spontaneous transmission between spouses (Linz et al. 2013). However, genomes from five patients contained 109, 135, 177, 562, and 1124 CNPs that spanned 34 kb, 45 kb, 87 kb, 321 kb, and 424 kb, respectively, of imported DNA (Ailloud et al. 2019). Genomes of sequentially taken isolates from the same hosts, from experimental infection of volunteers, and from family isolates in Japan contained between 10 and 441 CNPs indicating imported DNA fragments that were likely acquired during mixed infection with unrelated strains (Furuta et al. 2015; Kennemann et al. 2011; Linz et al. 2014b). The total, concatenated length of imported DNA fragments in those genomes ranged from 0.6 to 356 kb, with a median length of the imported fragments between 94 and 430 bp (range from 2 to 6,978 bp). At several genome positions, the pattern of polymorphisms suggested import of longer DNA fragments that were integrated into the genome as several smaller chunks, with on average 2.6 integrated fragments per imported DNA fragment (Furuta et al. 2015; Kennemann et al. 2011; Linz et al. 2014b). The recombination rate largely depends on the infection mode. In individuals carrying a single strain without transient co-colonization by a different strain, the bacterial population will be relatively homogeneous, with usually less than a (few) hundred polymorphisms between individual bacterial cells, even if isolated from different stomach compartments (Ailloud et al. 2019; Linz et al. 2013; Nell et al. 2018). The number of accumulated SNPs will reflect the time since the original infection, or alternatively, the time since the last major bottleneck or selective sweep that obliterated a large part of the effective population size. In addition to mutations, a very limited number of recombinant DNA fragments might accumulate due to DNA translocation of similar sequences from other chromosomal loci such as fragments of various OMP genes. Recombination with DNA from bacterial sister cells will very likely also occur but cannot be detected because of lacking sequence diversity. Those isolates show almost pure clonal descent and thus purely evolution by accumulation of mutations. Accordingly, the recombination rate in those patients is essentially (close to) zero (Didelot et al. 2013). In other patients, temporary mixed colonization results in accumulation of a few recombinant fragments. Other isolates can be highly divergent, with large stretches of identical DNA sequence interspersed by multiple

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imported DNA fragments. In these patients, the recombination rate is very high, with recombination accounting for the vast majority of the genetic changes and thus for a high degree of divergence from the original isolate (Ailloud et al. 2019; Didelot et al. 2013; Kennemann et al. 2011; Linz et al. 2014b). A further extreme is strain convergence in which an unrelated strain repeatedly accumulates DNA fragments from a co-colonizing strain. Evidence for strain convergence was observed during an analysis of family isolates from the UK in which an unrelated strain from a family member contained 418 DNA stretches with a mean length of 779 bp (range 201–7862 bp) that were identical to the genome of another family isolate (Krebes et al. 2014). These so-called regions of identity (RoIs) had a total length of over 325 kbp and hence replaced about 20% of the original genome by recombination (Krebes et al. 2014). Similarly, stomach colonization by two distinct strains in a Chinese patient with chronic gastritis resulted in over 150 recombination events between the two with an average length of 895 bp (Cao et al. 2015).

4.2 Evolution and Shuffling of Virulence Factors H. pylori is naturally competent for DNA uptake and incorporation into its genome, which contributes significantly to H. pylori’s genetic diversity (Dorer et al. 2011; Kennemann et al. 2011). Recombination is frequent throughout the genome, along with other events such as rearrangements, transpositions, insertions, and gene gain or loss (Kawai et al. 2011), and both mutation and recombination cause numerous changes in H. pylori genomes at a rapid rate. While mutations occur on average about three times more often than recombination events, the total number of exchanged nucleotides by recombination exceeds the ones caused by mutation by far. Although much of the genetic diversity may be neutral, some will affect the ability of these bacteria to colonize, persist, and/or affect disease in particular hosts. But what genes were affected by the genetic changes? A vaccine study (Nell et al. 2018), in which human volunteers were vaccinated with a mixture of VacA, CagA, and neutrophil-activating protein NAP and subsequently challenged with a cagPAI-positive strain (Malfertheiner et al. 2018), did not detect DNA imports in H. pylori re-isolated from the volunteers, indicating absence of mixed infection with an unrelated strain. However, the re-isolates possessed point mutations in the vaccine component gene vacA that resulted in three different premature stop codons and thus each in a truncated, non-functional protein. Given that those substitutions were found in H. pylori from the vaccine group, but not from the placebo group, these changes were interpreted as vaccine-elicited immune evasion. In addition, non-synonymous mutations in cagA, in the cagPAI gene cagW, and in the OMP genes hopF and hopG in isolates from both vaccine and placebo groups likely also indicated immune selection (Nell et al. 2018). In general, all studies of genome changes during infection as well as during transmission to new hosts reported significantly high frequencies of genetic changes, both point mutations and DNA imports, in OMP encoding genes, suggesting H. pylori

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adaptation to individual hosts (Eppinger et al. 2006; Furuta et al. 2015; Kennemann et al. 2011; Krebes et al. 2014; Linz et al. 2013, 2014b; Schuster et al. 2008). For example, sequentially taken strain pairs from patients with chronic H. pylori infection showed genetic changes in up to one third of all OMP genes, with particularly frequently altered genes of the hop and hof families of OMPs (Kennemann et al. 2011). Disproportionately frequent changes in various OMP genes were also observed in re-isolates from experimentally challenged human volunteers and rhesus macaques (Linz et al. 2014b) as well as from within family transmission (Krebes et al. 2014; Linz et al. 2013). Further, mutations and DNA translocations in fucosyltransferase gene fucT suggested that altered LPS O-antigen by fucosylation may contribute to immune evasion during host adaptation (Furuta et al. 2015; Linz et al. 2013). In addition to OMP genes, H. pylori isolates from Japanese children revealed frequent amino acid substitutions in many other virulence-related genes, including vacA and vacA-like genes, serine protease htrA, and in several cagPAI genes (Furuta et al. 2015). Strong diversifying selection for several components of the cagPAI-encoded T4SS, especially those predicted to be secreted or located at the bacterial surface, was also noted in a global collection of isolates from worldwide biogeographic sources (Olbermann et al. 2010). H. pylori genomes from worldwide biogeographic sources further showed evidence for selection of vacA and vacA paralogs (Montano et al. 2015), and a study on serine protease HtrA identified six codons under diversifying selection, three of which correlated with the frequency of severe gastric disease (Sharafutdinov et al. 2023). Given the significance of virulence factors such as CagA, VacA, HtrA and various membrane-bound OMPs in H. pylori pathogenicity, it is not surprising that these factors are under immune selection and undergo particularly frequent amino acid changes, which facilitates immune evasion and adaptation of the bacteria to individual hosts.

4.3 Recombination Between Different H. pylori Populations and Origin of Local H. pylori Subpopulations The population genetics of H. pylori captures both prehistoric and recent migration events. Large-scale population movements, particularly within the last five centuries, and subsequent recombination between isolates from previously unrelated bacterial populations have started to blur the population boundaries. For example, ancient and historic migrations from the Indian subcontinent carried hpEurope bacteria into Southeast Asian countries such as Thailand, Malaysia, and Cambodia, and massive immigration of Chinese, mostly from the Guangdong and Fujian provinces, within the last 200 years brought H. pylori of the hpEastAsia subpopulation hspEAsia into this area (Breurec et al. 2011a; Tay et al. 2009). Interstrain recombination has occurred during mixed colonization between hpEurope and hspEAsia strains, which resulted in the introgression of European cagA into hspEAsia strains, as well as in cagA with mosaic European and East Asian ancestry patterns (Breurec et al. 2011b).

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While introgression from hpEurope into hspEAsia strains happened frequently, as 9/32 (28%) of the analyzed hspEAsia strains from Cambodia contained Western (hpEurope) cagA, introgression of EastAsian cagA into hpEurope strains appeared to be rare as only 1/34 (2.9%) of strains assigned to hpEurope possessed an East Asian cagA. Similar to cagA, typing of vacA s1 alleles revealed a high prevalence (34%) of allele s1a in Cambodian hspEAsia strains, indicating introgression of the European vacA gene allele into East Asian H. pylori strains, which typically possess the s1c allele, and ongoing admixture (Breurec et al. 2011b). The primary predominant population in the Americas before Columbus was the hpNorthAsia subpopulation hspIndigenousAmericas (formerly hspAmerind). The European colonial expansion that started after the discovery of America resulted in a massive influx of hpEurope strains, and the transatlantic slave trade in the sixteenth to nineteenth centuries brought hpAfrica1 bacteria to North and South America (Falush et al. 2003b; Ghose et al. 2002; Yamaoka et al. 2002b). Admixture of hspIndigenousAmericas strains with hpEurope and hpAfrica1 strains post Columbus almost resulted in the disappearance of the original bacteria of the Indigenous peoples (Dominguez-Bello et al. 2008). Despite the expectation that high rates of admixture in both human and bacterial populations might erase local signals of population structure, genetic drift, local population bottlenecks, frequent recombination and selection resulted in the evolution of local gene pools and distinct local hpAfrica1 and hpEurope subpopulations, without substantial genetic input from the pre-Columbian hspIndigeousAsmericas bacteria. The distinct hpAfrica1 subpopulations include hspAfrica1NAmerica isolates in the US and Canada, and hspAfrica1Nicaragua and hspAfrica1MiscAmerica from Nicaragua, Mexico and Colombia. While the former are mostly composed of hspAfrica1WAfrica ancestry, the latter mostly possess hspAfrica1SAfrica ancestry. Local and recently evolved hpEurope subpopulations are hspEuropeColombia from Colombia, hspSWEuropeHonduras from Honduras, hspSWEuropeMexico from Mexico, and hspEuropePeru from Peru, all of which had high levels of hspSWEurope ancestry (Gutierrez-Escobar et al. 2020; Munoz-Ramirez et al. 2017, 2021; Thorell et al. 2017). H. pylori strains of the new subpopulations showed a high degree of differentiation in genes that encode proteins involved in pathogen-host interactions, particularly in known virulence factors. These included several cagPAI genes, the VacA toxin, and OMPs such as HofC, AlpB, and BabA (Table 3), that displayed stretches of un-proportionally high Asian ancestry. Other codons under strong positive selection were identified in the vacA mid-region that is involved in recognition of the host cell receptor, and in the cagA domains II and III that are involved in CagA binding and recognition of β1-integrin and in recruitment of PAR1 (Munoz-Ramirez et al. 2021; Thorell et al. 2017). A summary of the genes that were identified to be under positive selection on both a global scale and on a local scale within individual (sub-)populations is provided in Table 3.

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Table 3 Genes subjected to positive selection in individual H. pylori populations On a global scale • nusA: The transcription elongation factor gene. Montano et al. (2015) Crucial protein in DNA repair and damage tolerance • vspIM, bsp6IM: Modification methylase VspI, and modification methylase Bsp6I. Genes involved in methylation pattern • copA, copB: Copper transport proteins. Copper plays a role in the colonization of the stomach mucosa by H. pylori through the action of trefoil peptides. The presence of trefoil peptides, particularly TFF1, promotes H. pylori colonization of gastric epithelial cells and mucus • fliY, rpoN: Flagellar motor switch phosphatase, and RNA polymerase sigma-54 factor. Essential for cell motility and cell adhesion to the stomach mucosa On a local scale hpAfrica2: Montano et al. (2015) • hup: DNA-binding protein, protects DNA from stress damage • ngoBIM: Modification methylase NgoBI, involved in DNA methylation Kawai et al. (2011); Montano et al. (2015) hpAfrica1: • rlmN: Ribosomal RNA large subunit methyltransferase N. Genes involved in methylation pattern • fliI: Flagellum-specific ATP synthase. For proper colonization of the human stomach, cell motility and adhesion to the stomach mucosa are crucial factors • nixA, yhhG: High-affinity nickel-transport protein, and putative nickel-responsive regulator. The genes encode transport and regulation proteins of nickel EuroAsian (hpEurope + hpAsia2) • rimO, trmD: Ribosomal protein S12 methylthiotransferase, and tRNA guanine-N1–methyltransferase. Genes involved in methylation pattern • cadA: Cadmium zinc and cobalt-transporting ATPase • vacA: Vacuolating cytotoxin autotransporter

Montano et al. (2015)

(continued)

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Table 3 (continued) On a global scale Gutierrez-Escobar et al. (2020); America strains compared with non-America Munoz-Ramirez et al. (2021); Thorell et al. strains: • cagPAI: cag pathogenicity island. Numbers of (2017) non-synonymous mutations in functionality domains were diverged in 13 genes such as cagA, cagF, cagN, cagL, etc. • ppiC: A peptidyl-prolyl isomerase C. The protein encoded by this gene has two distinct domains, one of which is responsible for enzymatic activity and the other for chaperoning the host protein • vacA: Vacuolating cytotoxin autotransporter VacA • babA: Outer membrane protein BabA, adhesin • hofC: Outer membrane protein HofC, required for H. pylori colonization • alpB: It is required for colonization in experimental models and for efficient adhesion to gastric epithelial cells hspEuropePeru • pab1: Restriction endonuclease gene • AbiEii system: Involved in phage-infected cell abortion • fecA2: Protein involved in iron acquisition • tonB: Protein associated with nickel transport hspIndigenousAmericas Duncan et al. (2013); Montano et al. (2015) • dps: DNA protection during starvation protein/ neutrophil-activating protein NapA • ykpA, yecS: Uncharacterized ABC transporter ATP-binding protein, and probable amino-acid ABC transporter permease • flgL: Flagellar hook-associated protein 3. Involved in cell motility

5 Advances in NGS—From Multiple Contigs to Long Read-Based Complete Genomes The availability of high-throughput sequencing technologies has boosted the investigation of bacterial genomes, including their pathogenicity. A key advantage of these modern molecular methods is that they allow the identification of novel virulence factors and their genotypic variants of pathogenic bacteria including H. pylori (Engstrand 2009; Vital et al. 2022). Short-read sequencing technologies such as Illumina generate read length of up to 500 bp in length (after combination of the 300 × 300 bp paired reads) that require assembly into contigs. Currently, most bacterial sequences available in the RefSeq database were generated by Illumina sequencing (Segerman 2020). Despite the fact that most genomes are only available as contigs

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and are not completed, Illumina sequences are very accurate and have provided valuable insights into the genetic variation, evolution, host adaptation, and pathogenicity of H. pylori. In fact, most of the data described in the paragraphs above were based on Illumina genomes. The big disadvantage of the relatively short reads is the difficulty to resolve repetitive regions, including gene duplications, or structural variations (Ben Khedher et al. 2022; Hu et al. 2023). Recently, long-read sequencing technologies have emerged as a promising approach for obtaining complete genomes of H. pylori, offering several advantages over traditional short-read sequencing methods. Long-read sequencing technologies, such as Pacific Biosciences Single Molecule Real-Time (SMRT) sequencing and Oxford Nanopore sequencing, generate much longer sequencing reads compared to traditional short-read platforms. This allows for the sequencing of entire genomic regions without the need for de novo assembly. Since very recently, long-read sequencing technologies have capability to generate near-finished microbial genomes from isolates or metagenomes without short-read or reference polishing (Sereika et al. 2022). Obtaining complete genomes from long reads offers several advantages, including the ability to accurately identify genomic variations such as insertions, deletions, intra-chromosomal rearrangements, and gene duplications or duplications of entire genomic regions. For example, long-read sequencing facilitated the discovery of H. pylori strains with multiple cagA genes. This study showed that increased cagA copy numbers may represent a novel mechanism to enhance disease development (Su et al. 2019). Furthermore, complete genomes enable the identification of genetic variations in mobile genetic elements that play a significant role in antibiotic resistance (Fauzia et al. 2023). The disadvantages of the long-read sequencing technologies are the higher costs compared to traditional short-read sequencing technologies and the high requirements on DNA or RNA sample quality and quantity (Tedersoo et al. 2021).

6 Concluding Remarks Advances in NGS technologies, including 454 pyrosequencing, Illumina sequencing, PacBio SMRT long-read sequencing and Oxford Nanopore sequencing, have greatly enhanced our understanding of H. pylori genomics and provided a comprehensive view of the genetic variation and virulence factors associated with H. pylori pathogenesis. The availability of multiple genomes provided a clear picture of the presence and absence of genes, particularly of pathogenicity-related factors, within individual strains as well as in entire phylogeographic populations. Moreover, genome analyses enabled a reliable estimation of the H. pylori mutation and recombination rates during infection of the human host as well as of the length of the imported and incorporated DNA fragments. Furthermore, these projects highlighted several virulence genes that are under diversifying selection, which emphasized evasion of the host immune system as a crucial part of the adaptation process of H. pylori to a new host. Recent efforts even led to the identification of H. pylori genes and/or SNPs that

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are associated with the development of specific disease manifestations. While these GWAS were great achievements and provided proof of principle, these studies were performed on a limited number of genomes, and there is still a long road ahead with thousands of genomes from different high-cancer risk and low-cancer risk geographical sources to be analyzed. Importantly, a detailed functional characterization of the biological relevance of a particular SNP and of the mechanisms of how a particular SNP affects disease development has been provided for a first SNP, and there are more to be analyzed. Bacterial SNPs correlated with disease manifestations will be useful biomarkers for risk predictions in H. pylori infections. Overall, advances in H. pylori pathogenomics are expected to play a big role in the development future therapies that are tailored to individual patients, including the determination of antibiotic resistance in strains to minimize the failure rate during H. pylori eradication therapy. These findings have implications for personalized medicine, antimicrobial resistance surveillance, and the development of targeted therapeutic strategies. However, challenges remain in terms of cost, data analysis, and the standardization of sequencing protocols. Acknowledgements This work is supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan to Y.Y. (18KK0266, 19H03473, 21H00346 and 22H02871). BS and RIA were supported by the MEXT Scholarship Program.

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Gastric Cancer: The Microbiome Beyond Helicobacter pylori Melissa Mendes-Rocha, Joana Pereira-Marques, Rui M. Ferreira, and Ceu Figueiredo

Abstract Gastric cancer remains an important global health burden. Helicobacter pylori is the major etiological factor in gastric cancer, infecting the stomach of almost half of the population worldwide. Recent progress in microbiome research offered a new perspective on the complexity of the microbial communities of the stomach. Still, the role of the microbiome of the stomach beyond H. pylori in gastric carcinogenesis is not well understood and requires deeper investigation. The gastric bacterial communities of gastric cancer patients are distinct from those of patients without cancer, but the microbial alterations that occur along the process of gastric carcinogenesis, and the mechanisms through which microorganisms influence cancer progression still need to be clarified. Except for Epstein-Barr virus, the potential significance of the virome and of the mycobiome in gastric cancer have received less attention. This chapter updates the current knowledge regarding the gastric microbiome, including bacteria, viruses, and fungi, within the context of H. pylori-mediated carcinogenesis. It also reviews the possible roles of the local gastric microbiota, as well as the microbial communities of the oral and gut ecosystems, as biomarkers for gastric cancer detection. Finally, it discusses future perspectives and acknowledges limitations in the area of microbiome research in the gastric cancer setting, to which further research efforts should be directed. These will be fundamental not only to increase our current understanding of host-microbial interactions but also to facilitate translation of the findings into innovative preventive, diagnostic, and therapeutic strategies to decrease the global burden of gastric cancer.

M. Mendes-Rocha · J. Pereira-Marques · R. M. Ferreira · C. Figueiredo (B) i3S–Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Rua Alfredo Allen 208, 4200-135 Porto, Portugal e-mail: [email protected] Ipatimup–Institute of Molecular Pathology and Immunology of the University of Porto, Rua Júlio Amaral de Carvalho 45, 4200-135 Porto, Portugal M. Mendes-Rocha · C. Figueiredo Department of Pathology, Faculty of Medicine of the University of Porto, Alameda Prof. Hernâni Monteiro, 4200-319 Porto, Portugal © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 S. Backert (ed.), Helicobacter pylori and Gastric Cancer, Current Topics in Microbiology and Immunology 444, https://doi.org/10.1007/978-3-031-47331-9_6

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1 Introduction The human microbiome is the collection of microorganisms that reside in all body sites, comprising bacteria, archaea, fungi, microbial eukaryotes, and viruses, as well as their genes and their products (Hou et al. 2022). The total number of bacteria in the 70 kg “reference man” was estimated to be 3.8·1013 , which is about the same order of magnitude as the number of human cells (Sender et al. 2016). Our understanding of the human microbiome has considerably expanded as a result of the implementation of large-scale profiling techniques, particularly those based on next-generation sequencing (NGS) (Lloyd-Price et al. 2017). These include markerbased NGS, metagenomics, metatranscriptomics, metaproteomics, metabolomics, and culturomics. Each of these approaches provides different insights into the microbiome, including microbial composition and functional content at gene, transcript, protein, and metabolite levels (Knight et al. 2018), allowing to explore the underlying mechanisms of host-microbiome interactions and their contributions to health and disease. The microbiome is pivotal for the maintenance of the normal physiology and health of the host, being closely involved in a broad range of metabolic functions and in the development of the immune system (Hou et al. 2022). The composition of the microbiome differs between individuals and across distinct sites of the human body, being influenced by multiple factors, such as host genetics, diet, and life style (Gilbert et al. 2018). Disturbance of the microbiome homeostasis (dysbiosis) can lead to dysregulation of host physiological processes, being associated with numerous diseases, including cancer (Cullin et al. 2021). The relationship between microbes and cancer is not novel, but of all known microbial species, only 11 are recognized cancer causes by the International Agency for Research on Cancer, based on strong epidemiological, clinical, and functional data (IARC 2012). These include seven viruses, three macroparasites, and the bacterial species Helicobacter pylori. Together, these species are responsible for 2.2 million new cancer cases every year, representing 13% of all cancer cases (de Martel et al. 2020). In addition to the oncogenic microbial agents recognized by IARC, growing evidence suggests that additional microbes can contribute to cancer (Sepich-Poore et al. 2021). The microbiome, including both local and distant microbial communities, may influence cancer initiation and progression through various mechanisms (Cullin et al. 2021). Locally, microbes can target host cells by binding to their receptors or invading them, releasing toxins and virulence factors, producing or modulating local metabolites. They may also locally modulate the tumor immune microenvironment, affecting both the innate and adaptive immune responses. Remotely produced microbial or microbially-modulated host or dietary metabolites and bacterial vesicles may also impact carcinogenesis, by reaching the tumor site via systemic circulation (Cullin et al. 2021). These complex microbial-host interactions might activate oncogenic signaling pathways, downregulate tumor suppressor pathways, induce DNA damage, promote chronic inflammation, and modulate the immune microenvironment within the tumor, thereby influencing cancer development.

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Helicobacter pylori is the most important infectious cause of cancer worldwide, being responsible for 810,000 cancer cases in 2018 (de Martel et al. 2020). This bacterium plays a central role in gastric cancer (GC), a highly incident and leading cause of cancer globally (Sung et al. 2021). The plethora of mechanisms involved in H. pylori-mediated GC development are the subject of discussion of chapters “Helicobacter pylori-Induced Host Cell DNA Damage and Genetics of Gastric Cancer Development” and “Gastric Epithelial Barrier Disruption, Inflammation and Oncogenic Signal Transduction by Helicobacter pylori” of this book. Contrasting to the widely recognized role of H. pylori, the significance of the microbiome other than H. pylori in gastric carcinogenesis remains poorly understood. This chapter will focus on the GC microbiome beyond H. pylori. It will update the current knowledge on the relationship between GC and the gastric bacterial, fungal, and viral microbiome; it will examine the potential of the gastric, oral, and gut microbial communities as GC biomarkers; and it will discuss future challenges and limitations of microbiome research in the GC scenario.

2 The Microbiota of the Stomach The gastrointestinal (GI) tract is where the large majority of bacteria can be found. The oral cavity is estimated to harbor up to 1012 of bacteria, but along the GI tract, the persistence of these bacteria is prevented by a series of factors, which include the stomach acidity, bile acid production, the presence of digestive enzymes, as well as the presence of antimicrobial proteins. In addition to these, bacterial persistence is affected by chemical factors such as oxygen concentration and mucus production, and physical factors, including peristaltic movements and transit times (de Vos et al. 2022). The stomach, duodenum, and jejunum have an estimated 107 resident bacteria, the ileum has 1011 , and the colon, with the highest concentration, has approximately 1014 bacteria (Sender et al. 2016) (Fig. 1). Until the early 1980s, the stomach was considered as a sterile environment, due to its highly acidic conditions. This dogma changed soon after the discovery and in vitro isolation of H. pylori (Marshall and Warren 1984). In the years after, classical microbiological cultures were used to characterize the gastric microbiota, but this approach was difficult and insufficient to provide a comprehensive understanding of the microbiota (Thorens et al. 1996; Zilberstein et al. 2007). The use of alternative and improved techniques provided evidence of a more extensive and complex bacterial community within the stomach (Bik et al. 2006; Li et al. 2009; Delgado et al. 2013). Rather than being just a transient population of swallowed microorganisms, the gastric mucosal microbiota is actually distinct from that of the gastric lumen and of other regions of the GI tract (Schulz et al. 2018). Compared with the lower GI tract, the microbial communities of the upper GI tract, including that of the stomach, have lower microbial diversity. The taxonomic composition also changes along the GI tract, with Proteobacteria and Firmicutes being dominant phyla in the mucosal

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Fig. 1 The microbiome of the gastrointestinal tract. Estimated number of bacteria that reside in different locations of the gastrointestinal (GI) tract. Values are rounded for the upper bound order of magnitude and derive from bacterial concentrations and volume (Sender et al. 2016). The microbiota composition (at the phylum level) and diversity changes along the upper and lower GI tract are represented (Vuik et al. 2019; Vasapolli et al. 2019), as well as the most common genera found in the stomach (Rajilic-Stojanovic et al. 2020). Created with BioRender.com

microbiota of the upper, and Firmicutes and Bacteroidetes being dominant in the lower GI tract (Vasapolli et al. 2019; Vuik et al. 2019) (Fig. 1). In the stomach, the most frequently identified phyla include Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, and Fusobacteria. The genera most common in the stomach in addition to Helicobacter, are Streptococcus, Prevotella, Neisseria, Veillonella, Fusobacterium, and Haemophilus, the two dominant being Streptococcus and Prevotella (Bik et al. 2006; Li et al. 2009; Delgado et al. 2013; Parsons et al. 2017; Linz and Backert 2021). A recent systematic review confirmed that each of these genera was reported in over 67% of the included studies (Rajilic-Stojanovic et al. 2020). In addition to being predominant, Helicobacter alongside Streptococcus and Prevotella rank among the most transcriptionally active bacteria in the stomach (Parsons et al. 2017; Vasapolli et al. 2019) (Fig. 1).

3 The Microbiota of the Stomach and Gastric Cancer 3.1 H. pylori Infection and Gastric Cancer GC is one of the most incident and deadliest malignancies in the world. It ranks as the fifth most commonly diagnosed and the fourth leading cause of cancer-related death, according to the latest estimates (Sung et al. 2021). Approximately 90% of

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GCs are adenocarcinomas, which are derived from epithelial glandular structures (Karimi et al. 2014). Other types of cancer in the stomach include mucosa-associated lymphoid tissue (MALT) lymphomas and gastrointestinal stromal tumors (GIST). In this chapter, and unless otherwise specified, when we discuss GC, we will be referring to gastric adenocarcinoma. H. pylori infection is the major risk factor for GC, being responsible for approximately 90% of the cases worldwide (de Martel et al. 2020). In addition to the multitude of epidemiological, clinical, and mechanistic data supporting the role of H. pylori infection as key factor in GC development, successful eradication of the bacterium is effective in preventing GC (Yan et al. 2022). H. pylori colonizes the stomach of approximately 45% of all humans worldwide (Li et al. 2023). All infected individuals develop chronic gastritis, a condition that remains for their lifetime if the bacterium is not eradicated. Chronic gastritis as consequence of H. pylori infection is associated with two major histological types of GC, the diffuse and the intestinal types. The series of events that may culminate in intestinal-type GC is, however, better characterized. Some of the infected individuals may develop atrophic gastritis, gastric intestinal metaplasia, and dysplasia, which will increase their likelihood of developing GC (Correa 1992). This multistep process of carcinogenesis is multifactorial, in the sense that only a small minority of H. pyloriinfected patients will actually develop cancer. This is likely due to variations in H. pylori strain virulence, differences in the genetic susceptibility of the host, and environmental influences related to the lifestyle of the host (Pereira-Marques et al. 2019a). While the importance of H. pylori as an initial trigger to the multistep process of gastric carcinogenesis is fully established, the significance of the infection at later stages of the cascade is not so clear. On one hand, H. pylori eradication therapy is not always successful in preventing progression of the precancerous conditions and lesions (Yan et al. 2022). On the other hand, a progressive disappearance of H. pylori in the severely atrophic stomach and in intestinal metaplasia has been described (Kuipers 1998). Accordingly, analyses of the microbiome of GC patients showed significantly decreased relative abundance of Helicobacter in comparison to patients without GC (Castano-Rodriguez et al. 2017; Ferreira et al. 2018; Gantuya et al. 2020; Kadeerhan et al. 2021; He et al. 2022). Within the neoplastic stomach, Helicobacter has also reduced abundance in the tumor compared with the adjacent mucosa (Yu et al. 2017; Chen et al. 2019; Liu et al. 2019; Dai et al. 2021; Ai et al. 2023; Lehr et al. 2023). As H. pylori abundance decreases, and concurrently with the gradual gastric histopathological and physiological changes, it is likely that the stomach may become a less restricted microbial niche, which may be colonized by bacteria from other locations of the GI tract (Pereira-Marques et al. 2019a). These non-H. pylori bacteria may then serve as sustained stimuli, perpetuating chronic inflammation and generating a genotoxic environment, thereby facilitating gastric tumorigenesis (Fig. 2).

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Fig. 2 Model for the gastric bacterial microbiota in gastric carcinogenesis. H. pylori triggers a chronic inflammatory response in the gastric mucosa, which in some of the infected individuals lead to gastric histopathological and physiological changes, and to an altered gastric environment. In this less restricted microbial niche, H. pylori colonization decreases and other bacteria are able to thrive, leading to gastric dysbiosis. These new microbial members may contribute to sustain gastric chronic inflammation and genotoxicity, thereby promoting tumorigenesis (Pereira-Marques et al. 2019a, reproduced with permission from Springer Nature, https://link.springer.com/chapter/ 10.1007/5584_2019_366)

3.2 The Gastric Bacterial Microbiota in Gastric Carcinogenesis The relationship between the gastric bacterial microbiota and gastric carcinogenesis has been addressed by various studies (Table 1). Gastric dysbiosis and reduced microbial diversity may be implicated in the development of GC. Decreased microbial diversity in cancer patients compared with non-cancer controls has been noted (Aviles-Jimenez et al. 2014; Gunathilake et al. 2019; Nikitina et al. 2023), but this has not always been consistent (Castano-Rodriguez et al. 2017; Kadeerhan et al. 2021; He et al. 2022). Two comprehensive analyses in large cohorts of patients from Portugal and China also found reduced microbial diversity in intestinal metaplasia and GC (Coker et al. 2018; Ferreira et al. 2018). Accordingly, lower microbial richness and/or diversity in cancer compared with atrophic or non-atrophic gastritis were reported in several studies in Chinese patients (Wang et al. 2020c; Wu et al. 2021; Zhang et al. 2021a; Sun et al. 2022). A meta-analysis of the gastric microbiota that

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analyzed 16S rRNA gene sequencing datasets of 825 patients from six independent studies corroborated these findings (Liu et al. 2022). In the GC tissue, the microbial diversity has been reported as similar (Tseng et al. 2016; Yu et al. 2017; Wu et al. 2021; Ai et al. 2023; Nikitina et al. 2023) or as increased (Chen et al. 2019; Dai et al. 2021; Lehr et al. 2023) compared to the non-cancerous mucosa. In contrast, in a large-scale survey in Chinese patients exploring alterations of the microbiota among diverse stomach microhabitats, the bacterial richness was lower in the tumor and in the periphery of the tumor than in the distant normal tissue (Liu et al. 2019). Changes in the microbial profiles of the stomach in GC have also been reported, and patients with GC and chronic gastritis exhibit differences in the structure of the gastric microbial communities (Aviles-Jimenez et al. 2014; Castano-Rodriguez et al. 2017; Coker et al. 2018; Ferreira et al. 2018; Kadeerhan et al. 2021; He et al. 2022; Sun et al. 2022). Despite differences in the gastric microbial profiles between patients with GC and those with superficial gastritis, atrophic gastritis, or intestinal metaplasia (Coker et al. 2018), various studies point to similar gastric microbiota composition between precancerous lesions (Parsons et al. 2017; Coker et al. 2018; Hsieh et al. 2018). In contrast, others report significant differences in the structure of the microbial communities across the histological steps of the gastric carcinogenesis cascade (Liu et al. 2021a; He et al. 2022; Sun et al. 2022). Whether a progressive shift in the composition of the gastric microbiota occurs along the distinct stages of GC development remains to be elucidated. Accumulating evidence suggests variations in the GC microbiota related with taxa from the oral and intestinal microbial communities, but unique bacterial signatures associated with the cancerous stomach have not been identified. Nevertheless, some genera have been consistently reported as over-represented in the microbiota of GC patients. These included Lactobacillus (Aviles-Jimenez et al. 2014; Coker et al. 2018; Ferreira et al. 2018; Hsieh et al. 2018; Gantuya et al. 2020; Wang et al. 2020c; Wu et al. 2021; He et al. 2022; Sun et al. 2022; Nikitina et al. 2023), Fusobacterium (Coker et al. 2018; Hsieh et al. 2018; Gantuya et al. 2020; He et al. 2022) and Clostridium (Ferreira et al. 2018; Hsieh et al. 2018; Nikitina et al. 2023). In the transcriptionally active GC microbiota, these taxa were also found to be enriched (Castano-Rodriguez et al. 2017). Other genera commonly identified as enriched in the microbial profiles of patients with GC include Veillonella (Castano-Rodriguez et al. 2017; Wang et al. 2020c; He et al. 2022), Staphylococcus (Castano-Rodriguez et al. 2017; Nikitina et al. 2023), and Rhodococcus (Ferreira et al. 2018; Wu et al. 2021). Several studies showed depletion of Prevotella (Aviles-Jimenez et al. 2014; Ferreira et al. 2018; Wu et al. 2021; Nikitina et al. 2023) and Neisseria (Ferreira et al. 2018; Wu et al. 2021) in the microbial communities of the cancerous stomach. Reports are inconsistent regarding Streptococcus, which was identified both as enriched (Coker et al. 2018; Gantuya et al. 2020; Wang et al. 2020c; Nunes et al. 2021; Sun et al. 2022) and as depleted (Aviles-Jimenez et al. 2014; Ferreira et al. 2018; Wu et al. 2021; Nikitina et al. 2023) in the GC microbiota. Within the neoplastic stomach, the tumor tissues were shown to have enrichment of Streptococcus, Fusobacterium, Prevotella, Selonomonas, and Sphingomonas (Chen et al. 2019; Liu et al. 2019; Dai et al. 2021; Wu et al. 2021),

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Table 1 Summary of studies evaluating the gastric mucosal microbiota of gastric cancer versus non-cancer patients* References

Country

Diagnosis (N)

Diversity and taxonomy (genus) changes in gastric cancer patients

Ferreira et al. (2018)

Portugal

GC (54) Ga (81)

↓ α-diversity; β-diversity distinguished GC and Ga ↑ Lactobacillus, Citrobacter, Clostridium, Achromobacter, Rhodococcus ↓ Helicobacter, Neisseria, Streptococcus, Prevotella

Coker et al. (2018)

China

GC (20) SG (21) AG (23) IM (17)

↓ α-diversity in GC and IM vs SG; β-diversity distinguished GC from SG, AG, and IM ↑ Peptostreptococcus, Streptococcus, Parvimonas, Slackia, Dialister, Lactobacillus, and Fusobacterium (in GC vs SG); ↑ Peptostreptococcus, Dialister, and Mogibacterium (in GC vs SG, AG, and IM) ↓ Vogesella, Comamonadaceae, and Acinetobacter

Gunanthilake et al. (2019)

Korea

GC (268) HC (288)

↓ α-diversity; β-diversity distinguished GC and HC ↑ Helicobacter, Propionibacterium, and Prevotella ↓ Lactococcus

Gantuya et al. (2020)

Mongolia

GC (48) N (20) SG (20) AG (40) IM (40)

α-diversity: ↓ in GC and IM vs normal, and ↑ in IM vs AG and SG; β-diversity: NA ↑ Lactobacillus, Fusobacterium, Streptococcus ↓ Helicobacter

Wang et al. (2020c)

China

GC (29) HC (30) SG (21) IM (27) GIN (25)

↓ α-diversity in GIN and GC; β-diversity: GC and GIN clustered closely ↑ Lactobacillus, Veillonella, Prevotella, Streptococcus, Helicobacter

Nunes et al. (2021)

Portugal

EGC (31) N/SG (17) IM (12)

↑ α-diversity in IM; β-diversity: NA ↑ Streptococcus, Gemella

GC (64) SG (61) IM (55)

↑ α-diversity in GC vs SG; β-diversity: NA ↑ Lactobacillus, Prevotella, Fusobacterium, Vellionella, Neisseria, Sarcina, Alloprevotella, Gemela, Leptotrichia, Dolosigranulum, Selenomonas, Lachnoaerobaculum ↓ Helicobacter, Carnobacterium, Paenibacillus

He et al. (2022) China

(continued)

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Table 1 (continued) References

Country

Diagnosis (N)

Diversity and taxonomy (genus) changes in gastric cancer patients

Nikitina et al. (2023)

Lithuania

GC (76) HC (29)

↓ α-diversity; β-diversity distinguished GC and HC ↑ Lactobacillus, Clostridium sensu stricto, and Pseudomonas; Staphylococcus (in GC tissue vs HC); Rothia (RNA transcripts in GC tissue vs HC) ↓ Actinomyces, Atopobium, Granulicatella, Propionibacterium, Streptococcus, Veillonella, Rothia, Prevotella, Gemella, Methyloversatilis, Parvimonas, Reyranella, Sediminibacterium

* Only studies including ≥ 20 cancer cases are shown; Abbreviations: AG, atrophic gastritis; Dys, dysplasia; EGC, early gastric cancer; FD, functional dyspepsia; Ga, gastritis; GC, gastric cancer; GIN, gastric intraepithelial neoplasia; HC, healthy controls; IM, intestinal metaplasia; N, normal; NA, not available; NS, non-significant; SG, superficial gastritis

whereas Lactobacillus was found to be enriched in both cancer and non-cancerous tissues (Chen et al. 2019; Liu et al. 2019; Dai et al. 2021). The functions associated with the microbial changes of the neoplastic stomach are still far from being clear. This is in part due to the fact that the GC microbiota functions have been mainly predicted from 16S rRNA gene sequencing data, which provide a less comprehensive view of the microbial community than metagenomics or metatranscriptomics. Studies that used this indirect type of analysis have reported multiple pathways enriched in GC patients. These include pathways related to carbohydrate metabolism, digestion and adsorption, nucleotide metabolism and membrane transport (Castano-Rodriguez et al. 2017; Yu et al. 2017; Coker et al. 2018; Ferreira et al. 2018; Wang et al. 2020b; Wu et al. 2021; Liu et al. 2022; Park et al. 2022). Interestingly, various functional features related with bacterial production of carcinogenic N-nitroso compounds, namely nitrate reductase and nitrite reductase functions, were enriched in the GC microbiome (Tseng et al. 2016; Ferreira et al. 2018; Chen et al. 2019; Gantuya et al. 2020; Kadeerhan et al. 2021; Wu et al. 2021), which concurs with the hypothesis that non-H. pylori bacteria produce N-nitroso compounds at later stages of gastric carcinogenesis (Correa 1992). Still, these predictions are based on inference and should thus be interpreted with caution. In the gastric niche, the mucosal microbiome may impact GC development via secretion of microbial metabolites or by bacteria-mediated metabolization of host and dietary metabolites (Fig. 2). Alterations in the metabolome of GC have been reported (Dai et al. 2021; Yang et al. 2022b). Untargeted metabolomics combined with 16S rRNA-sequencing identified 150 differentially abundant metabolites that distinguished GC from non-cancerous tissues (Dai et al. 2021). Strong positive correlations were detected between metabolites enriched in cancer tissues, like D-glucosamine6-phosphate, N-acetyl-D-glucosamine-6-phosphate, and N-acetylneuramic acid and the abundance of Lactobacillus, Streptococcus, Prevotella, Faecalibacterium, and

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Fusobacterium. In contrast, the abundance of Helicobacter was negatively associated with these metabolites, suggesting a role of the former taxa in the synthesis of these compounds (Dai et al. 2021). In line with these results, another study identified four discriminative metabolites between proximal GC and matched normal tissue, and 30 discriminative metabolites between distal GCs and matched normal tissue. The abundance of bacteria enriched in distal cancer, including Streptococcus, Enterococcus, Lactobacillus, Acinetobacter, Muribaculaceae, Methylobacterium-Methylorubrum, and Faecalibacterium, was positively correlated with the enrichment of metabolites of the glutathione, purine, and multiple amino acids metabolism pathways (Yang et al. 2022b). Further and more direct searches for metabolites of the dysbiotic microbiome ought to be conducted, in order to identify their potential as promoters of gastric carcinogenesis.

3.3 The Microbiota in Animal Models of Gastric Cancer Many of the studies described in the previous section identified microbial profiles or features associated or correlated with GC. In vivo models, however, are a major resource to evaluate the contribution of the microbiome to GC. Transgenic insulingastrin (INS-GAS) mice spontaneously develop gastric tumors at 20 months of age, or at 6 months when infected with H. pylori (Fox et al. 2003). Experiments using germ-free (GF) INS-GAS mice infected with H. pylori showed that animals developed gastritis and gastric intraepithelial neoplasia (GIN) more rapidly than uninfected GF mice, but the onset of GIN occurred only in few mice and at 11 months of infection. In contrast, in specific pathogen-free (SPF) INS-GAS mice infected with H. pylori, all animals developed GIN at 6 to 7 months (Lofgren et al. 2011). Later on, it was shown that infection with H. pylori in GF INS-GAS mice colonized with a restricted microbiota of three species, namely, Lactobacillus murinus, Bacteroides sp., and Clostridium sp., resulted in GIN development at a similar extent to that of SPF animals infected with H. pylori (Lertpiriyapong et al. 2014). The K19-Wnt1/C2mE (Gan) mice express Wnt1, COX-2, and PEG2 and develop tumors resembling the intestinal subtype of GC in humans (Oshima et al. 2011). In this model, GF Gan mice had smaller tumors than SPF Gan animals. Moreover, depletion of the microbiota with antibiotics in SPF Gan mice resulted in suppression in the growth of gastric tumors, whereas the repopulation of the gut microbiota and infection with Helicobacter reversed the suppression of tumor growth detected in GF animals (Oshima et al. 2011). These data highlight the importance of the resident microbiota to GC development. In a recent study, GF INS-GAS mice were co-infected with H. pylori and Staphylococcus salivarius or S. epidermidis, respectively, isolated from Colombian patients from high- and low-risk GC regions (Shen et al. 2022). Animals co-infected with H. pylori and S. salivarius developed higher inflammation scores, hyperplasia and dysplasia, and high expression of pro-inflammatory cytokines, compared to mice

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infected with H. pylori alone, or to mice co-infected with H. pylori and S. epidermidis. The gastric tissues of animals co-infected with H. pylori and S. salivarius contained higher levels of FoxP3, a regulator of T-cell function, and of Ki67, a marker of proliferative cells (Shen et al. 2022). Changes of the gastric microbiome upon Helicobacter infection were also evaluated in mice with different genotypic backgrounds, including C57BL/6 WT, MyD88 deficient (myeloid differentiation primary response gene 88, Myd88−/− ), TRIF (mice deficient in the Toll/interleukin-1 receptor (TIR) domain-containing adaptorinducing interferon-β, Trif Lps2 ), and MyD88 and TRIF double knockout (Myd88−/− / Trif Lps2 , DKO) mice (Bali et al. 2021). Infection with Helicobacter felis resulted in decreased alpha diversity in all mouse models and led to distinct microbiota structures. Differences in the microbiota profiles were mostly shaped by the abundance of the orders Campylobacterales (to which Helicobacter belongs) and Lactobacillales. Specifically, Myd88−/− and DKO mice had low H. felis abundance and high abundance of Lactobacillales at 3 and 6 months of infection compared to Trif Lps2 and WT mice. Interestingly, low abundance of H. felis facilitated the dominance of other members of the microbiome. Increased abundance of Lactobacillales was associated with increased severity of epithelial metaplasia in Myd88−/− and in DKO mice (Bali et al. 2021). A causal role for the gastric microbiota in gastric carcinogenesis has been recently demonstrated in GF C57BL/6 mice after transplantation of the gastric microbiota from patients with different disease states (Kwon et al. 2022). GF mice transplanted with gastric microbiota of patients with intestinal metaplasia or GC developed severe histopathological changes, namely loss of parietal cells, intense foci of inflammation, and increased expression of proliferative markers. One year after transplantation, histopathological alterations worsened and were characterized by dysplastic features. Shared abundance of taxa across human donors and mice recipients with intestinal metaplasia and GC included Bacteroides, Haemophilus, Lactobacillus, and Veillonella. The recapitulation of premalignant lesions in GF mice after gastric microbial transplantation from patients with gastric diseases provided strong evidence linking the gastric microbiota to the causality of GC (Kwon et al. 2022). Taken together, data from in vivo models show that microbes other than Helicobacter, including those of the resident microbiota, are important for gastric carcinogenesis. Although these are invaluable data, further research is needed to understand the underlying mechanisms exerted by bacteria to promote GC.

3.4 The Gastric Non-bacterial Microbiome in Gastric Cancer While bacteria are the most extensively studied component of the microbiome, little is known about the fungi (i.e., the mycobiome) and the viruses (i.e., the virome) besides Epstein-Barr virus (EBV), and their potential impact in GC. Studying the mycobiome and the virome in cancer tissues is challenging, due to the high proportion of non-microbial host genetic material, resulting in lack of sensitivity to detect the

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very low proportions of fungal or viral sequences, and due to poor representation or absence of well-characterized gastric fungal and viral sequences from reference databases (Dohlman et al. 2022; Elbasir et al. 2023).

3.4.1

The Mycobiome in Gastric Cancer

Though the impact of the mycobiome in GC is poorly understood, two recent studies provided an extensive overview of the mycobiomes associated with tumors in a variety of human cancers (Dohlman et al. 2022; Narunsky-Haziza et al. 2022). Both report the presence of fungal sequences in cancer, albeit at a much smaller proportion compared to bacteria, with fungal community compositions that differ between cancer types. Dohlman and colleagues explored whole-genome sequencing data of different cancers from the Cancer Genome Atlas (TCGA), including 321 GC cases, to determine their fungal composition (Dohlman et al. 2022). After removal of contaminants and false-positive signals, Candida albicans, C. tropicalis, and Saccharomyces cerevisiae were among the most frequently found species in GC. In the comparison of tumor and normal tissues, Candida was significantly and uniquely enriched in gastric tumor tissues. The bacterial populations present in Candida-associated tumors included Streptococcus, Clostridium, and Lactobacillus and were less likely to include H. pylori. The presence of Candida in gastric tumors was associated with positive enrichment of genes related to cytokine interactions, host immunity, and inflammation, and with significant up-regulation of genes involved in cytosolic DNA sensing, Toll-like receptor signaling, and Nod-like receptor signaling. High levels of C. tropicalis in gastric tumors were associated with decreased patient survival, suggesting Candida may represent a prognostic biomarker (Dohlman et al. 2022). This data fits well with that of Zhong and co-workers, who characterized the mycobiota by internal transcribed spacer (ITS) sequencing, showing that C. albicans was significantly increased in GC tissues, and distinguished them from non-cancerous tissues with an AUC of 0.74 (Zhong et al. 2021). Yang and colleagues also characterized the gastric fungal microbiome in GC patients and 11 healthy individuals (Yang et al. 2022a). GC had reduced abundance of Rozellomycota and a higher Basidiomycota to Ascomycota ratio, previously reported to reflect fungal dysbiosis. At lower taxonomic levels, Apiotrichum, Cutaneotrichosporon, Sarocladium, and Malassezia were more abundant in GC, and Rhizopus, Rhodotorula, Apodus, and Cystobasidium were more abundant in the stomach of healthy controls. In receiver operating characteristic (ROC) analysis these taxa showed diagnostic potential with AUCs over 0.88. A positive correlation was identified between fungi enriched in GC and the expression of pro-inflammatory factors, including TNF, CXCL9, CXCL10, and CXCL11; and between fungi enriched in healthy controls and the expression of factors associated with anti-inflammatory properties, including IL-4, IL-6, and CCL17 (Yang et al. 2022a). Taken together, and although not clarifying its role in pathogenesis, these data suggest a relationship between the mycobiome and GC.

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The Virome in Gastric Cancer

When discussing viruses in the context of GC, the best known is EBV. Less than 10% of the gastric carcinoma cases are EBV-positive, and these tumors have distinct epidemiological, histopathological, and molecular features. They are more frequent in male patients, tend to occur in non-antral gastric subsites, frequently exhibit diffuse-type histology, have an intense lymphoid infiltration, and have a more favorable prognosis than other GC subtypes (Cancer Genome Atlas Research 2014; Figueiredo et al. 2017). EBV-positive gastric tumors have extensive DNA hypermethylation, known as CpG island methylator phenotype. EBV-induced hypermethylation not only contributes to maintaining the EBV type I or II latency programs in GC but also leads to the down-regulation and silencing of multiple tumor suppressor genes, cell cycle genes, and factors associated with cell differentiation, thus promoting a highly proliferative and poorly differentiated cell population (Stanland and Luftig 2020; Damania et al. 2022). EBV-positive GCs also have high gene expression scores for CD8 T-cells, M1-macrophages, and IFN-γ signatures (Liu et al. 2018). These strong immune signatures together with other molecular features, which include programmed death-ligand 1 (PD-L1) overexpression, make EBV-positive tumors one of the GC subtypes that may benefit from therapy with immune checkpoint inhibitors (Kim et al. 2018). Studies on the relationship between viruses other than EBV and GC are sporadic. A systematic review with meta-analysis identified significant associations between GC and hepatitis B virus (HBV), with an odds ratio (OR) of 1.39 (95% confidence interval [CI] 1.11–1.75), cytomegalovirus (CMV) with an OR of 2.25 (95% CI 1.14– 4.43), human papillomavirus (HPV), with an OR of 1.63 (95% CI 1.05–2.54), and John Cunningham virus (JCV), with an OR of 2.52 (95% CI 1.26–5.04) (Wang et al. 2020a). Another meta-analysis showed infection with Human T-cell lymphotropic virus type 1 (HTLV-1) associated with a lower relative risk of GC of 0.45 (95% CI 0.28–0.71) (Schierhout et al. 2020). Still, a large proportion of the included studies did not adjust for various GC risk factors, namely, H. pylori infection, so these data have to be interpreted with caution. Although HBV is considered as an hepatotropic virus, some studies point for an association between HBV infection and extra-hepatic cancers, including GC. The comparison of the primary incidence of cancers among 5,773 patients with HBV in the USA cancer registries, with that of the general population, revealed higher incidence of various cancer types, including GC, in HBV-infected patients, with a standardized rate ratio of 7.75 (95% CI 7.71–8.20) (Spradling et al. 2022). Another study in the US elderly population, which analyzed 1,825,316 people with first cancers diagnosed in the SEER registries and 200,000 randomly selected, cancerfree, and age-, sex-, and race-matched controls, reported a positive association of HBV and GCs, with an adjusted OR of 1.19 (95% CI 1.03–1.37) (Mahale et al. 2019). Similar results were reported in China, with hepatitis B surface antigen (HBsAg) positivity being associated with GC, with an OR of 1.24 (95% CI 1.06–1.45) (Wei et al. 2017). HBV DNA was identified in gastric tissues, and HBV core antigen was detected in gastric lymphocytes, normal gastric glands, and cancer cells by

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immunohistochemistry. The mechanisms that may connect HBV infection with GC are unknown. Overall, the relationship between EBV and GC is clear in the sense that positive tumors represent a separate molecular subtype, but the influence of EBV in the precancerous stomach is still unclear. If and how other viruses are involved in GC remain to be elucidated.

4 The Non-gastric Microbiome and Gastric Cancer Various studies have investigated the relationships of the oral and of the gut microbiota with that of GC (Table 2), as they may represent a proxy for cancer detection, and thus a non-invasive approach for diagnosis.

4.1 The Oral Microbiome and Gastric Cancer The main aim of studies on the relationship between the oral microbiota and GC has been the identification of bacterial markers for cancer detection. Various microniches of the oral cavity have been addressed, including the tongue coating, saliva, and subgingival plaque. In the oral microbiota of GC cases, and compared to healthy controls, a trend towards decreased microbial diversity has been identified, suggestive of dysbiosis (Hu et al. 2015; Wu et al. 2018; Huang et al. 2021; Yang et al. 2022c; Zhang et al. 2022a) (Table 2). The genera enriched in the oral microbiota of GC patients include Streptococcus, which has been reported in the tongue coating and in the salivary microbiota (Wu et al. 2018; Huang et al. 2021; Zhang et al. 2022a), Pseudomonas in the tongue coating, saliva and subgingival plaque (Sun et al. 2018; Xu et al. 2021), and several other genera identified in single studies. Fusobacterium (Hu et al. 2015; Huang et al. 2021), as well as both Neisseria and Porphyromonas were found depleted in GC patients (Hu et al. 2015; Wu et al. 2018; Huang et al. 2021), but Neisseria has been also identified as enriched in the oral microbiota of GC patients and of patients with gastric intestinal metaplasia (Wu et al. 2022; Yang et al. 2022c). The relationship between the tongue coating microbiota, the serum metabolic features, and inflammatory cytokines was explored in a comparison of GC patients and healthy controls (Xu et al. 2021). A model combining Peptostreptococcus, Peptococcus, Porphyromonas, Megamonas, Rothia, and Fusobacterium distinguished GC patients from controls, with an AUC of 0.85. Peptostreptococcus, Peptococcus, Porphyromonas, and Parvimonas, which were enriched in healthy controls, were negatively correlated with serum lysophospholipid metabolites and eicosapentaenoic acid. Porphyromonas, Parvimonas, and Capnocytophaga were negatively correlated with serum IL-17α levels. The authors suggest that the loss of particular microbiota may be responsible for an altered inflammatory and metabolome profile in GC (Xu et al. 2021). A metagenomics study of the tongue coating of patients at different

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Table 2 Studies evaluating the oral or the gut microbiota of gastric cancer versus non-cancer patients References

Country

Diagnosis (N)

Sample type

Diversity and taxonomy changes in gastric cancer patients

Hu et al. (2015)

China

GC (74) HC (72)

Oral Tongue coating

↓ α-diversity in GC with thick tongue coating; β-diversity: NA ↑ Actinobacteria ↓ Proteobacteria, Neisseria, Haemophilus, Fusobacterium, Porphyromonas

Sun et al. (2018)

China

GC (37) HC (13)

Oral Saliva, subgingival plaque

α- and β-diversity: NA ↑ Pseudomonadaceae, Dethiosulfovibrionaceae, Paraprevotellaceae, Veillonellaceae, Actinomycetaceae

Wu et al. (2018)

China

GC (57) HC (80)

Oral Tongue coating

↓ α-diversity; β-diversity distinguished GC and HC ↑ Streptococcus ↓ Neisseria, Prevotella, Porphyromonas

Liang et al. (2019)

China

GC (20) HC (22)

Gut α-diversity: ↓ diversity and ↑ Fecal samples richness; β-diversity: NA ↑ Enterobacteriaceae, Escherichia/Shigella, Veillonella, Clostridium ↓ Bacteroidaceae

Qi et al. (2019)

China

GC (116) HC (88)

Gut ↑ α-diversity; β-diversity: Fecal samples distinguished GC and HC; ↑ Prevotella, Escherichia/ Shigella, Klebsiella, Lactobacillus, Streptococcus, Alistipes, Veillonella, Bifidobacterium, Ruminococcaceae, Christensenellacea, Parabacteroides ↓ Lachnoclostridium, Roseburia, Lachnospira, Faecalibacterium

Wu et al. (2020)

China

GC (134) HC (58)

Gut α-diversity: NS; β-diversity: NA Fecal samples ↑ Veillonella, Megasphaera, Prevotella, Streptococcus salivarius, Bifidobacterium dentium, Lactobacillus salivarius (continued)

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Table 2 (continued) References

Country

Diagnosis (N)

Sample type

Diversity and taxonomy changes in gastric cancer patients

Huang et al. (2021)

China

GC (99) SG (101) AG (93)

Oral Saliva

↓ α-diversity; β-diversity distinguished GC from SG and AG ↑ Streptococcus, unclassified Streptophyta ↓ Fusobacterium, Haemophilus, Neisseria, Parvimonas, Peptostreptococcus, Porphyromonas, Prevotella

Liu et al. (2021b)

China

GC (38) HC (35)

Gut α-diversity: NS; β-diversity: Fecal samples distinguished GC and HC ↑ Proteobacteria, Escherichia, Desulfovibrio ↓ Roseburia, Faecalibacterium

Park et al. (2021)

Korea

GC (181) HC (272)

Gut α-diversity: NS; β-diversity: Fecal samples distinguished GC and HC ↑ Firmicutes, Streptococcus, Subdoligranulum, Enterobacter, Lactobacillus, Klebsiella, Ruminiclostridium ↓ Bacteroidetes, Prevotella

Xu et al. (2021)

China

GC (181) HC (112)

Oral Tongue coating

Yu et al. (2021)

China

GC (49) HC (49)

Gut ↑ α-diversity; β-diversity: Fecal samples distinguished GC and HC ↑ Lactobacillus, Streptococcus, Fusobacterium ↓ Roseburia, Blautia

Zhang et al. (2021b)

China

GC (62) HC (61)

Gut ↑ α-diversity; β-diversity: Fecal samples distinguished GC and HC ↑ Leptotrichia, Fusobacterium Prevotella, Porphyromonas, Lactococcus, Streptococcus, Bacillus, Selenomonas, Anaerococcus, Aggregatibacter, Haemophilus, Neisseria, Nitrospira, Actinomyces, Bifidobacterium, etc

α-diversity: ↓ diversity and ↑ richness; β-diversity: NS ↑ Lactococcus, Megamonas, Geobacillus, Pseudomonas, Lactobacillus, Carnobacterium, Faecalibacterium, Leuconostoc

(continued)

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Table 2 (continued) References

Country

Diagnosis (N)

Sample type

Diversity and taxonomy changes in gastric cancer patients

Yang et al. (2022c)

USA

GC (165) HC (323)

Oral Mouth wash

↓ α-diversity; β-diversity: NS ↑ GC risk: Neisseria mucosa, Prevotella pleuritidis ↓ GC risk: Mycoplasma orale, Eubacterium yurii

Zhang et al. (2022a)

China

GC (76) HC (70)

Oral Mouth swab Gut Fecal samples

↓ α-diversity; β-diversity: distinguished GC and HC ↑ Streptococcus, Gemella, Herbaspirillum ↓ Haemophilus, Neisseria α-diversity: NS; β-diversity: distinguished GC and HC ↑ Escherichia-Shigella, Streptococcus ↓ Faecalibacterium, Romboutsia

Zhang et al. (2022b)

China

GC (22) HC (30)

Gut α-diversity: NS; β-diversity: NA Fecal samples ↑ Streptococcus, Lactobacillus ↓ Prevotella

Abbreviations: AG, atrophic gastritis; GAC, gastric adenocarcinoma; GC, gastric cancer; GIST, gastrointestinal stromal tumors; HC, healthy controls; NA, not available; NS, non-significant; SG, superficial gastritis

stages of the gastric carcinogenesis cascade, however, did not reveal major differences in the microbial composition or functions between patient groups (Cui et al. 2019). Analysis of the salivary microbiota of patients with superficial gastritis, atrophic gastritis and GC identified Streptococcus and Bifidobacterium in GC, Bacteroides and Haemophilus in atrophic gastritis, and Peptostreptococcus in superficial gastritis, as the representative genera responsible for the differences among stages of carcinogenesis (Huang et al. 2021). Their microbial classifier had high accuracy in distinguishing cancer from non-cancer patients, with an AUC of 0.91. The aforementioned data hint at a potential use of the oral microbiota for diagnosis of GC and/or precancerous lesions, but their retrospective nature, the fact that they were mostly conducted in Asians and with low-resolution microbial characterization, strongly limits their conclusions. Previous studies have associated oral health conditions like periodontal disease and tooth loss with upper GI tract cancers, including GC and gastric precancerous lesions (Salazar et al. 2012; Lo et al. 2021). A recent prospective study used metagenomics to explore the relationship between the oral microbiome and GC, in prediagnostic oral samples from Asian, African American, and European American populations (Yang et al. 2022c). Asian GC patients had significantly lower microbial diversity than controls, but this was not observed in either African Americans or European Americans. Still, the overall oral microbial richness was associated with

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decreased GC risk. H. pylori was not detected in the oral microbiome of any patient, despite its high positive serological rate among participants. Of the various periodontal pathogens investigated, only P. gingivalis was associated with an increased GC risk among Asians, with an OR of 1.37 (95% CI: 1.01–1.86). The meta-analysis of the combination of cohorts identified Neisseria mucosa and Prevotella pleuritidis associated with increased risk of GC [ORs of 1.31 (95% CI 1.03–1.67) and 1.26 (95% CI 1.00–1.57), respectively] and Mycoplasma orale and Eubacterium yurii associated with decreased risks of GC [ORs of 0.74 (95% CI 0.59–0.94) and 0.80 (95% CI 0.65– 0.98), respectively]. The analyses of the microbial gene families and metabolic pathways revealed associations between tricarboxylic acid (TCA) cycles (Krebs cycle) II and VII and increased GC risk, and between hexitol metabolism-related gene families and pathways and decreased GC risk. Another metagenomics study involving 89 patients with intestinal metaplasia and 89 matched controls in the USA, identified Peptostreptococcus stomatis, Neisseria elongata, and N. flavescens positively associated, and Lactobacillus gasseri, Streptococcus sanguinis, Shuttleworthia satelles, Achromobacter xylosoxidans, and Kingella oralis inversely associated with intestinal metaplasia (Wu et al. 2022). Lipopolysaccharide and ubiquinol biosynthesis pathways enriched in intestinal metaplasia patients were correlated with N. elongata and N. flavescens, whereas sugar degradation pathways under-represented in intestinal metaplasia were correlated with L. gasseri, S. mutans, S. sanguinis, and S. parasanguinis. Interestingly, the analysis of the gastric microbiota of the same individuals, revealed various taxa in both the oral and gastric microbiota, including Bacilli, L. gasseri, S. mutans, S. parasanguinis, and S. sanguinis, consistently associated with lower odds of intestinal metaplasia. These data are suggestive of the involvement of various bacteria of the oral microbiome in the etiology of gastric precancerous lesions and GC.

4.2 The Gut Microbiome and Gastric Cancer Examinations of the gut microbiome composition in the setting of GC have mostly aimed to identify fecal bacterial markers for the detection of GC (Table 2). Some of the genera frequently enriched in the gastric mucosa of GC patients are also found enriched in the gut microbiota, such as Lactobacillus and Streptococcus (Qi et al. 2019; Wu et al. 2020; Park et al. 2021; Yu et al. 2021; Zhang et al. 2021b, 2022b; Zhou et al. 2022), Fusobacterium (Yu et al. 2021; Zhang et al. 2021b, 2022b; Li et al. 2022), and Prevotella (Qi et al. 2019; Zhang et al. 2021b; Li et al. 2022). Increased abundances of Escherichia/Shigella and of Veillonella have also been reported in the gut microbiota of GC patients (Liang et al. 2019; Qi et al. 2019; Wu et al. 2020; Liu et al. 2021b; Zhang et al. 2022a). Both Lactobacillus and Streptococcus were enriched in the gut microbiota of Chinese GC patients compared with healthy controls, and Streptococcus could predict GC in a random forest model with AUCs > 0.77, suggesting its potential as a marker

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for GC diagnosis (Yu et al. 2021). Interestingly, and compared to patients with nonliver metastasis, the gut microbiota of those with metastatic liver disease had enrichment of Streptococcus. Streptococcus anginosus and S. constellatus were recently identified as having significantly increased proportions in stools and tumor tissues of cancer patients (Zhou et al. 2022). Quantitative PCR analyses revealed the highest abundance of S. anginosus and S. constellatus in patients with early cancers, where the fecal signature combination reached a sensitivity of 91.1% with a specificity of 64%. A sensitivity of 81.4% with 73.4% specificity was shown for advanced GC detection. Another study of Chinese patients reported the potential value of other Streptococcus species, namely S. salivarius and S. mitis, together with Lactobacillus salivarius, Bifidobacterium dentium, Megasphaera, Prevotella, and Desulfovibrio, which discriminated GC patients from healthy controls with AUCs > 0.70 (Wu et al. 2020). Desulfovibrio was also identified as a potential fecal microbial GC biomarker associated with disease staging (Liu et al. 2021b). The authors experimentally showed that Desulfovibrio-promoted hydrogen sulfide secretion stimulated the production of inflammatory molecules such as nitric oxide, IL-1β, and IL-18 in cancer cells, suggesting a potential mechanism by which this bacterium contributes to GC progression through inflammation. Putative changes in the gut microbiota at various points along the gastric carcinogenesis cascade have also been investigated. No significant differences in the gut microbial alpha-diversity or beta-diversity were observed between individuals with a normal stomach, patients with gastritis, and patients with intestinal metaplasia from Linqu in China (Gao et al. 2018). However, a study comparing non-atrophic, mild atrophic, and severe atrophic gastritis in Japanese patients identified a significant increase in the relative abundance of Lactobacillus in the gut microbiota of patients with severe atrophy (Iino et al. 2018). These results concur with those reported using a rat model of chemically induced GC, in which the animals develop chronic gastritis, atrophic gastritis, intestinal metaplasia, intraepithelial neoplasia, and GC at specific times of exposure (Yu et al. 2020). In this model, gut microbial dysbiosis has been identified, with significant decrease in diversity accompanied by an increase in species richness, along with a consistent increase in the abundance of Lactobacillus along the steps of gastric carcinogenesis. Together, while the above findings suggest possible fecal microbial GC biomarkers, studies are not without limitations and additional validation in large cohorts of patients is necessary.

5 Concluding Remarks Despite the growing number of studies on the microbiome and its impact on GC, there are still many open questions and limitations that need to be addressed in future research. While it is clear that the cancerous stomach has a microbiota distinct from that of the stomach with chronic gastritis, the microbial alterations that occur along the process of gastric carcinogenesis still need to be clarified. Only few studies have analyzed gastric precancerous lesions and the majority included low numbers of

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cases. Despite the differences between the microbiota of the cancerous and noncancerous stomachs, a gastric microbial signature common to all cancers has not been identified. Likewise, in the case of the oral and gut microbiomes, a consistent microbial signature associated to GC remains to be identified. Generalization of results has been difficult so far, and the lack of universal gastric, oral, or fecal microbial markers remains a challenge for GC microbiome-based diagnostics. Adding to the retrospective and descriptive nature of the majority of studies, interindividual and interpopulation microbial differences can contribute to the variability of the findings. The microbiome exhibits considerable variation with age, host genetics, host lifestyle, and environmental exposures, and additional differences are observed between ethnic groups and geographic regions (Gilbert et al. 2018). It is therefore critical to control the potential confounding effects of such factors. Since the majority of microbiome studies in GC focus on patients of Asian origin, future studies should include patients from other origins or multiple cohorts from different geographic areas. An additional hurdle leading to inconsistent results in microbiome studies is technical variability. Discrepancies may arise from the multiple steps of analyses, including sample collection and processing, sequencing, and bioinformatics analysis (Knight et al. 2018). Measures to mitigate the impact of contaminations should be implemented as they can have severe impact on low microbial biomass microbiome studies, as is the case of gastric tissue specimens (Eisenhofer et al. 2019). Strong efforts should also be made to promote rigorous reporting of microbiome experimental and analytical methods, and to foster raw data availability and detailed metadata (Scott et al. 2019). This will be essential to facilitate reproducibility and to effectively use data to advance knowledge on the microbiome and GC. The majority of studies characterizing the gastric microbial communities rely on marker-based sequencing data obtained from short 16S rRNA gene amplicons, which limits taxa identification to the genus level. Metagenomics or metatranscriptomics could be envisioned as means to explore the GC bacterial microbiome in greater detail, allowing identification of species and detection of its functional content, including microbial toxins, virulence factors, and enzymes that can influence carcinogenesis. This approach would additionally provide insights into the understudied gastric mycobiome and virome. Still, the characterization of low microbial biomass samples remains challenging, due to the high content of host genetic material that may interfere with microbiome analysis (Pereira-Marques et al. 2019b). The issue of causality versus consequence in GC is still undisclosed for microbes besides H. pylori. Future endeavors in functional studies aimed to identify carcinogenic microbial-host interactions, involving local and remote microbial communities and specific microbes and their metabolites, will deepen our understanding of microbe-driven molecular mechanisms underlying GC. Moreover, international, concerted efforts need to be undertaken to conduct large longitudinal studies in clinically and microbially well-characterized cohorts, to fully address microbiome dynamics and its impact on GC development. Multidisciplinary research efforts will be necessary to dissect the complex microbial-host interactions that occur along gastric carcinogenesis and to generate knowledge to support the design of

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microbiome-based preventive, diagnostic, and therapeutic strategies aimed to reduce GC burden. Acknowledgements MM-R has a fellowship from Fundação para a Ciência e a Tecnologia (FCT) (2021.06828.BD). RMF has a FCT researcher position under the Individual Call to Scientific Employment Stimulus (CEECIND/01854/2017). The team is also funded by national funds through FCT (PTDC/BTM-TEC/0367/2021 and 2022.02141.PTDC).

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Helicobacter pylori-Induced Host Cell DNA Damage and Genetics of Gastric Cancer Development Steffen Backert, Bodo Linz, and Nicole Tegtmeyer

Abstract Gastric cancer is a very serious and deadly disease worldwide with about one million new cases every year. Most gastric cancer subtypes are associated with genetic and epigenetic aberrations caused by chromosome instability, microsatellite instability or Epstein-Barr virus infection. Another risk factor is an infection with Helicobacter pylori, which also triggers severe alterations in the host genome. This pathogen expresses an extraordinary repertoire of virulence determinants that take over control of important host cell signaling functions. In fact, H. pylori is a paradigm of persistent infection, chronic inflammation and cellular destruction. In particular, H. pylori profoundly induces chromosomal DNA damage by introducing double-strand breaks (DSBs) followed by genomic instability. DSBs appear in response to oxidative stress and pro-inflammatory transcription during the S-phase of the epithelial cell cycle, which mainly depends on the presence of the bacterial cag pathogenicity island (cagPAI)-encoded type IV secretion system (T4SS). This scenario is closely connected with the T4SS-mediated injection of ADP-glycero-β-D-manno-heptose (ADP-heptose) and oncoprotein CagA. While ADP-heptose links transcription factor NF-κB-induced innate immune signaling with RNA-loop-mediated DNA replication stress and introduction of DSBs, intracellular CagA targets the tumor suppressor BRCA1. The latter scenario promotes BRCAness, a disease characterized by the deficiency of effective DSB repair. In addition, genetic studies of patients demonstrated the presence of gastric cancer-associated single nucleotide polymorphisms (SNPs) in immune-regulatory and other genes as well as specific pathogenic germline variants in several crucial genes involved in homologous recombination and DNA repair, all of which are connected to H. pylori infection. Here we review the molecular mechanisms leading to chromosomal DNA damage and specific genetic aberrations S. Backert (B) · B. Linz · N. Tegtmeyer (B) Division of Microbiology, Department of Biology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstr. 5, 91058 Erlangen, Germany e-mail: [email protected] N. Tegtmeyer e-mail: [email protected] B. Linz e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 S. Backert (ed.), Helicobacter pylori and Gastric Cancer, Current Topics in Microbiology and Immunology 444, https://doi.org/10.1007/978-3-031-47331-9_7

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in the presence or absence of H. pylori infection, and discuss their importance in gastric carcinogenesis.

1 Introduction Gastric cancer (GC) is associated with substantial rates of morbidity and mortality in the global human population. The currently announced GLOBOCAN statistics reported 1,089,103 new cases of GC in the year 2020, accounting for about 5.7% of the overall cancer burden worldwide (Sung et al. 2021). The incidence rates vary among geographical regions and are especially high in some countries of East Asia and Eastern Europe, while the rates in Africa, North Europe and North America are commonly the lowest (Thrift et al. 2023). Altogether, these incidences are responsible for 769,793 annual deaths in the world, ranking this malignancy the fourth most prominent source of cancer-related deaths (Sung et al. 2021). Interestingly, the overall associated death rates are about twofold higher in males compared to females. In most cases, GC is diagnosed at later stages, which represents a serious medical challenge associated with a poor prognosis for the patients. Thus, GC still represents an eminent and perilous cancer type in humans. The majority of these cancers comprises adenocarcinomas that can be categorised by different parameters. For example, histologically GC has been divided by Lauren into the major diffuse and intestinal subtypes, respectively. The development of the intestinal type of adenocarcinomas follows the well-known Correa cascade, as characterized by the successive progression of specific lesions in the stomach (Correa 2013). On the other hand, diffuse type adenocarcinomas exhibit a more aggressive performance. With regard to the anatomic location, GC has been commonly found in the lower stomach (non-cardia subtype) and less often in the upper stomach (cardia subtype). The latter classification distinguishes two distinct clinical scenarios, although often described as one entity. In addition, a thorough molecular study in patients defined four categories, namely GC either (i) with chromosome instability (about 50%), (ii) with microsatellite instability (about 21%), (iii) with genomic stability (about 20%) or (iv) with Epstein-Barr virus (EBV) infection (about 9%) (Cancer Genome Atlas Research Network 2014). All these categories are characterized by specific markers, such as DNA double strand breaks (DSBs) associated with genomic aberrations, DNA repair gene mutations, epigenetic modifications such as methylation of genes, EBV-DNA presence or missing genomic changes, respectively. In general, GC development has been described as a multifactorial and highly complex disease. In addition to the predisposing genetic circumstances, high salt consumption, alcohol, smoking, lack of vitamin C and infection with Helicobacter pylori also impact cancer development (Fig. 1). In fact, chronic H. pylori infection has been regarded as the primary trigger of the non-cardia type of stomach cancer. H. pylori is a Gram-negative bacterial pathogen that colonizes the harsh milieu in the antrum of the human stomach in about half of the world population (Li et al. 2023). H. pylori isolates show a high degree of genetic variability, which is associated

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Fig. 1 Contribution of human germline mutations, other genetic aberrations, physiological pre-conditions, Helicobacter pylori virulence factors, environmental parameters and EpsteinBarr Virus (EBV) infection to the pathogenesis and development of gastric adenocarcinoma. Pathogenic germline variants of the indicated 9 genes are strongly linked to enhanced risk of GC in patients. These constraints merge with the other shown factors and establish a situation in the stomach exemplified by prolonged gastric inflammation, oxidative stress, DNA damage, epigenetic changes, oncogenic signaling, pH upregulation and overgrowing microbiota. Such a scenario typically promotes GC development. For more details and references, see text

with different pathogenicity potentials (Gobert and Wilson 2022; Nabavi-Rad et al. 2023). The development of GC depends on the expression of the vacuolating cytotoxin A (VacA) allele s1m1 and the cytotoxin-associated genes (cag) pathogenicity island (PAI). The cagPAI represents a genetic locus with up to 31 genes, which is present in highly virulent H. pylori isolates and missing in less virulent strains. Genetic, functional and other analyses have demonstrated that the cagPAI encodes a type-IV secretion system (T4SS), belonging to a group of versatile molecular transporter complexes that are able to translocate effector molecules into target cells (Fischer et al. 2020; Sheedlo et al. 2022). This T4SS has been shown to deliver the oncoprotein CagA (Tegtmeyer et al. 2017; Imai et al. 2021), the LPS metabolite ADP-heptose (Gall et al. 2017; Pfannkuch et al. 2019; Faass et al. 2021; Maubach et al. 2021) and genomic DNA (Varga and Peek 2017) into eukaryotic cells. While injected CagA induces a complex signaling network to manipulate cell polarity, adhesion, motility, proliferation, cell cycle progression, receptor endocytosis, cytoskeletal dynamics, anti-apoptosis and iron acquisition (for details see chapters 2, 8 and 9 of this book), ADP-heptose triggers pro-inflammatory signal transduction via transcription factor NF-κB (for details see chapter 8 of this book) and chromosomal DNA activates toll-like receptor (TLR)-9-mediated anti-inflammatory signaling (for details see chapter 4 of this book). On the other hand, VacA triggers cell vacuolization and apoptosis, and inhibits T cell proliferation (Foegeding et al. 2016; Sharafutdinov et al. 2021). Numerous other H. pylori genes were also shown to impact the clinical outcome of the infection with this pathogen. For example, the bacterium expresses various outer membrane proteins that act as adhesins to establish binding to host cell

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receptors, including SabA, BabA, HopQ, AlpA, AlpB and OipA (Bugaytsova et al. 2017; Moonens et al. 2018; Bonsor and Sundberg 2019). In addition, recognised pathogenicity-linked processes include the release of reactive oxygen and nitrogen species, urease-mediated chemotaxis and inflammasome activation, epithelial barrier disruption by serine protease HtrA and the introduction of DSBs in host genomic DNA (Johnson and Ottemann 2018; Pachathundikandi et al. 2019; Müller and He 2023; Sharafutdinov et al. 2023). Here we discuss our current understanding of H. pylori-induced chromosomal DNA damage and the genetic basis of GC progression (summarized in Fig. 2).

Fig. 2 Schematic presentation of molecular mechanisms in H. pylori-induced epithelial cell damage that promote gastric cancer development. (1) The delivery of bacterial virulence factors such as oncoprotein CagA, serine protease HtrA (171L variant), ADP-heptose (ADPH) and vacuolating cytotoxin A (VacA, s1m1 allele) contributes to gastric carcinogenesis. (2) Extracellular HtrA opens the tight and adherens junctions by cleavage of occludin, claudin-8 and E-cadherin. (3) Epithelial barrier disruption is associated with raised iron acquistion by H. pylori. (4) Transfected CagA can bind the cytoplasmic tail of E-cadherin to release β-catenin from the complex, which triggers nuclear cell proliferation signaling. (5) Translocation of ADPH induces inflammation through activating transcription factor NF-κB. (6) This and other events are associated with the onset of oxidative stress and (7) the induction of DNA double-strand breaks (DSBs). This genotoxic stress combined with erroneous DNA repair in infected cells enhances (8) genome instability, (9) microsatellite instability and (10) telomere shortening. (11) Intracellular CagA also induces the mRNA downregulation of specific DNA repair genes, and increases (12) oncogenic signaling and (13) anti-apoptotic effects. The latter is antagonized by the delivery of pro-apoptotic VacA. (14) In addition, H. pylori activates proto-oncogenes and (15) triggers promoter hypermethylation in the host, which can (16) inactivate tumor suppressors and other genes. Together, these activities contribute significantly to tumor progression initiated by H. pylori. For more details and references, see text

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2 Genetic Predisposition of the Host, Induction of DNA Damage and Microbial Factors 2.1 Host Genetic Factors and Mutations in Gastric Cancer The majority of GC cases appears sporadically. However, there are also some hereditary variants of the disease, which are connected with a family history that is independent of H. pylori infection. Among them are the hereditary diffuse GC type, familial intestinal GC, familial adenomatous polyposis, gastric adenocarcinoma and proximal polyposis of the stomach, as well as the Lynch, Li–Fraumeni and Peutz– Jeghers syndromes (Correa 2013; Thrift et al. 2023). These rather rare GC forms correlate with mutations in crucial adherens junction components, most notably the E-cadherin gene CDH1 and α-Catenin gene CTNNA1, which control proper epithelial cell lining and link to the actin cytoskeleton (Blair et al. 2020; Gullo et al. 2021). Other involved mutations were detected in exon 1B of the APC (adenomatous polyposis coli) gene and in the IL12RB1 gene encoding interleukin 12 receptor subunit beta-1 (Li et al. 2016; Vogelaar et al. 2015). While the APC gene encodes a multifactorial protein with roles in the Wnt signaling cascade, cell cycle regulation, cell-to-cell adhesion, cytoskeletal regulation and apoptosis, IL-12 holds a crucial function in the interplay of innate and adaptive immune responses. For example, dendritic and phagocytic cells commonly produce IL-12 upon contact with microbial pathogens, which controls cytotoxic functions of NK cells and T cells, e.g. by producing IFNγ. In addition, infection with cagPAI-positive H. pylori provoked the anomalous expression of AID (activation-induced cytidine deaminase) in gastric epithelial cells via NF-κB activation (Matsumoto et al. 2007). This enzyme normally elicits somatic hypermutation in immunoglobulin genes, thus ensuring antibody diversification in B cells. In epithelial cells, AID expression leads to the generation of mutated protooncogenes such as TP53, MYC, or KRAS, associated with genetic instability and development of neoplasia (Matsumoto et al. 2007; Shimizu et al. 2014). Finally, recent studies on a large scale cohort of patients in Japan has shown the presence of 9 specific genetic germline variations of CDH1, MLH1, MSH2, MSH6 and APC genes as well as the BRCA1, BRCA2, ATM and PALB2 genes involved in DNA repair through homologous recombination (Usui et al. 2023). A possible risk mechanism for the latter genes is genome instability through reduced DNA damage repair capacity in the cells (Fig. 2). Together, these variants provoke a genetic predisposition that directly connects to GC appearance in H. pylori-positive persons. These data imply that in patients carrying either of the above pathogenic variants, the H. pylori infection should be assessed and eradicated.

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2.2 Common SNPs in Gastric Cancer Development In addition to the above described gene variants, single nucleotide polymorphisms (SNPs) in several genes were also found to correlate with GC development. SNPs represent natural genetic changes at the nucleotide level, which appear with different incidences among ethnic groups (McLean and El-Omar 2014). Various SNPs in specific genes have been described to modulate the mRNA expression and resulting protein function, correlating with gastric cancerogenesis. In particular, the first identified SNPs associated with an increased GC risk were characterized in the IL-1 gene cluster, comprising the IL-1α, IL-1β and IL-1RN genes (El-Omar et al. 2000). In the following years, gastric disease-associated SNPs were also described in dozens of other host factors involving chemokine and cytokine genes (e.g. IL-2, IL-6, IL-8, IL10, IL-17A, IFN-γ, TNF etc.), pattern recognition receptor (PRR)-associated genes (e.g. NOD1, NOD2, CD14, TLR1, TLR2, TLR4, TLR5, TLR9, MD-2, LBP, TIRAP, NLRX1 etc.), inflammasome-related genes (e.g. NLRP3, NLRP12, ASC, CARD8, CASP1 etc.), DNA repair-associated genes (e.g. ERCC2, OGG1, MSH2, MLH1, XRCC1, XRCC3 etc.), and others (Castaño-Rodríguez et al. 2014; McLean and ElOmar 2014; Pachathundikandi et al. 2015; Rudnicka et al. 2019). The correlation of SNPs and other mutations in immune-regulatory genes with GC development have been also investigated in regard to histological and anatomical subtypes, presence or absence of H. pylori and geographic region using meta-analysis and systematic review of the Human Genome Epidemiology (HuGE) project (Persson et al. 2011). In addition, genome-wide association studies (GWAS) were performed to pinpoint genes with SNPs in patients with different gastric disease outcomes (Badr et al. 2021; Tanikawa et al. 2018; Hishida et al. 2019). For example, 61 oncogenes and 37 specific other genes were identified in GWAS and validated against a compiled signature group of 55 genes to identify GC signaling pathways (Badr et al. 2021). Two GWAS reports examined SNPs in H. pylori genes in relation to GC development (Berthenet et al. 2018; Tuan et al. 2021). These studies revealed increased GC risk with SNPs in babA and some cagPAI genes, but their potential function remained unknown. Another study showed that an alanine to threonine point mutation in the EPIYA-B motif of oncoprotein CagA was inversely associated with GC risk, as this amino acid change reduced CagA phosphorylation, IL-8 production, and loss of polarity in host cells (Zhang et al. 2015). More recently, three GC-related SNPs were identified in H. pylori protease gene htrA (Sharafutdinov et al. 2023). One of these SNPs was studied in detail and showed that 171S to 171L mutation in HtrA enhanced its proteolytic activity on tumor-suppressor E-cadherin and occludin in the adherens and tight junctions, respectively. 171L-type HtrA (but not 171S-type)-expressing bacteria triggered impetuous cell disruption, enhanced delivery of CagA, increased pro-inflammatory responses and cell proliferation as well as massive induction of DSBs in the host genome (Sharafutdinov et al. 2023). Collectively, these observations emphasize the existence of cancer-related SNPs not only in host genes, but also in H. pylori, which could be used as potential new biomarkers for GC risk assessment during H. pylori infection.

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2.3 Role of Microsatellite Instability and EBV Infection Besides the above described GC-associated genetic variations, microsatellites distributed all-over the host genome are also susceptible to mutations. Microsatellites are short tandem DNA repeat sequences of up to 6 bases. Mutations in these microsatellite sequences can arise due to defects in the cellular mismatch repair (MMR) machinery during DNA recombination and replication, which can modify the number of these repeats (Vilar and Gruber 2010). This phenotype is called microsatellite instability (MSI) and represents another central feature of various sporadic GCs in the antrum (Baretti and Le 2018; Puliga et al. 2021). The MMR system comprises various DNA repair genes such as MLH1 and MLH3 (MutL homologs 1 and 3), PMS1 and PMS3 (post-meiotic segregation increased proteins 1 and 3) as well as MSH2, MSH3 and MSH6 (MutS homologs 2, 3 and 6), which excise and correct small nucleotide mispairings. Consequently, any failures in these MMR enzymes may result in the lack of successful DNA repair (Baretti and Le 2018; Puliga et al. 2021). Cancers carrying a strongly elevated MSI status have been assigned as MSIhigh (Boland and Goel 2010). These MSI-high cancers are commonly hypermethylated with a CpG island methylation level of about 10%, leading to downregulation of MLH1 gene expression (Cancer Genome Atlas Research Network 2014). By comparison, healthy gastric cells revealed a promoter CpG island methylation frequency of approximately 1–2%. In addition, MSI-high cancers are associated with specific germline mutations detected in the PMS2, MLH1, MSH2 or MSH6 genes (Boland and Goel 2010). Thus, both genetic and epigenetic aberrations are finally responsible for the occurrence of MSI-high cancers. Remarkably, although hyper-mutated, MSI-high tumors exhibit the best prognosis for patients and the smallest rate of reappearance after surgery (about 22%) compared to the other GC subtypes (Cristescu et al. 2015; Liu et al. 2017). In addition, MSI-high cancers have been characterized by elevated inflammation and other immune activities, thus may represent promising anti-cancer immunotherapy targets (Colotta et al. 2009; Kim et al. 2018). A growing body of evidence suggests that a substantial number of other gastric tumors arise due to infection with carcinogenic EBV (Hirabayashi et al. 2023). EBV belongs to the herpesviruses and carries a linear, double-stranded DNA genome of 172 kb, encoding about 85 genes. The virus triggers typical diffuse type GCs, which can be found in the cardia and corpus. EBV-positive GC exhibits various discrete epigenomic and genomic properties compared to other GC subtypes (Stanland and Luftig 2020). Infection with EBV typically establishes a CpG island hypermethylator phenotype with a frequency of about 19% (Cancer Genome Atlas Research Network 2014). This EBV-mediated hypermethylation in the human genome does not appear randomly. Similar to MSI-high-type gastric tumors, recurrent EBV targets are distinct DNA repair determinants such as MLH1, MSH2 and MSH6 genes (Stanland and Luftig 2020). Additionally, EBV-triggered hypermethylation downregulates dozens of other genes that enhance tumorigenesis, such as major tumor suppressors, cell differentiation regulators and cell cycle controlling proteins. For example, the EBV latent membrane protein LMP2A stimulates DNA methyltransferase (DNMT1)

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transcription through activating the nuclear STAT3 (Signal transducer and activator of transcription 3) signaling cascade (Hino et al. 2009). Subsequently, DNTM1 methylates the promoter of PTEN (Phosphatase and Tensin homolog), a dual-specificity phosphatase and tumor suppressor, thus activating the phosphatidylinositol 3-kinase (PI3K)-Akt signal transduction pathway (Hino et al. 2009; Luongo et al. 2019). In this way, β-catenin stabilization and activation of Wnt via PI3K-Akt signaling is achieved by EBV. In addition, several reports investigated frequently mutated genes in EBV tumors such as the tumor suppressor gene ARID1A (AT-rich interactive domain 1A), a subunit of the chromatin remodeling complex (Saito et al. 2020) as well as the amplification of gene copy numbers, e.g. in chromosome 9p24.1, which results in the constitutive expression of programmed death ligands PD-L1 and PDL2, as well as Janus kinase JAK2 (Cancer Genome Atlas Research Network 2014). It also appears that EBV encodes multiple miRNAs that exhibit critical functions in GC progression, which are currently under investigation (Kase et al. 2021). Finally, it has been repeatedly postulated that co-infection of EBV with H. pylori might have a possible role in disease outcome (Stanland and Luftig 2020; Zavros and Merchant 2022), which should be studied in more detail in the future.

2.4 Role of Bacteria-Triggered Oxidative Stress Responses In addition to the above described aberrations, oxidative stress is another important determinant of GC progression (Panieri and Santoro 2016; Liu et al. 2023). H. pylori-infected epithelial cells exhibit a strong pro-inflammatory response triggered by the T4SS, which attracts macrophages and neutrophils that produce reactive oxygen and nitrogen species (ROS and RNS) in vitro and in vivo (Sokolova and Naumann 2019). This scenario generates considerable oxidative stress both to the host and bacterium, with the primary goal to kill the intruding microbe. In particular, ROS and RNS irreversibly oxidize cellular biomolecules such as DNA and others. In this way, they exhibit direct DNA damaging capabilities due to crosslinking of DNA strands, induction of DNA single-strand breaks (SSBs) and the production of oxidized bases such as 8-hydroxy-2, -deoxyguanosine (8-OHdG), which is considered as a biomarker for oxidative DNA damage (Naumann et al. 2017). Consequently, these ROS- and RNS-mediated effects are responsible for damage of chromosomal and mitochondrial DNA in the host, associated with mutations and instability of both genomes, finally leading to tumorigenesis (Machado et al. 2013; Uehara et al. 2013). In this context, typical DNA oxidation products are G/C to T/A as well as A/T to G/C mutations, which have been detected in the gastric mucosa of mice infected with H. pylori (Sheh et al. 2010). To detoxify at least part of the ROS and RNS, the bacteria and host cells express certain anti-stress factors, including catalases, thiolreductases, superoxide dismutases and other enzymes (Toh and Wilson 2020). In contrast, a collection of host cell factors can even enhance ROS production, such as spermine oxidase (SMOX), NAPDH oxidase (NOX) and small Rho GTPase Rac1. These enzymes are activated by H. pylori infection and during GC development

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(Murray-Stewart et al. 2016; den Hartog et al. 2016; Tegtmeyer et al. 2021). In this context, a negative regulator of oxidative stress is the Apurinic/apyrimidinic (AP) endonuclease-reduction/oxidation factor 1 (APE1) by modulating Rac1 and NOX during H. pylori infection (den Hartog et al. 2016). In line with the above observations, miR-124 that normally acts as a potent suppressor of SMOX transcription and tumorigenesis, is diminished in gastric biopsies of cancer patients with H. pylori infection (Murray-Stewart et al. 2016). In vitro, infection with H. pylori induces the expression of SMOX in a CagA-dependent fashion, which triggers the oxidation of spermine to spermidine (Chaturvedi et al. 2011). This reaction also produces hydrogen peroxide (H2 O2 ) associated with oxidative stress, thus promoting DNA damage (Gobert and Wilson 2017). In addition, various other pathways have been described in the activation of oxidative stress such as the expression of the cation transport regulator 1 (Wada et al. 2018), expression of γ-glutamyl-transpeptidase that also generates H2 O2 and 8-OHdG in H. pylori-infected gastric epithelial cells (Gong et al. 2010), activation of NOX and ROS production by NapA (Neutrophilactivating protein A) of H. pylori (Satin et al. 2000), and the detection of 8-OHdG in serum samples of H. pylori-infected patients (Yeniova et al. 2015). In addition, it has been reported that chronic inflammation and oxidative DNA damage triggered by H. pylori cause the overexpression of poly (ADP-ribose) polymerase-1 (PARP-1) and shortening of telomers by the non-homologous end joining (NHEJ) recombination mechanism (Lee et al. 2016). Finally, oxidative stress induced in cells infected by H. pylori is ultimately connected to apoptotic signal transduction, as it results in elevated pro-apoptotic Bax and downregulated anti-apoptotic Bcl-2 expression in gastric epithelial cells (Sokolova and Naumann 2019). Taken together, H. pylori induces oxidative stress both in epithelial and immune cells that is connected to the onset of various signal transduction ways including DNA mutagenesis, important in gastric disease development.

2.5 Bacterial Induction of DNA Double-Strand Breaks Numerous publications have now documented convincingly that H pylori infection of gastric epithelial cells profoundly induces DSBs, associated with massive DNA damage in the nucleus (Toller et al. 2011; Hanada et al. 2014; Hartung et al. 2015; Koeppel et al. 2015; Bauer et al. 2020; Imai et al. 2021; Kolinjivadi et al. 2022). Early studies have demonstrated DSB-mediated chromosomal DNA damage by microscopic visualisation of metaphase chromosomes and pulsed field gel electrophoresis of linear cleavage products (Toller et al. 2011). The authors also showed that this genome damage triggered DNA repair reactions typical for DSBs as discussed below. The induction of DSBs was predominantly observed with cagPAIpositive clinical strains compared to less active cagPAI-negative isolates (Hanada et al. 2014). Screening of a H. pylori mutant library demonstrated a crucial function of T4SS genes for DSB stimulation (Hartung et al. 2015). Interestingly, inhibition of the pro-inflammatory transcription factor NF-κB abrogated the induction of

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DSBs, suggesting that mRNA production is somehow involved. Furthermore, two nucleotide excision repair enzymes (the endonucleases XPF and XPG) promoted the introduction of DSBs, which on the other hand activated NF-κB and cellular survival responses (Hartung et al. 2015). Koeppel and co-workers reported distinct DNA damage configurations during H. pylori infection, often near the telomers and in regions with strong transcriptional activity (Koeppel et al. 2015). Thus, such regions in the chromosome are more susceptible to genetic aberrations than others, which may promote GC development. A more recent study demonstrated that H. pylori promotes DSBs that arise in S-phase cells exhibiting strong NF-κB activity (Bauer et al. 2020). This signaling was initiated by T4SS-injected ADP-heptose, which activated the ALPK1 > TIFA > NF-κB signal transduction cascade. In this way, H. pylori triggered replication stress during formation of RNA/DNA-hybrids, socalled RNA-loops, during NF-κB-dependent transcription in S-phase cells (Bauer et al. 2020). Furthermore, the transcription factor USF1 was reported to stabilise tumor-suppressor p53, a guardian of genomic integrity, upon exposure to genotoxic stress. Interestingly, H. pylori induced USF1 downregulation, causing the deregulation of p53, and thus diminished genome stability (Costa et al. 2020). Infection of SMOX −/− knockout mice also demonstrated that this enzyme promotes DNA damage and inflammation (Sierra et al. 2020). In addition, transfected CagA inactivates partitioning kinase Par1b through direct interaction, thus preventing the phosphorylation of tumor suppressor BRCA1 (breast cancer gene 1), subverting its import in the nucleus (Imai et al. 2021). This scenario promotes BRCAness, a disease characterized by the deficiency of efficient DSB repair through homologous recombination. Furthermore, the interaction of intracellular CagA with PAR1b activated the Hippo signal transduction pathway that evades apoptosis to ensure survival of DSB-exposed cells (Imai et al. 2021). Taken together, H. pylori triggers DSBs in a T4SS-dependent manner, and when not properly repaired because of blocked DNA repair enzymes, this leads to accumulation of mutations in the genome that ultimately promote gastric carcinogenesis.

2.6 DNA Damage Repair Reactions As outlined above, H. pylori infection triggers DNA damage in the host in various ways, indirectly through the induction of oxidative stress and directly by introducing DSBs. In this way, DNA damage occurs and activates various responses such as the BER (Base excision repair) and MMR signaling cascades (den Hartog et al. 2016; Strickertsson et al. 2014). While the BER pathway represents a crucial machinery to repair oxidative DNA damage (van der Veen and Tang 2015), the MMR pathway repairs inaccurate deletion, insertion, and mispairing of bases (Basso et al. 2007). In addition, the DNA damage response (DDR) generally activates the ATM (Ataxia telangiectasia mutated) and ATR (Ataxia-telangiectasia and Rad3-related) DNA repair cascades (Priya et al. 2023). ATM and ATR kinases typically identify such DNA damage and stimulate signaling factors with roles in cell cycle checkpoint,

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cell death and DSB repair control. Homologous recombination (HR) and NHEJ represent two crucial mechanisms for the repair of DSBs. HR uses homologous sequences as template and occurs as an error-free DNA repair mechanism, while NHEJ is an error-prone trail, sometimes resulting in chromosomal translocations and deletions. Han and co-workers described that the long noncoding RNA SNHG17 functions as a decoy for micro RNA 3909, which in turn controls the mRNA expression of Rad51, shifting DSB repair from HR towards NHEJ during H. pylori infection (Han et al. 2020). Toller and co-workers described that H. pylori-induced genome damage triggered a typical DNA repair reaction through ATM, engagement of repair protein 53BP1 (p53-binding protein 1) and MDC1 (Mediator of DNA damage checkpoint protein 1) as well as phosphorylation of H2AX (Histone H2A variant protein X) (Toller et al. 2011). Further studies demonstrated the overexpression of ATM in infected gastric biopsies and in cultured cell lines (Hanada et al. 2014). In this way, DSBs are mostly repaired, for example after killing of the cell-attached bacteria using antibiotics. However, the DNA repair capacities are saturated during the course of infection (Toller et al. 2011). Using RNAseq and other methods, it was then shown that the H. pylori T4SS compromised various DNA repair enzymes at the transcriptional level (Koeppel et al. 2015). The authors compared the mRNA expression of 179 genes in the DDR cascades. They found 58 genes, such as ATR, p53, MLH1, and NBS1, to be downregulated by about twofold, and only a small fraction of 11 DDR genes was upregulated. H. pylori was also shown to downregulate the DNA repair proteins PMS2 and ERCC1 (Raza et al. 2020), and DNA glycosylase NEIL2, associated with increased DNA damage and inflammation (Sayed et al. 2020). Similar downregulation of DDR enzymes was reported to proceed in a CagA-dependent manner during infection (Kontizas et al. 2020). In addition, it was shown that ectopic expression of CagA downregulated the mRNA production of various crucial other DNA repair factors, including BRCA1, BRCA2, FANCI and FANCD2, which further contributes to genome instability and increased cancer risk (Kolinjivadi et al. 2022). Together, these experiments showed that H. pylori infection mainly suppresses DNA repair functions in host cells, thus enhancing DNA aberrations involved in GC development.

2.7 Therapeutic Options H. pylori infects the gastric mucosa of approximately 50% of the world’s population, but thankfully causes GC only in a small percentage of the infected people (1–2%). In addition to the genetic predisposition of the host and the genotype of the infecting pathogen, several environmental and cultural factors also affect the onset of malignant changes, such as diet, smoking and consumption of alcohol (Fig. 1). Cigarette smoke not only increases the risk for cancer in the respiratory system; several studies accumulated evidence for smoking causing GC (Karimi et al. 2014; Ladeiras-Lopes et al. 2008; Praud et al. 2018). When ingested, the carcinogenic substances in cigarette smoke can also irritate the gastric mucosa, and thus contribute to inflammatory stress. Risk of GC not only correlates with the duration of regular

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smoking, but also with the number of cigarettes smoked per day. Conversely, the risk of developing cancer decreases with time after quitting smoking (Praud et al. 2018). Likewise, frequent consumption of alcohol, particularly hard liquor, has been linked to GC through the alcohol by-product acetaldehyde that has been classified as a cancer-promoting agent. Alcohol exacerbates inflammation through increased generation of ROS, which contribute to the activation of carcinogens. In addition, frequent consumption contributes to folate deficiencies, and thus decreased protection against aberrant DNA methylation, which also increases cancer risk. Like smoking, the effect of alcohol was dose dependent (Jelski et al. 2008; Na and Lee 2017; Rota et al. 2017). Similarly, poor diet is correlated with H. pylori-associated GC, particularly high-salt diet intake and frequent intake of salt-preserved food, including processed meats (Morais et al. 2022; Wu et al. 2021). H. pylori and salt synergistically increase the expression of cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS), which are also thought to be involved in the promotion of GC development (Toyoda et al. 2008), and expression of H. pylori oncoprotein CagA was elevated during growth under high-salt conditions (Loh et al. 2007). Conversely, regular consumption of fruit and vegetables, particularly white vegetables, resulted in a decreased cancer risk (Fang et al. 2015, Kobayashi et al. 2002). Unfortunately, our modern life style is often associated with consumption of processed food, including cured meats, and salt-preserved foods, but inadequate amounts of fresh fruits and vegetables, and smoking is popular in many countries. Yet, a balanced diet that includes fresh fruits and vegetables, only moderate salt consumption, avoidance of excessive alcohol intake and the avoidance of tobacco smoking can greatly contribute to the prevention of GC by not exacerbating H. pylori-induced inflammation. Several studies showed that eradication of H. pylori infection, particularly in patients with gastritis, reduced the progression of precancerous lesions and lowered the risk of GC (Correa et al. 2000). For a more detailed discussion of treatment and surgery we refer the reader to chapter 11 of this book. Here, we focus on treatment with natural compounds such as antioxidants, because besides anti-H. pylori treatment, suppression of oxidative stress may be one of the key points for prevention and treatment. Thus, consumption of antioxidants that can potentially neutralize H. pylori-triggered ROS and RNS might be beneficial to the host. For example, canolol, a phenolic compound from crude canola oil, is known for scavenging oxygen radicals, which prevents the oxidation of canola oil (Galano et al. 2011). Analysis of the anti-inflammatory effects of canolol as food supplement on H. pylori infection in a Mongolian gerbil model revealed significantly attenuated gastritis, lower IL-1β, TNF, COX-2 and iNOS expression and lower gastrin levels in the gastric mucosa, and much fewer animals of the canolol-fed group developed gastric adenocarcinoma compared to the control group, showing that canolol effectively suppressed inflammation, cell proliferation and carcinogenesis (Cao et al. 2008). Likewise, curcumin, a polyphenol from turmeric, has potent anti-inflammatory, anti-oxidant, and anti-tumour effects with pleiotropic properties (Kwiecien et al. 2019). Curcumin increases the expression of catalase and superoxide dismutase, both of which have anti-oxidative effects by neutralizing ROS, and decreases expression of pro-inflammatory markers such as TNF, COX-2, iNOS and NF-κB (Czekaj et al. 2016; Morsy and El-Moselhy 2013).

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Thus, by regulation of several cell signaling pathways, curcumin inhibits cancer proliferation. These pathways include cell proliferation cascades (e.g. c-myc, cyclin D1, NF-κB), cell survival pathways (e.g. Bcl-2, Bcl-xL, c-IAP1), caspase activation, tumor suppressor pathways (p53, p21), death receptor (DR4, DR5), as well as the modulation of tyrosine kinases (e.g. JNK, Akt, AMPK) (Ahmad et al. 2016; Bahrami and Ferns 2021; Kwiecien et al. 2019). Other natural anti-inflammatory compounds are capsaicin from chillies and piperine from peppers, both of which were shown to reduce expression of TNF and to inhibit in vitro proliferation of H. pylori-infected gastric cells (Toyoda et al. 2016). Along with the pro-inflammatory cytokines, capsaicin reduced NF-κB activation and the expression of NF-κB-dependent miRNAs mir21 and mir223 that are reported to be upregulated in GC (Saha et al. 2022), while piperine suppressed the expression of IL-1β, IFN-γ, IL-6, and iNOS (Toyoda et al. 2016). Cinnamaldehyde from cinnamon exhibits anti-inflammatory effects on H. pylori-infected gastric epithelial cells by suppressing H. pylori-induced activation of NF-κB and IL-8 (Muhammad et al. 2015). Given that active inflammation is an important milestone in the progression to GC, inhibition or at least delay of the process is an important checkpoint for the outcome of the disease. Not surprisingly, those and other natural compounds are part of the traditional medicine for the treatment of a multitude of symptoms, including the particular illness of the digestive tract. Over the past years a few synthetic cancer inhibitor drugs were approved, some of which are suitable for the treatment of GCs. Among those are PARP inhibitors, drugs that target PARP-1 that is overexpressed following H. pylori-triggered oxidative DNA damage and chronic inflammation (Lee et al. 2016). PARP inhibitors such as the FDA-approved olaparib (Bochum et al. 2018) competitively inhibit NAD+ at the catalytic site of PARP1 and PARP2. Since those enzymes are essential for the above discussed BER pathway, particularly to the repair of SSBs in the DNA, inhibition of this pathway triggers the accumulation of SSBs, which progresses to the formation of DSBs. While those DSBs are usually repaired via the HR pathway, cells that lost the function of BRCA1/2 (HR-deficient cells) cannot repair the damage, which leads to cell apoptosis. PARP inhibitors further inhibit the repair by NHEJ, which also results in cell death (Gupta et al. 2018). In addition, PARP inhibitors are thought to hamper the Microhomology-mediated end joining (MMEJ) repair pathway, which may contribute to the death of cancer cells that are unable to perform the DNA repair (Tomasini et al. 2021). Finally, Olaparib, which is administered orally in GC patients, can activate DDR pathways as well as reactivate DNA checkpoints, and can be combined with other substances to improve treatment efficiency (Wang and Xie 2022).

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3 Concluding Remarks The role of host cell DNA damage during the onset of GC is developing into a complex picture. Most of the processes appear to be long-term, and gastric complications usually develop at an advanced age. While the emerging picture is already exhibiting multiple facets of the interaction between the invading pathogen and the host epithelium, it is increasingly clear that our puzzle is still far from complete. Despite the exceptional progress that has been made over the past decade towards the understanding of the mechanisms of cancer, there is still a long road ahead. Further studies on the mechanisms of H. pylori-triggered cancer development, including on the importance and repair of SSBs and DSBs, are essential to move the field forward. For example, further experiments are necessary to clarify the exact DDR mechanisms in H. pylori-associated development and progression of the disease. This will pave the way for the development of better therapeutics and treatment options, including targeted therapies for individual patients. Ultimately, the orchestrated efforts of scientists, physicians and public health authorities in prevention, screening, research and treatment of GC will lead to a decline in the incidence of the disease. Acknowledgements This work was supported by grants of the Deutsche Forschungsgemeinschaft (DFG) to S.B. (BA 1671/16-1) and N.T. (TE776/3-1).

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Gastric Epithelial Barrier Disruption, Inflammation and Oncogenic Signal Transduction by Helicobacter pylori Michael Naumann, Lorena Ferino, Irshad Sharafutdinov, and Steffen Backert

Abstract Helicobacter pylori exemplifies one of the most favourable bacterial pathogens worldwide. The bacterium colonizes the gastric mucosa in about half of the human population and constitutes a major risk factor for triggering gastric diseases such as stomach cancer. H. pylori infection represents a prime example of chronic inflammation and cancer-inducing bacterial pathogens. The microbe utilizes a remarkable set of virulence factors and strategies to control cellular checkpoints of inflammation and oncogenic signal transduction. This chapter emphasizes on the pathogenicity determinants of H. pylori such as the cytotoxin-associated genes pathogenicity island (cagPAI)-encoded type-IV secretion system (T4SS), effector protein CagA, lipopolysaccharide (LPS) metabolite ADP-glycero-β-Dmanno-heptose (ADP-heptose), cytotoxin VacA, serine protease HtrA, and urease, and how they manipulate various key host cell signaling networks in the gastric epithelium. In particular, we highlight the H. pylori-induced disruption of cell-tocell junctions, pro-inflammatory activities, as well as proliferative, pro-apoptotic and anti-apoptotic responses. Here we review these hijacked signal transduction events and their impact on gastric disease development.

M. Naumann (B) · L. Ferino Institute of Experimental Internal Medicine, Medical Faculty, Otto Von Guericke University, Leipziger Str. 44, 39120 Magdeburg, Germany e-mail: [email protected] L. Ferino e-mail: [email protected] I. Sharafutdinov · S. Backert (B) Dept. Biology, Division of Microbiology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstr. 5, 91058 Erlangen, Germany e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 S. Backert (ed.), Helicobacter pylori and Gastric Cancer, Current Topics in Microbiology and Immunology 444, https://doi.org/10.1007/978-3-031-47331-9_8

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1 Introduction H. pylori is a human-specific pathogen that inhabits the hostile environment of the stomach in approximately 50% of the world population (Malfertheiner et al. 2023). The majority of individuals are infected by the bacterium early in childhood. Without intervention such as antibiotic therapy, colonization can persist for decades. Although infection by the pathogen is asymptomatic in most persons, a small subgroup of infected individuals can develop gastric disorders that include chronic gastritis, peptic ulceration and stomach cancer (Thrift et al. 2023; Zavros and Merchant 2022). The histological progression of H. pylori infection from early stages of chronic inflammation proceeds through the occurrence of metaplasia, dysplasia up to gastric cancer (GC) (Correa and Houghton 2007). In about 10 to 20% of colonized people the infection eventually results in ulcer disease, while 1 to 2% develop GC and in Abl > Cortactin > Vav2 pathway leading to Rac1 activation and actin polymerisation (Sharafutdinov et al. 2021a; Tegtmeyer et al. 2021a; Knorr et al. 2021). Additionally, it was shown that CagA interacts with p120-catenin and receptor c-Met, resulting in the suppression of the cell-invasive phenotype (Oliveira et al. 2009). However, the proposed CagAtriggered deregulation of AJs with regard to β-catenin activation is controversial. For example, disruption of E-cadherin in the AJs in cultured cells was also seen during infection with ΔcagA, ΔcagE and ΔvacA mutants (Bebb et al. 2006; Sokolova et al. 2008; Weydig et al. 2007). In addition, suppression of β-catenin phosphorylation through the kinases Akt1 and GSK-3β and TCF/LEF-dependent transcription (Fig. 3b) was reported as a process dependent on the cagPAI, but not cagA (Sokolova et al. 2008). Similarly, nuclear translocation of p120-catenin required the cagPAI, but not cagA, and relieved Kaiso-triggered repression of mmp-7 mRNA (Ogden et al. 2008). Thus, further studies are required to pinpoint the exact functions of CagA in this scenario.

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Fig. 3 H. pylori infection-mediated control of β-catenin signaling. a T4SS-mediated injection of H. pylori CagA induces the formation of the E-cadherin-CagA complex (1). The adherens junction (AJ) component β-catenin is subsequently released from the AJs, followed by its nuclear translocation (2). In the nucleus, β-catenin promotes TCF/LEF-dependent transcription of cell proliferation genes including proto-oncogenes such as c-myc and cyclin d1 (3). This activity can be augmented through nuclear translocation of p120-catenin (ctn). In the nucleus, p120ctn binds Kaiso, which relieves Kaiso-dependent suppression of TCF/LEF (4). In addition, H. pylori activates the Wnt signaling cascade, comprising adenomatous polyposis coli (APC), axin, glycogen synthase kinase 3 beta (GSK-3β) and casein kinase 1 (CK1), which control β-catenin functions through its phosphorylation, nuclear recruitment and disintegration (5). b An important other H. pylori factor, OipA, was described to mediate the conversion of the lipid phosphatidylinositol-4,5-biphosphate (PIP2) to phosphatidylinositol-3,4,5-triphosphate (PIP3) through EGF receptor and PI3-kinase activation (6). This signaling promotes 3-phosphoinositide dependent protein kinase-1 (PDK1) recruitment to the plasma membrane, which stimulates Akt kinase activity associated with inhibition of phosphatase PTEN (7). Akt upregulates the expression of proto-ongene MDM2, a nuclear E3 ubiquitinligase, which regulates the degradation of tumor suppressor p53 (8). Further, Akt can stabilize β-catenin through downregulation of GSK3β-mediated phosphorylation of β-catenin (9). Activated Akt phosphorylates FOXO1/3 followed by their nuclear translocation, where they act as transcriptional controllers, inhibiting pro-apoptotic genes (10). Finally, Akt can activate the mTOR complex 1 (mTORC1), maintaining host protein biosynthesis via the translation regulators eEF2and 4E-BP1 (11). In turn, Akt and mTORC1 functions are regulated by the kinase AMPK as indicated (12). In addition, injected CagA can activate Rac1-dependent actin polymerization via interactions with Abl kinase, actin-binding protein cortactin and guanine exchange factor Vav2, resulting in cell motility and enhanced disruption of AJs (13). For more details see text

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2.4 Regulation of Oncogenic Signaling in Epithelial Cells The above discussed H. pylori-induced epithelial cell damage is intimately connected with strong pro-inflammatory responses. The cag T4SS has been described as the major inducer of different signaling pathways. Some of the activated pathways, such as NF-κB and mitogen-activated protein kinase (MAPK) signaling, including c-Jun N-terminal kinase (JNK) and p38 have been disclosed mechanistically (Fig. 4a,b). The most prominent T4SS-induced signaling is represented by the classical and alternative NF-κB cascades (Fig. 4a), which lead to the transcription of target genes that directly affect inflammation and cell survival such as IL-8 or cellular inhibitor of apoptosis protein 2 (cIAP2), respectively (Sokolova et al. 2013; Lim et al. 2022). In order to reveal the mechanism of H. pylori-triggered NF-κB activation, it was first shown that the translocation of heptose-1, 7-bisphosphate (HBP) into host cells via the T4SS induces alpha-protein kinase 1 (ALPK1) and NF-κB signaling (Gall et al. 2017; Stein et al. 2017; Zimmermann et al. 2017). In further studies, however, it was demonstrated that instead of HBP, another intermediate LPS metabolite, ADP-heptose, is the main effector to trigger NF-κB activation in response to infection with various Gram-negative pathogens (Zhou et al. 2018; Pfannkuch et al. 2019). Once translocated into the host cell, ADP-heptose is recognized by the serine/threonine kinase ALPK1 (Zhou et al. 2018) that phosphorylates tumor necrosis factor receptor-associated factor (TRAF)-interacting protein with forkheadassociated domain (TIFA) at threonine 9 causing TIFA oligomerization (Stein et al. 2017; Maubach et al. 2021; Garcia-Weber et al. 2023). Binding of the TNF receptor associated factor 2 (TRAF2) or TRAF6 to TIFA finally leads to classical or alternative NF-κB activation (Maubach et al. 2021). TIFA-associated TRAF6 forms K63-linked polyubiquitin chains that serve as a scaffold for the recruitment of transforming growth factor TGF-β-activated kinase 1 (TAK1) via the TAK1-binding proteins 2 and 3 (TAB2/3) during H. pylori infection (Fig. 4a). Activated TAK1 in turn activates the inhibitor of the nuclear factor κB kinase (IKK) complex (Maubach et al. 2021). IKK-dependent phosphorylation and degradation of IκBα allows nuclear translocation of the dimeric transcription factor RelA/p50, which leads to target gene transcription (Sokolova et al. 2013). In addition to TAK1, the mitogen-activated protein kinase kinase kinase 3 (MEKK3) and tyrosine kinase Src (c-Src) are also activated upon H. pylori infection and phosphorylate IKKβ to activate NF-κB (Sokolova et al. 2014; Rieke et al. 2011). Although the detailed mechanisms of kinase activation remain unclear, it was demonstrated that MEKK3 activation is strictly T4SS-dependent (Sokolova et al. 2014), and T4SS-dependent engagement of the actin-binding protein cortactin seems to be required for c-Src activation (Knorr et al. 2021; Tegtmeyer et al. 2021b). Apart from classical NF-κB, ADP-heptose also triggers the alternative NF-κB pathway (Fig. 4a). Thereby, interaction of TRAF2 and TIFA leads to the displacement of cIAP1 from TRAF2 within the NF-κB-inducing kinase (NIK) regulatory complex, followed by transient cIAP1 degradation. The NIK regulatory complex consists of TRAF2, TRAF3, NIK and either cIAP1 or cIAP2. The E3 ubiquitin ligases cIAP1/2

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Fig. 4 Regulation of transcription factor NF-κB activity and MAP kinase pathways during infection with H. pylori. a T4SS-dependent translocation of ADP-heptose (ADPH) induces ALPK1 activation and TIFA oligomerization, which in turn leads to the induction of classical or alternative NF-κB signaling (1). In classical NF-κB, TIFA induces TAK1, which phosphorylates IKKβ within the IKK complex, which is built up by IKKα, IKKβ and NEMO (2). IKKα then phosphorylates IκBα, leading to its ubiquitinylation and proteasomal degradation. Therefore, the dimeric transcription factor RelA/p50 is translocated into the nucleus and induces target gene transcription. In alternative NF-κB signaling, TIFA drives the accumulation of NIK, which then phosphorylates IKKα, which in turn phosphorylates p100 (3). The phosphorylation of p100 serves as signal for its proteasome-dependent processing into p52. The RelB/p52 translocates into the nucleus, where it induces target gene transcription. b H. pylori infection induces MAPKKK activation, which leads to the phosphorylation of MAPKKs. Amongst these MAPKKs, MKK4 and MKK7 phosphorylate JNK, MKK3 and MKK6 phosphorylate p38, while MEK1 and MEK2 phosphorylate ERK (1). The activated MAP kinases JNK, p38 and ERK translocate into the nucleus where they phosphorylate the transcription factors c-Jun, ATF-2 and TCF, respectively (2). This leads to the transcription of further transcription factors of the Fos and Jun families (3). Upon c-Jun phosphorylation, the heterodimeric AP-1 complex is formed by c-Jun:c-Fos. AP-1 then binds to the AP-1 site within the DNA to induce target gene transcription (4). For more details see text

control NIK turnover by constitutive K48-polyubiquitinylation. Accordingly, cIAP1 depletion leads to the accumulation of NIK in the cytoplasm to advance alternative NF-κB signaling by activation of IKKα (Maubach et al. 2021) (Fig. 4a). IKKα mediated phosphorylation of p100 causes its proteasome-dependent processing into p52.

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Thereafter, RelB/p52 dimers translocate into the nucleus and induce the transcription of anti-apoptotic genes (Feige et al. 2018; Lim et al. 2022). In addition to NF-κB, H. pylori infection activates the transcription factor activator protein-1 (AP-1), which controls cell proliferation, apoptosis and immune responses (Naumann et al. 1999). The dimeric AP-1 transcription factor comprises different subfamilies, out of which Fos:Jun dimers display the highest transactivation capacity. Transcriptional activation and DNA binding of AP-1 are regulated by the MAPK pathway (Atsaves et al. 2019). Stimulation of G protein-coupled receptors (GPCR) by certain environmental factors, such as pro-inflammatory cytokines, activate Rho GTPases, leading to the phosphorylation of a mitogen-activated protein kinase kinase kinase (MAPKKK), like p21 activated kinase (PAK1), apoptosis signal-regulating kinase 1 (ASK1) or TAK1. These kinases activate mitogen-activated protein kinase kinases (MAPKK), which specifically phosphorylate mitogen-activated protein kinases (MAPK). The MAPKs are grouped into extracellular signal-regulated kinases (ERK) and stress-activated JNK as well as p38. While ERKs are activated by MEK1 and MEK2, JNKs are phosphorylated by MKK4 and MKK7, and p38 is targeted by MKK3 and MKK6 (Fig. 4b). Activated MAPKs are translocated into the nucleus where they phosphorylate target proteins like AP-1 transcription factors (Garcia-Hernandez et al. 2022). In regard to H. pylori infection, it was demonstrated that JNK and p38 signaling are T4SS-dependent, while ERK signaling relies on other H. pylori-mediated stimuli (Al-Ghoul et al. 2004). In general, it was observed that H. pylori infection not only triggers AP-1 activation, but also the expression of several other AP-1 transcription factors such as c-Jun, JunB, JunD, c-Fos and Fra-1 (Ding et al. 2008). As for AP1 activation, H. pylori T4SS triggers the activation of the Rho GTPases Rac1 and Cdc42, which in turn stimulates PAK1 (Churin et al. 2001). Activated PAK1 probably activates MKK4, which was shown to phosphorylate JNK upon H. pylori infection. Active JNK phosphorylates c-Jun, leading to the induction of AP-1 (Naumann et al. 1999). Apart from PAK1, Hayakawa et al. (2013) demonstrated a reciprocal interaction of the stress-activated protein kinases TAK1 and ASK1 in JNK signaling. Thereby, a rapid activation of p38 via TAK1 inhibited the formation of reactive oxygen species (ROS) to prevent ROS-dependent activation of ASK1. However, ROS-triggered activation of ASK1 caused prolonged JNK signaling to induce apoptosis of infected cells. While studying the impact of H. pylori infection in the adult gastric epithelial cell line GES-1 as well as human stomach fetal epithelium (HSFE), Yang et al. (2022) observed differential JNK and p38 activation in children compared to adults. They demonstrated a higher JNK activation in the adult tissue accompanied by elevated Lewis antigen expression and bacterial colonization. In contrast, fetal epithelial cells showed pronounced p38 signaling, which suppressed JNK signaling. Therefore, Lewis antigen expression and bacterial colonization was lower in fetal epithelium. Other studies revealed that activation of JNK and p38 signaling triggers AP1 activation in infected gastric epithelial cells, causing the upregulation of matrix metalloproteinases MMP-3, -9 and -10 (Bae et al. 2021; Karayiannis et al. 2023). This finding is corroborated by immunohistochemical analysis of human samples

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from H. pylori-associated gastritis revealing elevated MMP-10 expression compared to healthy tissue (Lv et al. 2019). Since MMP-10 cleaves several extracellular matrix components, as well as other pro-MMPs, its upregulation can lead to the formation of gastric lesions. Interestingly, pre-treatment of cells with β-carotene prior to H. pylori infection inhibited the activation of JNK and p38, and prevented MMP-10 expression (Bae et al. 2021). A recent study by Sharafutdinov et al. (2021b) demonstrated that cortactin overexpression in AGS cells is induced via JNK signaling and depends on a functional T4SS.

2.5 Apoptotic Cell Death Signaling Programmed cell death has crucial physiological functions that include organ maintenance by tissue renewal or immune cell selection. However, extensive cell death is triggered upon infection, in chronic inflammation or during tissue damage and may have deleterious consequences. The main pathway of programmed cell death is apoptosis, which can be mediated via the extrinsic or intrinsic pathway (Fig. 5). The intrinsic pathway is triggered by toxic reagents or DNA damage, and leads to mitochondrial outer membrane permeabilization (MOMP) mediated by proapoptotic proteins, like Bcl-2-associated X protein (BAX), Bcl-2 homologous antagonist (BAK) or p53 upregulated modulator of apoptosis (PUMA). Consequently, the socalled apoptosome is formed by the cytoplasmic cytochrome c, procaspase-9 and activator factor-1 (Apaf-1) (Bertheloot et al. 2021). This leads to the activation of caspase-9, which in turn activates the executor caspases-3 and -7. The extrinsic pathway, in contrast, is initiated upon binding of death ligands to their correspondent death receptors. This induces oligomerization of death receptors. The subsequent recruitment of adaptor proteins as well as initiator caspases-8 and -10 induce caspase activation. The initiator caspases then activate caspases 3, -6, and -7 followed by cell death (Bertheloot et al. 2021). Depending on the trigger, H. pylori-induced apoptosis is mediated either via the intrinsic or extrinsic pathway (Fig. 5). The virulence factor VacA is a main trigger for the intrinsic apoptosis pathway as it disrupts mitochondrial integrity causing MOMP, which leads to cytochrome c release (Galmiche et al. 2000; Oldani et al. 2009). The counteracting pro- and anti-apoptotic cell signaling cascades in response to H. pylori infection, discussed in the following sections, pose an enigma of the H. pylori-mediated effects on gastric mucosal injury (Lim et al. 2023).

2.5.1

Intrinsic Apoptosis

It was reported that H. pylori triggers intrinsic apoptosis by the induction of ROS formation, endoplasmic reticulum stress response and PUMA expression (Dang et al. 2020; Chaithongyot and Naumann 2022; Zhu et al. 2017) (Fig. 5). PUMA is a critical initiator of the intrinsic apoptosis pathway, as it causes mitochondrial membrane

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Fig. 5 Apoptotic cell death signaling induced by H. pylori infection. H. pylori infection regulates intrinsic and extrinsic apoptosis pathways to either induce or suppress apoptosis. Concerning the intrinsic apoptosis pathway, H. pylori induces the upregulation of PUMA (1), the formation of reactive oxygen species (ROS), (2) and delivery of the bacterial virulence factor VacA (3). PUMA, ROS as well as VacA induce MOMP. As a result, released cytoplasmic cytochrome C engages Apaf1 and caspase-9 to form the apoptosome, causing the onset of apoptosis (4). In contrast, translocation of CagA induces PI3K, followed by PKB1 activation. PKB1-mediated phosphorylation of XIAP induces its E3 ubiquitin ligase activity, leading to the ubiquitinylation and proteasomal degradation of the anti-apoptotic protein Siva1 (5). Moreover, sequential binding of CagA to Siva1 and ULF enhances ULF-dependent ubiquitinylation and degradation of the pro-apoptotic protein ARF (6). Both CagA dependent mechanisms suppress the onset of apoptosis via the intrinsic pathway. Further, ADP-heptose (ADPH) triggers the nuclear translocation of RelB/p52, followed by the transcription of anti-apoptotic target genes (7). Moreover, ADPH induces nuclear translocation of RelA/p50. USP48-mediated deubiquitinylation of RelA stabilizes nuclear RelA levels to prolong transcription of TNFAIP3, which encodes the deubiquitinylase A20 (8). A20-mediated deubiquitinylation of caspase-8 ultimately inhibits apoptosis (9). H. pylori also triggers the activation of PKC, which phosphorylates c-Abl. Phosphorylated c-Abl interacts with 14-3-3 leading to the suppression of c-Abl-mediated cleavage of caspases-8, preventing apoptosis (10). For more details see text

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permeabilization (Bertheloot et al. 2021). The immunohistochemical analysis of human gastric tissue displayed elevated levels of PUMA expression in injured tissue compared to healthy tissue. It was further demonstrated that H. pylori infection induces PUMA expression via the classical NF-κB pathway in AGS cells and mice. Interestingly, knockdown of PUMA in AGS cells and mice prevented H. pyloriinduced apoptosis. Hence, PUMA is a critical contributor to the pathogenesis of H. pylori-mediated gastritis by promoting apoptosis in infected gastric epithelial cells (Dang et al. 2020). An early response to the infection with microbial pathogens is the formation of ROS (Andrieux et al. 2021). Apart from its antimicrobial role, ROS also induces DNA damage in host cells that eventually causes oncogenic mutations. In order to prevent the proliferation of oncogenic cells, apoptosis is induced via the intrinsic pathway (Lim et al. 2023). Interestingly, the formation of ROS was prompted in AGS and NCI-N87 cell lines upon H. pylori infection, which was attributed to cullin-1 dependent K48 polyubiquitinylation and degradation of the deubiquitinylase STAM-binding protein like 1 (STAMBPL1) (Chaithongyot and Naumann 2022). Due to the increased proteasomal degradation of STAMBPL1, its anti-apoptotic target gene survivin was destabilized, causing an increase in apoptosis (Chaithongyot and Naumann 2022). Zhu et al. (2017) reported that the vacuolating cytotoxin (VacA) induces endoplasmic reticulum stress response, followed by autophagy and apoptosis. There, it seems like autophagy is upstream of intrinsic apoptosis since treatment of infected cells with autophagy inhibitor 3-methyladenine or downregulation of autophagy effector proteins protected cells from apoptosis. Interestingly, transfection of VacA (Oldani et al. 2009) or treatment of cells with recombinant VacA (Yuan et al. 2021) is sufficient for the induction of apoptosis. Palrasu et al. (2020) reported an involvement of PI3K/Akt signaling in inhibition of H. pylori-associated apoptosis (Fig. 5). Initially they observed a downregulation of the pro-apoptotic protein Siva1 in the gastric epithelium of infected mice. Mechanistically, they revealed that CagA-dependent activation of the PI3K/Akt pathway leads to phosphorylation of the E3 ligase X-linked inhibitor of apoptosis protein (XIAP) at serine 87. Phosphorylated XIAP mediates the ubiquitinylation of its substrate protein Siva1, leading to its proteasomal degradation. In contrast, depletion of XIAP in infected epithelial cells stabilized Siva1 protein levels. Furthermore, siRNA-mediated Siva1 downregulation inhibited intrinsic apoptosis and DNA damage responses (Palrasu et al. 2020). An extension of this work demonstrated that H. pylori infection also causes the downregulation of ADP-ribosylation factor (ARF), a key regulator of apoptosis (Palrasu et al. 2022). Interestingly, ARF downregulation correlated with the upregulation of the E3 ubiquitin ligase for ARF (ULF) and downregulation of Siva1. Herein, sequential binding of CagA to the E3 ligases Siva1 and ULF targets ARF for proteasomal degradation, preventing infected cells from apoptosis (Palrasu et al. 2022). Thus, CagA mediates the degradation of the pro-apoptotic proteins Siva1 and ARF by enhanced activation of their corresponding E3 ubiquitin ligases XIAP and ULF.

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Extrinsic Apoptosis

The bacterial virulence factors T4SS and CagA were shown to be crucial mediators of pro- as well as anti-apoptotic mechanisms. In a study by Lim et al. (2022), H. pylori infection of gastric epithelial cells induced the activation of the dimeric transcription factor RelB/p52 via the alternative NF-κB pathway. Activation of RelB/ p52 triggered the expression of the anti-apoptotic genes baculoviral IAP repeat containing 2 (BIRC2), BIRC3 and B-cell lymphoma 2-related protein A1 (BCL2A1). Interestingly, cells depleted in the deubiquitinylase A20 augmented the activation of alternative NF-κB signaling and contained increased amounts of anti-apoptotic proteins compared to wt cells. Immunoprecipitation of A20 with the components of the NIK regulatory complex unveiled that H. pylori infection promotes the binding of A20 to TIFA. The following TIFA displacement from the NIK regulatory complex restores the ability of NIK-degradation and leads to shutdown of alternative NF-κB signaling. Accordingly, A20 disrupts alternative NF-κB signaling and promotes H. pylori-associated apoptosis (Lim et al. 2022). T4SS- and NF-κB-dependent regulation and release of death receptor ligands such as TNF-related apoptosis-inducing ligand (TRAIL) leads to activation of caspase 8 and extrinsic apoptosis (Lin et al. 2014). However, it was recently observed that the nuclear deubiquitinylase USP48 interacts with constitutive photomorphogenesis 9 signalosome subunit 1 (CSN1) to specifically remove K48-polyubiquitin chains from RelA. Consequently, nuclear RelA is protected from proteasomal degradation, and RelA/p50 target gene transcription is prolonged, followed by de novo protein synthesis (Jantaree et al. 2022a). A20, one of the newly synthesized proteins, removes K63-polyubiquitin chains from its target protein procaspase-8. The removal of the polyubiquitin chain prevents the autocatalytic cleavage of procaspase-8 into active caspase-8, which itself averts apoptosis (Lim et al. 2017). The importance of USP48 was emphasized by the fact that USP48 knockdown AGS cells had increased numbers of apoptotic cells (Jantaree et al. 2022b). In addition, a recent study using RNAseq revealed a putative role of H. pylori HtrA in mediating the anti-apoptotic host response, probably via more efficient CagA delivery into the host cells (Sharafutdinov et al. 2022). Anti-apoptotic mechanisms in H. pylori infection have also been shown by Posselt et al. (2019) who reported that H. pylori regulates the phosphorylation of the nonreceptor tyrosine kinase c-Abl by activation of protein kinase C (PKC). Phosphorylation at threonine 735 of c-Abl induces an interaction with 14-3-3 that leads to the retention of fully activated c-Abl in the cytoplasm, causing cytoskeletal rearrangements such as cell elongation and migration. Interestingly, the nuclear exclusion of phosphorylated c-AblT735 prevented the activation of caspase-8, averting the onset of extrinsic apoptosis and eventually activates the intrinsic apoptosis pathway and initiator caspases in a feedback loop.

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2.6 Impact of the Microenvironment on Gastric Carcinogenesis The tumor cells and the surrounding stroma, which includes non-cancerous epithelial and endothelial cells, immune cells, blood cells, fibroblasts and the extracellular matrix, constitute the tumor microenvironment (Navashenaq et al. 2022). The crosstalk of tumor cells with fibroblasts leads to the development of cancer-associated fibroblasts (CAFs) that promote carcinogenesis. The release of cytokines and TGFβ from tumor cells induce the transformation of fibroblasts into CAFs, which on their own release cytokines that activate a strong inflammatory response in the tumor environment. Moreover, matrix metalloproteases produced by CAFs degrade the extracellular matrix and cause epithelial-mesenchymal transition (EMT) and metastasis (Navashenaq et al. 2022). As H. pylori infection modulates the crosstalk of gastric epithelial cells and fibroblasts in the inflammatory milieu, infection might thereby contribute to carcinogenesis (Jantaree et al. 2022a; Krzysieka-Maczka et al. 2020; Shen et al. 2020). Co-culture of gastric fibroblasts with gastric epithelial cells suppressed apoptosis in epithelial cells in an NF-κB-dependent manner (Jantaree et al. 2022a). Although the mediators of the cellular crosstalk remain to be identified, it was already revealed that CSN-associated USP48 directs NF-κB/RelA stabilization allowing for the prolonged transcription of target genes, like A20, in gastric epithelial cells. Further, the A20-mediated deubiquitinylation of caspase-8 suppresses apoptosis (Jantaree et al. 2022b). Krzysiek-Maczka et al. (2020) tested the impact of released factors from fibroblasts on epithelial cells by H. pylori infection of rat fibroblasts for 72 h. After the removal of bacteria, fibroblasts were cultivated for additional 96 h before supernatants of these fibroblasts (herein referred to as H. pylori-AGF supernatant) were collected. Interestingly, growth of rat gastric epithelial cells for 24 h in the H. pylori-AGF supernatant induced morphological changes in the epithelial cells towards an elongated mesenchymal-like shape with long and thin protrusions. It was shown that the observed EMT-like changes in non-cancerous epithelial cells were stimulated by the release of TGF-β from fibroblasts upon H. pylori infection. Moreover, the H. pylori-AGF supernatant induced the reprogramming of non-cancerous rat gastric epithelial RGM1 cells into Lgr5+ /Oct4+ stem-like neoplastic cells. In general, Lgr5+ cells display stem-like features, while Oct4 controls the pluripotency and dedifferentiation of cells (Krzysiek-Maczka et al. 2020). Thus, the impact of H. pylori on fibroblasts intensifies the malignant transformation of the gastric epithelium. In addition to modulation of gastric epithelial cells by factors secreted by fibroblasts, cells in the tumor microenvironment have also been shown to interact directly with fibroblasts. The vascular adhesion molecule 1 (VCAM1) was first identified as an adhesion receptor expressed on endothelial cells that was required for the recruitment of immune cells (Osborn et al. 1989). Interestingly, VCAM1 expression is upregulated in gastric fibroblasts via the JAK/STAT pathway upon H. pylori infection. VCAM1 expressed on the fibroblast cell surface interacts with integrin αvβ1/ 5 expressed on GC cells. The direct interaction of both cell types seems to promote

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tumor invasion, as gastric epithelial cells were more invasive in vitro and in vivo in the presence of VCAM1 expressing CAFs compared to VCAM1-knockdown CAFs (Shen et al. 2020). In addition to fibroblasts, infiltration of immune cells also contributes to H. pyloriassociated gastric carcinogenesis. It was shown that the expression of insulin growth factor binding protein 7 (IGFBP7) correlates with the progression of GC and is upregulated upon TGF-β treatment of cells (Jin et al. 2020). A study by Zhao et al. (2021) showed that the expression of IGFBP7 was enhanced in H. pylori-associated GC compared to GC patients without H. pylori infection. Moreover, IGFBP7 protein levels also correlated with immune infiltration of macrophages, dendritic cells, CD4+ , CD8+ and neutrophils. Although the underlying mechanism of IGFB7 regulation in H. pylori infection remains to be clarified, the upregulated IGFBP7 expression seems to promote H. pylori associated carcinogenesis by supporting an inflammatory milieu. This assumption was supported by findings of Li et al. (2022), who observed immune cell dependent changes in the gastric epithelium. The study revealed that the expression of programmed cell death ligand 1 (PD-L1) in gastric epithelial cells was induced by cytokines released from infiltrating immune cells. Infection of gastric epithelial cells did not induce PD-L1 expression, while gastric epithelial cells cultured in the supernatant of infected peripheral blood mononuclear cells showed enhanced STAT1 activation and elevated PD-L1 expression. Thereby, STAT1 signaling seems to promote PD-L1 expression (Li et al. 2022). Since PD-L1 ligand inhibits CD8+ T-cell activation and proliferation (Sheppard et al. 2004), the anti-tumor response mediated by CD8+ cells is suppressed and facilitates tumor immune escape and tumor progression. This is in line with the observation that patients with low CD8+ immune infiltration accompanied by low STAT1 activation and PD-L1 expression showed worse survival compared to patients with high CD8+ infiltration (Li et al. 2022). Although H. pylori infection mainly elicits pro-carcinogenic responses, H. pyloridependent induction of protective mechanisms that inhibit carcinogenesis were also reported (Navashenaq et al. 2022). As described, ASK1 is activated by H. pylori induced ROS (Hayakawa et al. 2013). Further, it was observed that ASK1-dependent JNK signaling suppresses H. pylori-induced inflammation, because activation of this pathway prevented the infiltration of immune cells (Hayakawa et al. 2020). Moreover, the knockdown of ASK1 in H. pylori infected cells caused enhanced activation of NFκB and recruitment of macrophages, neutrophils and T-cells to the gastric mucosa of infected mice. As a result, an increased release of apoptotic ligands and proinflammatory cytokines such as TNF and interferon gamma (IFN-γ) was detected. As mentioned before, IFN-γ alters signaling cascades in the gastric gland leading to hyperplasia (Kapalczynska et al. 2022). Consequently, activation of ASK1 upon H. pylori infection protects from tumorigenesis by suppressing the immune response.

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3 Concluding Remarks In this chapter we reviewed multiple key mechanisms of H. pylori to control signaling in gastric epithelial, fibroblast and immune cells. Most notably, the development of GC in H. pylori-positive patients is characterized by disturbance of the epithelial cell lining and loss of cell polarity. In particular, the H. pylori-triggered interruption of TJs and AJs represents a hallmark of the infection process. This scenario is evoked by a collection of diverse bacterial effector molecules that hijack central host signal transduction modules that in turn are intimately connected with cancer-promoting activities. Early signaling events comprise the activation of receptor kinases such as EGFR and c-Met as well as cytoplasmic kinases including ERK, JNK, p38, Akt and GSK-β, and the stimulation of pro-inflammatory transcription factors NF-κB and AP-1. This is followed by the complex activation of cell proliferative signaling via β-catenin, induction of cell cycle arrest and cellular survival strategies. In recent years, we obtained vast information about the communication between H. pylori and its human host during infection, and details of the involved signal transmission networks. Thus, H. pylori represents a paradigm for contact-dependent crosstalk in the host. Future studies are required to also investigate the finetuning among the multiple signaling platforms and how this knowledge can be used to combat H. pylori infections and related diseases such as GC. Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft by grants to M.N. (RTG 2408, 361210922) and to S.B. (BA 1671/16-1).

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Impact of the Helicobacter pylori Oncoprotein CagA in Gastric Carcinogenesis Masanori Hatakeyama

Abstract Helicobacter pylori CagA is the first and only bacterial oncoprotein etiologically associated with human cancer. Upon delivery into gastric epithelial cells via bacterial type IV secretion, CagA acts as a pathogenic/pro-oncogenic scaffold that interacts with and functionally perturbs multiple host proteins such as pro-oncogenic SHP2 phosphatase and polarity-regulating kinase PAR1b/MARK2. Although H. pylori infection is established during early childhood, gastric cancer generally develops in elderly individuals, indicating that oncogenic CagA activity is effectively counteracted at a younger age. Moreover, the eradication of cagA-positive H. pylori cannot cure established gastric cancer, indicating that H. pylori CagAtriggered gastric carcinogenesis proceeds via a hit-and-run mechanism. In addition to its direct oncogenic action, CagA induces BRCAness, a cellular status characterized by replication fork destabilization and loss of error-free homologous recombinationmediated DNA double-strand breaks (DSBs) by inhibiting cytoplasmic-to-nuclear localization of the BRCA1 tumor suppressor. This causes genomic instability that leads to the accumulation of excess mutations in the host cell genome, which may underlie hit-and-run gastric carcinogenesis. The close connection between CagA and BRCAness was corroborated by a recent large-scale case–control study that revealed that the risk of gastric cancer in individuals carrying pathogenic variants of genes that induce BRCAness (such as BRCA1 and BRCA2) dramatically increases upon infection with cagA-positive H. pylori. Accordingly, CagA-mediated BRCAness plays a crucial role in the development of gastric cancer in conjunction with the direct oncogenic action of CagA.

M. Hatakeyama (B) Institute of Microbial Chemistry, Laboratory of Microbial Carcinogenesis, Microbial Chemistry Research Foundation, 3-14-23 Kamiosaki, Shinagawa-Ku, Tokyo 141-0021, Japan e-mail: [email protected] Institute for Genetic Medicine, Hokkaido University, Kita 15, Nishi 7, Kita-Ku, Sapporo 060-0815, Japan © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 S. Backert (ed.), Helicobacter pylori and Gastric Cancer, Current Topics in Microbiology and Immunology 444, https://doi.org/10.1007/978-3-031-47331-9_9

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1 Introduction Gastric cancer is the fifth most common malignancy and the third leading cause of cancer-related deaths worldwide. Importantly, more than half of all gastric cancer cases are from East Asian countries (represented by Japan, China, and South Korea). According to Lauren’s classification, gastric cancer is classified into two major types: intestinal type and diffuse type. Intestinal-type gastric cancer is a well-differentiated adenocarcinoma, histopathologically characterized by the formation of abnormal glands with irregular tubular structures, multiple lumens, and reduced stromal regions (Lauren 1965). This type of gastric cancer typically arises through sequential pathological gastric mucosal changes, from atrophic gastritis to intestinal metaplasia, and then to dysplasia, known as Correa’s cascade (Correa 1992). Diffuse-type gastric cancer spreads throughout the stomach mucosa without forming glands and easily metastasizes to remote organs and tissues. Diffuse-type gastric cancer comprises of poorly differentiated tumor cells. A variant of diffuse-type gastric cancer rapidly invades the stomach wall with extensive fibrosis, making it thick, hard, and rubbery. This morphological variant is called scirrhous gastric cancer or linitis plastica, and its prognosis is extremely poor (Jung et al. 2016). Diffuse-type gastric cancer occurs at a younger age than intestinal-type gastric cancer does. A small portion (1–3%) of gastric cancers are hereditary diffuse-type gastric cancers (HDGC) caused by germline mutations in the CDH1 or CTNNA1 genes encoding E-cadherin or αcatenin, respectively (Guilford et al. 1998). In HDGC, the tumor often develops in patients younger than 40 years of age. Helicobacter pylori is a microaerophilic, spiral-shaped Gram-negative bacterium that specifically colonizes the human stomach mucosa. While H. pylori is not a commensal bacterium, it is highly adapted to the human stomach environment and is estimated to infect approximately half of the entire human population (Bravo et al. 2018). In most cases, H. pylori infection is established in early childhood (less than 5 years of age). Bacterial infections occur via oral or fecal–oral routes, primarily within families. Once successfully colonized, H. pylori infection persists in the stomach throughout its lifetime unless the bacterium is artificially eradicated with antibiotics. Immediately after its discovery, H. pylori was found to play a major etiological role in chronic atrophic gastritis and peptic ulcers (Anand and Graham 1999). Subsequent clinico-epidemiological studies also indicated a close relationship between H. pylori infection and gastric cancer. Based on this, the International Agency for Research on Cancer, World Health Organization (IARC/WHO) classified H. pylori as a group I carcinogen, a definite cause of cancer in humans, in 1994 (Møller et al. 1995). Additional large-scale prospective cohort studies confirmed the association. Artificial infection studies using rodents including mice, rats, and Mongolian gerbils with H. pylori infection independently also provided supportive evidence for the role of the bacterial pathogen in the development of gastric cancer (Ansari and Yamaoka 2022). It is now recognized that H. pylori is the single greatest risk factor for the development of gastric cancer, acting as an etiologic agent for more than 80% of all human gastric cancers, including both intestinal-type and diffuse-type gastric

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cancers (IARC Helicobacter pylori Working Group 2014). Notably, however, this percentage appears to be substantially underestimated in East Asian countries, where gastric cancer is among the highest in the world, given that the frequency of gastric cancer without a history of H. pylori infection is extremely low (less than 5%) in this geographic area. Consistent with the causative role of H. pylori in the development of gastric cancer, large-scale intervention studies provided evidence that eradication of H. pylori by antibiotics is beneficial for preventing gastric cancer. Furthermore, patients with early gastric cancer who received H. pylori eradication treatment had reduced rates of metachronous gastric cancer development (Choi et al. 2018). In this chapter, the role of the first-identified bacterial oncoprotein, H. pylori CagA, in the development of gastric cancer in individuals infected with H. pylori is discussed.

2 The cag Pathogenicity Island and Type IV Secretion System (T4SS) Based on the presence or absence of the cytotoxin-associated gene A (cagA) in the bacterial genome, H. pylori is divided into cagA-positive and cagA-negative strains. The cagA gene encodes the bacterial protein CagA, which varies in its size from 130 to 145 kDa due to the amino acid sequence variation in its C-terminal region (Covacci et al. 1993). While CagA was originally considered to act as a bacterial toxin, subsequent studies revealed that the acute cytotoxic activity of H. pylori was caused by another bacterial toxin termed vacuolating toxin A (VacA). The cagA gene is localized at the 3’ end of the cag pathogenicity island (cagPAI), a 40-kilobase DNA segment that was thought to be introduced into the H. pylori genome by a horizontal DNA transfer from an unknown donor (Censini et al. 1996). Approximately 30–40% of H. pylori strains isolated in Western countries do not carry the cagPAI, and thus are cagA-negative, whereas almost all of the H. pylori isolates in East Asian countries possess the cagPAI and are cagA-positive. The cagPAI DNA segment contains 27–31 genes including cagA, depending on the strains. Of these, at least 18 genes encode proteins that serve as components of a bacterial type IV secretion system (T4SS), a syringe-like structure that secretes macromolecules such as DNA and protein across two bacterial membranes (outer and inner membranes) in Gram-negative bacteria. The association between CagA and gastric cancer was first reported in 1995 (Blaser et al. 1995). The importance of cagA-positive H. pylori infection in the development of gastric cancer was subsequently corroborated by a number of epidemiological and other studies (Uemura et al. 2001; Choi et al. 2018).

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3 T4SS-Mediated Delivery of CagA into Gastric Epithelial Cells The initial step in H. pylori colonization in the human stomach is stable adhesion of the bacterium with the luminal surface of gastric epithelial cells, a process that is mediated through a number of bacterial adhesins including BabA/B, SabA, OipA, HopZ, and AlpA/B (Matos et al. 2021). BabA/B and SabA adhesins are outer membrane proteins (OMPs) that bind to Lewis b (Leb) and sialyl-Lewis x (sLex) antigens, respectively. The stable adhesion initiates and facilitates the formation of the pilus-like T4SS syringe on the surface of H. pylori, the tip of which reaches the surface of attached gastric epithelial cells to form a conduit that connects the bacterium and host cytoplasm for CagA delivery. The conduit formation is initiated by the interaction of CagL, a component of T4SS, with the host plasma membrane protein integrin-β1 in a manner that is dependent on the Arg-Gly-Asp (RGD) motif present in CagL (Kwok et al. 2007). Integrin-β1 also interacts with other T4SS components such as CagI and CagY as well as CagA independently of the presence of an RGD motif (Koelblen et al, 2017). These Cag proteins may cooperatively bind to integrin to stabilize T4SS-gastric epithelial cell interaction. In addition, the CagA protein exposed on top of the T4SS pilus interacts with the plasma membrane phospholipid phosphatidylserine (PS), which is usually localized to the inner membrane leaflet but is exposed to the outer membrane leaflet upon direct contact with H. pylori, to facilitate CagA delivery into the cells (Murata-Kamiya et al. 2010). Since translocation of CagA is attenuated by treatment of gastric epithelial cells with hydroxymethylglutaryl (HMG)-CoA reductase inhibitors, statins, cholesterol enriched in membrane lipid rafts also appears to play a role in the T4SS-mediated CagA delivery. The requirement of multiple proteins as well as membrane lipids in the delivery of CagA across the plasma membrane suggests that the process may not be simply due to physical puncture of the plasma membrane by T4SS, but rather involves complicated biophysical/biochemical events that contribute to the conduit formation. Recent studies have revealed that the H. pylori HopQ adhesin protein interacts with the amino-terminal immunoglobulin-like domain of human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), specifically CEACAM1, 5 and 6, with high affinity, and this protein–protein interaction is indispensable for the H. pylori-mediated CagA delivery (Javaheri et al. 2016; Königer et al. 2016; Nguyen et al. 2023). In contrast to human CEACAMs, HopQ hardly binds to mouse CEACAM1. Since the mouse genome lacks Ceacam5 and Ceacam6, no strong HopQ-CEACAM interaction could occur in the H. pylori-infected mouse stomach, suggesting that H. pylori T4SS cannot efficiently deliver CagA into mouse gastric epithelial cells (Shrestha et al. 2022). Accordingly, while mice have been routinely used for H. pylori infection experiments, the lack or marked reduction in the injection of CagA into stomach cells make this animal an inappropriate model for studying the role of H. pylori-delivered CagA in gastric pathogenesis. On the other hand, transgenic expression of CagA in mice circumvents this serious problem and thus provides a suitable mouse model for studying the role CagA in the development

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of gastric cancer. The importance of CagA delivery into gastric epithelial cells in the development of gastric cancer was corroborated by the finding that systemic expression of CagA in cagA-transgenic mice caused spontaneous development of gastrointestinal and hematological malignancies (Ohnishi et al. 2008). The results provided compelling evidence that CagA acts in mammals as an oncoprotein, making researchers recognize CagA as the first bacterial oncoprotein.

4 Molecular Structure of the CagA Protein CagA consists of a structured N-terminal region (~70% of the entire protein) and an intrinsically disordered/unstructured C-terminal tail (~30% of the entire protein) (Hayashi et al. 2012; Kaplan-Türköz et al. 2012). The 100 kDa N-terminal CagA consists of three domains, termed Domains I-III. The variation of the molecular weight of CagA is due to the sequence polymorphism in its C-terminal region. Domain II contains a basic patch composed of clustered basic amino acid sequences, which electrostatically interacts with the acidic phosphatidylserine (PS) and thereby tethers CagA to the plasma membrane. The H. pylori CagA protein has no significant homology in its primary structure with known proteins in both eukaryotes and prokaryotes. Upon delivery into gastric epithelial cells, however, CagA undergoes tyrosine phosphorylation by host cell kinases in the Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs that are present in variable numbers (in most cases, three EPIYA motifs) in the C-terminal polymorphic region (EPIYA-repeat region) (Covacci and Rappuoli 2000; Hatakeyama 2004). Based on the sequence spanning each of the EPIYA motifs, four distinct EPIYA segments have been defined in the EPIYA-repeat region of CagA: EPIYA-A, EPIYA-B, EPIYA-C, and EPIYA-D. H. pylori cagA-positive strains, except those isolated in East Asia, produce the CagA protein with EPIYA segments that are arranged as EPIYA-A (32 amino acids), EPIYA-B (40 amino acids), and EPIYA-C (34 amino acids) segments in that order and are referred to as Western CagA or ABC-type CagA (Hatakeyama 2004). Furthermore, among Western CagA species, the EPIYA-C segment variably duplicates in tandem, typically between 1 and 3 times. CagA produced in East Asian H. pylori isolates also possesses the EPIYA-A and EPIYA–B segments but does not contain the EPIYA-C segment. Instead, it has a distinct EPIYA motif-containing segment, termed EPIYA-D. Accordingly, the EPIYA-repeat region of East Asian CagA is arranged in the order of EPIYA-A, EPIYA-B, and EPIYA-D segments (East Asian CagA or ABD-type CagA). Since a single CagA protein never contains both EPIYA-C and EPIYA-D segments, the presence of EPIYA-C is the hallmark of Western CagA, whereas the presence of EPIYA-D (~45 amino acid) is the hallmark of East Asian CagA. In addition to the two major CagA subtypes, variable alignments of these EPIYA segments create further diversity among CagA isolates. Distinct EPIYA segments are tyrosine-phosphorylated by different host kinases (Mueller et al. 2012). Src family kinase (SFK) members expressed in gastric epithelial

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cells (c-Src, Fyn, Lyn, and Yes) specifically phosphorylate the EPIYA-C and EPIYAD segments, while c-Abl can phosphorylate all of the EPIYA segments. Furthermore, a single CagA protein delivered into gastric epithelial cells undergoes tyrosine phosphorylation on 1 or 2 EPIYA segments but never on 3 segments. More specifically, “EPIYA-A and EPIYA-C” and “EPIYA-B and EPIYA-D” are preferably phosphorylated in combination in Western CagA and East Asian CagA, respectively. Hence, there may be a stepwise event in which the EPIYA-C or EPIYA-D segment is phosphorylated by SFKs at the initial stage of H. pylori infection, followed by phosphorylation of the EPIYA-A or EPIYA-B segment by c-Abl at later time points. Tyrosinephosphorylated CagA in the EPIYA segments is dephosphorylated by SHP1. SHP1, the one and the only SHP2 homologue in mammals, is primarily expressed in hematopoietic cells, where it negatively regulates immune cell activation. While less abundant, it is also expressed in gastrointestinal cells. Like SHP2, SHP1 forms a physical complex with CagA in gastric epithelial cells, although the interaction is independent of EPIYA tyrosine phosphorylation. The interaction potentiates SHP1 phosphatase activity that dampens EPIYA-tyrosine phosphorylation of CagA (Saju et al. 2016). Hence, SHP1 is the long-sought tyrosine phosphatase that counteracts phosphorylation-dependent CagA actions, although the level of SHP1 expression in gastric epithelial cells falls short of sufficiently neutralizing the oncogenic CagA action and thereby preventing CagA-mediated gastric carcinogenesis. In addition to the EPIYA motif, the C-terminal disordered CagA region contains another repeatable sequence motif, originally designated as the CagAmultimerization (CM) motif, as CagA can multimerize (dimerize) through this motif. The CM motif, comprising 16 amino acid residues, is located distal to the EPIYA-C or EPIYA-D segment (Ren et al. 2006). Although the CM motif is fairly conserved, there are 5 amino acid alterations between the East Asian and Western CagA species. Based on this variation, the CM motif of Western CagA is termed CMW and that of East Asian CagA is termed CME . Remarkably, the amino-terminal (N-terminal) 16-amino-acid stretch that constitutes the EPIYA-C segment (34 amino acids) is identical to that of CMW . Accordingly, multiplication of the EPIYA-C segment in Western CagA also increases the number of CM motifs. In contrast, the East Asian EPIYA-D segment, which hardly duplicates, does not contain a CM-like sequence.

5 Direct Oncogenic Action of CagA (Tyrosine Phosphorylation-Dependent) The potential of host cell-delivered CagA in signal perturbation, which may contribute to neoplastic cell transformation, was first demonstrated by the finding that phosphorylated CagA specifically interacts with at least nine Src homology 2 (SH2) domain-containing proteins (Selbach et al. 2009). One of the most important interaction partners is the SHP2 tyrosine phosphatase. The CagA-SHP2 interaction is mediated through tyrosine-phosphorylated EPIYA-C or EPIYA-D segments of CagA

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and SH2 domains of SHP2 (Higashi et al. 2002). Physiologically, SHP2 is required for full activation of the pro-mitogenic/pro-oncogenic RAS-ERK MAP kinase signaling pathway and is also involved in cell morphogenesis and cell motility. Deregulation of SHP2 by CagA is of great significance in terms of carcinogenesis because gain-offunction mutations in PTPN11, the gene encoding SHP2, have been found in a variety of human malignancies. The finding that transgenic expression of a CagA mutant that cannot undergo EPIYA tyrosine phosphorylation and is thus incapable of binding SHP2 failed to develop neoplastic lesions supports the importance of CagA phosphorylation in its oncogenic action. Most reported PTPN11 mutations in human cancers are missense mutations in exons 3 and 8, which encode the N-SH2 domain and the phosphatase domain, respectively. Crystal structure analysis of SHP2 suggests that such mutations subvert the autoinhibitory intramolecular interaction that occurs between the N-SH2 domain and the C-terminal phosphatase domain, thereby constitutively activating the catalytic function. Binding of tyrosine-phosphorylated CagA to the SH2 domains also induces a conformational change in SHP2, which relieves its autoinhibitory composition and results in aberrant activation of SHP2. The CagA-SHP2 interaction, mediated by the tyrosine-phosphorylated EPIYA-C or EPIYA-D segment, is one of the key interactions by which CagA exerts its prooncogenic action. The sequence flanking the phosphotyrosine (pY) residue of the EPIYA-D segment perfectly matched the consensus high-affinity binding sequence for the SHP2 SH2 domain, whereas the sequence flanking the pY residue of the EPIYA-C segment differed from the consensus sequence by a single amino acid at the pY + 5 position. Consistently, the SHP2-binding affinity of East Asian CagA (ABD type) is approximately 100-fold stronger than that of Western CagA (ABC type) (Hayashi et al. 2017). A co-crystallization study of CagA and SHP2 further revealed that while the EPIYA-C segment binds to SHP2 N-SH2 primarily through the phosphotyrosine residue in the EPIYA motif, the interaction of the EPIYA-D segment with SHP2 N-SH2 utilizes not only phosphotyrosine in the EPIYA motif but also aspartic acid, which is located at the pY + 5 position. The dual interaction underlies the 100-fold stronger interaction between East Asian CagA (ABD type) and SHP2 than that between Western CagA (ABC type) and SHP2. Consistent with this, clinico-epidemiological studies have revealed that gastric cancer is more closely associated with East Asian CagA-producing strains than with Western CagA-producing strains in geographical regions where two distinct strains co-circulate. Furthermore, in Western countries, a number of clinico-epidemiological studies have indicated that infection with H. pylori strains carrying Western CagA with two or more EPIYA-C segments is a greater risk for the development of gastric carcinoma than is infection with H. pylori carrying CagA with a single EPIYA-C segment. Because SHP2 binding is the only known CagA activity for which the magnitude is associated with the number of EPIYA-C segments, the degree of CagA-SHP2 interaction may link the number of EPIYA-C segments with gastric cancer risk. Indeed, it was revealed that the strength of CagA-SHP2 binding was elevated again by approximately 100-fold upon duplication of the CagA EPIYA-C segment from one segment to two segments in Western CagA, most likely due to monomeric interaction vs. dimeric interaction of CagA with SHP2, which possesses two CagA-binding SH2 domains (Nishikawa

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and Hatakeyama 2017). Divalent interactions dramatically stabilize protein–protein interactions through the avidity effect (or entropic effect). These findings thus provide a molecular basis for the role of EPIYA-C duplication as a distinct risk factor for gastric cancer in Western countries. Tyrosine-phosphorylated EPIYA-A and EPIYA-B segments serve as binding sites for C-terminal Src kinase (CSK) (Tsutsumi et al. 2003). CSK is responsible for inhibitory tyrosine phosphorylation of SFKs at the C-terminal tail. Thus, CagA-mediated CSK activation counteracts CagA-SHP2 signaling by downregulating CagA phosphorylation on EPIYA-C and EPIYA-D via the inhibition of SFKs. Tyrosine-phosphorylated EPIYA-B segment also binds to the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K) and activates the lipid kinase activity (Zhang et al. 2015). Prevalent Western CagA proteins carry a single nucleotide polymorphism (SNP) in EPIYA-B, which diminishes the PI3K-binding activity of CagA. Additionally, CagA interacts with Crk adaptor proteins in a tyrosine phosphorylationdependent manner, although the responsible EPIYA segment remains to be determined. Through this interaction, CagA disrupts adherens junctions by perturbing the adaptor function of Crk in cell signaling (Suzuki et al. 2005). CagA also binds to Grb2 via the EPIYA-containing region, thereby deregulating the RAS-ERK signaling pathway to stimulate cell proliferation (Mimuro et al. 2002). Despite the fact that Grb2 possesses an SH2 domain, the CagA-Grb2 interaction is reportedly independent of CagA tyrosine phosphorylation.

6 Direct Oncogenic Action of CagA (Tyrosine Phosphorylation-Independent) The oncogenic scaffold function of CagA is not necessarily mediated by tyrosinephosphorylated EPIYA segments. The CM motif serves as a binding site for PAR1b, a member of the PAR1 family that is predominantly expressed in gastric epithelial cells (Saadat et al. 2007). PAR1 was originally discovered as one of the Partitioningdefective (PAR) proteins (PAR1 to PAR6), which are indispensable for the establishment of cell polarity during early embryonic development in C. elegans. In mammals, PAR1 was independently discovered as a microtubule affinity-regulating kinase (MARK) that regulates microtubule stability by phosphorylating microtubuleassociated proteins such as MAP1, MAP2, and tau. The mammalian PAR1 family comprises four isoforms, PAR1a/MARK3, PAR1b/MARK2, PAR1c/MARK1, and PAR1d/MARK4. In polarized epithelial monolayer, PAR1b is located in the basolateral membrane, where it mediates the establishment and maintenance of apicalbasal polarity. PAR1b also inhibits GEF-H1, a RhoA-specific GEF, through phosphorylation, causing downregulation of RhoA and thereby inhibiting actin stress fiber formation. Hence, the kinase modulates the cytoskeleton by regulation of not only microtubule dynamics but also actin rearrangements. CagA is capable of binding to all PAR1 members, with the highest affinity for PAR1b. The CagA CM

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motif, comprising 16 amino acids, directly binds to the kinase catalytic domain of PAR1 and thus inhibits the kinase activity. CagA-mediated PAR1b kinase inhibition disrupts the gastric epithelial monolayer by evoking junctional defects, while inducing the loss of apical-basal polarity. The CM motif also interacts with the ligand-activated HGF receptor c-Met to potentiate c-Met-dependent stimulation of PI3K/AKT signaling (Suzuki et al. 2009). This, in turn, promotes prooncogenic Wnt signaling and nuclear factor κB (NF-κB) signaling, which induces cancerpromoting inflammatory responses. Additionally, CagA associates with the cytoplasmic domain of E-cadherin through the CM motif and thereby disrupts the Ecadherin/β-catenin complex, causing deregulated activation of Wnt signaling. CagA also interacts with GSK-3β via the disordered C-terminal tail. Through complex formation, CagA sequesters GSK-3 β to the insoluble fraction, causing Wnt signal activation by inhibiting GSK-3β (Lee et al. 2014). The structured N-terminal region of CagA also interacts with host proteins, which may additionally contribute to the neoplastic transformation of gastric epithelial cells. N-terminal CagA binds to the tumor suppressor RUNX3 to stimulate proteasomemediated degradation of RUNX3 by recruiting an unidentified E3 ubiquitin ligase (Tsang et al. 2010). N-terminal CagA also enhances proteasome-dependent degradation of a p53 tumor suppressor by physically associating with apoptosis-stimulating protein of p53 2 (ASPP2) (Buti et al. 2011). Furthermore, CagA-stimulated Akt phosphorylates and activates human double minute 2 (HDM2) and ARF-binding protein 1 (ARF-BP1) E3 ligases, which promote p53 degradation in conjunction with ASPP2. Since ARF (p14ARF) is a negative regulator of both HDM2 and ARF-BP1, the genetic loss of ARF in gastric epithelial cells may further potentiate CagA/ASPP2mediated p53 degradation. Collectively, these findings indicate that N-terminal CagA promotes the survival of CagA-delivered cells by impeding pro-apoptotic signaling through the inhibition of p53 and RUNX3. More recently, N-terminal CagA was found to interact with van Gogh-like (VANGL), a key regulator of the noncanonical Wnt/Planar cell polarity (PCP) signaling pathway, and thereby causes mislocalization of VANLG from the plasma membrane to the cytoplasmic compartment. This perturbs Wnt/PCP signaling that directs collective cell migration, which is critically involved in the formation and maintenance of the gastric stem cell niche, especially in pyloric glands, resulting in the aberrant expansion of pyloric gland base cells (Takahashi-Kanemitsu et al. 2023). As mentioned above, sequence polymorphisms exist in the CM motif between Western CagA and East Asian CagA (Nishikawa and Hatakeyama 2017). Western CagA typically possesses 2–4 repeats of the CMW motif, as it usually contains 1–3 tandem repeats of the EPIYA-C segment, followed by a single CMW immediately distal to the last EPIYA-C repeat. East Asian CagA, in contrast, does not possess a CM motif sequence within the EPIYA-D segment but contains a single CME immediately downstream of EPIYA-D. An increase in the number of CMW motifs in a single Western CagA synergistically augments PAR1b binding, which is proportional to the degree of stress fiber formation and disruption of tight junction function. For instance, Western CagA containing four CMW motifs exhibits a PAR1b-binding affinity that is more than 30-fold higher than that of Western CagA containing a single CMW .

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In addition, East Asian CagA (ABD type), which contains a single CME , shows a PAR1b-binding affinity that is comparable to that of Western CagA (ABC type), which contains two CMW motifs (Nishikawa and Hatakeyama 2017). Accordingly, CM polymorphism is a determinant of the magnitude of CagA-mediated perturbation of the cytoskeleton, both microtubule-mediated and actin-mediated, which may also influence the potential of individual CagA in promoting gastric carcinogenesis.

7 Hit-and-Run Mechanism of CagA-Mediated Gastric Carcinogenesis The CagA protein is intermittently injected into gastric epithelial cells during chronic infection with cagA-positive H. pylori. Once gastric cancer is established, however, tumor cells no longer require H. pylori infection or CagA delivery to maintain their neoplastic cell phenotype. This indicates that gastric carcinogenesis, initiated by cagA-positive H. pylori infection, should go through a “hit-and-run” process in which cancer precursor cells eventually lose dependency on cagA-positive H. pylori during multistep gastric carcinogenesis (Hatakeyama 2014). Such a hit-andrun mechanism may require functional compensation of CagA by genetic mutations and/or epigenetic alterations of cancer-related genes in the host genome. From this standpoint, the hit-and-run process should involve mechanisms that induce genomic instability to elicit excess gene mutations. This notion is supported by the results of recent studies demonstrating that in vitro infection of gastric epithelial cells with H. pylori induces DNA double-strand breaks (DSBs) (Toller et al. 2011; Hanada et al. 2014; Hartung et al. 2015; Murata-Kamiya and Hatakeyama 2022). However, in those studies, H. pylori mutants possessing functional T4SS but lacking the cagA gene were still capable of effectively inducing DSBs in gastric epithelial cells, indicating that CagA is indispensable for DSB induction. In contrast, two recent studies revealed a direct role of CagA in the induction of DSBs in gastric epithelial cells in both transfection and in vitro infection systems (Zamperone et al. 2019; Imai et al. 2021). CagA-mediated DSB induction requires the CagA CM sequence, which inhibits PAR1b kinase activity via complex formation. DSBs are the most dangerous form of DNA damage, in which the phosphate backbones of the two complementary DNA strands are broken simultaneously. Unrepaired DSBs induce apoptosis or premature cell senescence, whereas incorrect DSB repair leads to various types of mutations, including insertions, deletions, and translocations, thereby causing genomic instability that promotes carcinogenesis. Obviously, excess DNA damage contributes to the development of hit-and-run gastric carcinogenesis, in which CagA-dependent stages move on to CagA-independent stages.

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8 CagA-Mediated Induction of BRCAness that Elicits Genomic Instability The CagA-PAR1b complex, which is responsible for DSB induction, is formed at the inner plasma membrane. Because DSBs occur in the nucleus, CagA must induce DNA damage in an indirect manner, which is triggered by CagA-mediated PAR1b kinase inhibition. Indeed, subsequent studies revealed that PAR1b serves as a kinase that phosphorylates the BRCA1 tumor suppressor, which shuttles between the cytoplasm and nucleus. Phosphorylation of BRCA1 at serine-616, the residue comprising the distal nuclear localization signal (NLS), allows cytoplasmic-to-nuclear translocalization of BRCA1 (Imai et al. 2021). Thus, the inhibition of PAR1b kinase activity by CagA prevents the nuclear delivery of BRCA1. Recent studies have also revealed that a nuclear protein complex composed of BRCA1, BRCA2, PALB2, and RAD51 (BRCA complex), which was originally identified as a molecular machinery that mediates error-free homologous recombination (HR)-mediated DSB repair, plays a key role in the protection of stalled DNA replication forks that prevent fork collages and subsequent generation of DSBs. With the loss of the BRCA complex in the nucleus, CagA-expressing cells give rise to DBS formation owing to fork destabilization. “BRCAness” is defined as a cellular status induced by malfunctioning of the BRCA complex, which is characterized by (1) destabilized replication forks and (2) loss of HR-mediated error-free DSB repairs (Lord and Ashworth 2016). Cells suffering from BRCAness produce excess DSBs, which can only be repaired by error-prone mechanisms, most notably by microhomology-mediated end-joining (MMEJ), which is mediated by DNA polymerase θ (POLQ), thereby inducing genomic instability that hyperaccumulates mutations in the genome. BRCAnessassociated genome mutational signatures are characterized by a unique single-base substitution (SBS) termed SBS3, which is characterized by relatively equal distributions of single-base substitutions, and an insertion/deletion (ID) termed ID6, which is characterized by small deletions of >5 bp with extended stretches of overlapping microhomology at breakpoint junctions. Hence, sustained and reversible induction of BRCAness by H. pylori CagA generates unique genomic mutation signatures, which underlie the genomic instability that promotes hit-and-run gastric carcinogenesis (Fig. 1).

9 Induction of DSBs by H. pylori in a CagA-Independent Manner Infection of gastric epithelial cells with H. pylori can also induce DSBs independent of CagA (Hartung et al. 2015; Bauer et al. 2020). Metabolic precursors of H. pylori lipopolysaccharide (LPS), D-glycero-β-D-manno-heptose 1,7-bisphosphate (HBP) and ADP-β-D-manno-heptose (ADP-heptose), are delivered to host cells through

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Fig. 1 CagA induces DNA double-strand breaks (DSBs) that cause genomic instability in host cells. CagA-mediated PAR1b kinase inhibition prevents phosphorylation of the BRCA1 tumor suppressor, which is required for the cytoplasmic-to-nuclear translocation of BRCA1 to protect stalled DNA replication forks from DSB induction while promoting error-free homologous recombination (HR)-mediated DSB repair. In CagA-delivered gastric epithelial cells, the lack of BRCA1 in the nucleus gives rise to a cellular status termed BRCAness, which is noted for excess DSB formation that is repaired by error-prone non-HR mechanisms, specifically microhomology-mediated end-joining (MMEJ), which generates BRCA mutation signatures (SBS3 + ID6) in the genome

T4SS. Delivered HBP and ADP-heptose are recognized by alpha-kinase 1 (ALPK1) causing activation of TRAF-interacting protein with forkhead-associated domain (TIFA)-mediated innate immune response that activates NF-κB. When NF-κB activation occurs during the S-phase of the cell cycle, active transcription results in R-loop formation, which is a three-stranded nucleic acid structure composed of an RNA/DNA hybrid. R-loops cause replication fork stalling, in which the nucleotide excision repair endonucleases XPG (also known as ERCC5) and XPF (also known as ERCC4) cut the DNA strand in the RNA/DNA hybrid, producing a single-strand gap that is converted into a DSB by replication or additional strand breaks. Whereas the formation of H. pylori-induced DSBs depends on NF-κB activation, inhibition of NF-κB signaling that completely suppresses the production of IL-8 can reduce the levels of DSBs by only half. In addition, treatment of cells with the canonical NF-κB activator TNF does not induce DSBs (Bauer et al. 2020). These results indicate that NF-κB activation is not a major factor in the induction of DSBs in the host cell genome. Since CagA-mediated DSB induction is not influenced by the inhibition of NF-κB signaling, CagA and ADP-heptose may independently give rise to DSB generation in gastric epithelial cells. Nevertheless, it should be emphasized that CagA exacerbates DSB formation triggered by both CagA-dependent and -independent mechanisms through BRCAness induction.

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10 Cellular Status of p53, a Game Changer of CagA Action H. pylori infection is in most cases established during early childhood and persists for a lifetime unless eradicated by antibiotics, indicating that the CagA oncoprotein is occasionally injected into gastric epithelial cells of child. Nonetheless, a vast majority of individuals infected with cagA-positive H. pylori do not develop gastric cancer at a young age. Instead, they induce degenerative mucosal lesions, such as gastritis and ulceration, rather than hyperproliferative/procancerous lesions, indicating the presence of a system that impedes the oncogenic action of CagA in the stomach mucosa of young people (Saito et al. 2010). This cell-autonomous tumor suppressor system appears to be mediated primarily by the p53 tumor suppressor, which is activated in response to BRCAness-induced DSB formation and/or oncogenic stress induced by CagA-SHP2-deregulated aberrant RAS activation in normal gastric epithelial cells. p53-mediated growth inhibition (G1 cell cycle arrest)/premature senescence in CagA-delivered normal gastric epithelial cells may be beneficial for the stable colonization of H. pylori in the stomach by reducing acid secretion while providing nutrients released from dead cells in young people. As long as the p53 system is functionally active, the expansion of cells exposed to oncogenic CagA action is effectively prevented. Importantly, inactivation of the p53 tumor suppressor system has been shown to occur relatively frequently in normal non-transformed gastric epithelial cells of elderly people, in most cases through aging-associated inactivation of TP53. This notion is consistent with the fact that gastric cancer, especially the intestinal type, is a disease in older people that is clinically diagnosed five or more decades after the onset of H. pylori infection. Long-term infection with cagA-positive H. pylori results in sequential histological changes (gastritis-atrophy-metaplasia-dysplasia) in the stomach mucosa known as Correa’s cascade, the endpoint of which is typically intestinal-type gastric cancer. Although TP53 is mutated in approximately half of all human cancers, mutation is found in more than 70% of intestinal-type gastric cancers (Cancer Genome Atlas Research Network 2014), indicating that this mutation is of particular importance in the development of intestinal-type gastric cancer induced by cagA-positive H. pylori. Mechanistically, loss of the p53 tumor suppressor system enables expansion of CagA-induced BRCAness cells (with excess mutations), which is most likely driven by CagA-deregulated mitogenic signaling pathways such as RAS-ERK signaling, PI3K-AKT signaling, and/or Wnt signaling.

11 Role of BRCAness in the Development of Gastric Cancer In addition to H. pylori, mutation signature studies of cancer genomes have indicated a molecular connection between BRCAness and gastric cancer. Of thirty-four different human cancer types analysed, only four displayed the BRCA mutation signature that is characterized by the presence of SBS3 and ID6, as above described (Alexandrov

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et al. 2015). These included breast, ovarian, pancreatic, and gastric cancers. Of these, a substantial fraction of breast, ovarian, and pancreatic cancers has been shown to carry germline loss-of-function mutations (recently referred to as “pathogenic variants”) in BRCA1 or BRCA2 genes. Hence, gastric cancer is the only human cancer that displays the BRCA mutation signature, despite the lack of mutations in the BRCA1 or BRCA2 gene. It is highly possible that the BRCA signature was engraved in the genome during the chronic exposure of cancer precursor cells to CagA, which induces reversible BRCAness in the cell. The close association between BRCAness and the development of gastric cancer strongly supports the notion that BRCAness plays a key role in the development of gastric cancer (especially intestinal-type gastric cancer). If this is true, individuals who carry pathogenic variants of the BRCA1 or BRCA2 gene may have an increased gastric cancer risk because they have quantitatively and/or qualitatively reduced function of the BRCA complex and thereby trigger BRCAness more easily than do healthy individuals. A major hurdle in approaching this important question is that the proportion of carriers of BRCA1 or BRCA2 pathogenic variants in the general population is only about are approximately 0.2%, making it extremely difficult to perform epidemiological studies with reliable population sizes. Nevertheless, the results of several previous studies, including case reports, supported the idea that gastric cancer risk is elevated in carriers of pathogenic BRCA1/BRCA2 variants. This notion was consolidated by a recent large-scale case–control study conducted in Japan (11,000 gastric cancer cases and 44,000 controls), which provided compelling evidence that carriers of pathogenic variants of BRCAness-inducing genes (BRCA1, BRCA2, PALB2, and ATM) have an increased risk of gastric cancer (Usui et al. 2023). Importantly, all of these BRCA gene products are physically or functionally associated with BRCA1 and are involved in homologous recombination-mediated DSB repair as well as replication fork stabilization. Thus, functional impairment of the BRCA complex predisposes cells to BRCAness and BRCAness-associated genomic instability. A more striking finding obtained from this study was that the gastric cancer risk was greatly increased when pathogenic BRCA variant carriers were also infected with H. pylori, presumably East Asian cagA-positive strains in most cases since the clinical samples were collected at the Aichi Cancer Center in Nagoya, Japan. As a result, the cumulative risk of gastric cancer development (by 85 years of age) in individuals carrying one of the pathogenic BRCA variants (BRCA1, BRCA2, ATM, or PALB2) with H. pylori infection increased up to 45.5%, whereas the risk in individuals without H. pylori infection was less than 5% regardless of the presence of pathogenic variants.

12 Concluding Remarks H. pylori CagA is the first identified bacterial oncoprotein that, upon delivery into gastric epithelial cells, serves as an oncogenic scaffold that directs multistep gastric carcinogenesis. Notably, however, it generally requires more than half a century from

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the establishment of cagA-positive H. pylori colonization in the stomach to the development of gastric cancer, suggesting that there must be a mechanism that strongly counteracts the oncogenic CagA action in young people with cagA-positive H. pylori infection. In this regard, p53 appears to be a major barrier that prevents CagA-injected cells from neoplastic transformation, and a promising game-changing event in gastric carcinogenesis is the inactivation of p53 in gastric epithelial cells, which could be achieved by various mechanisms such as aging-associated TP53 mutation, amplification of HDM, and inactivation of ARF. It remains unclear whether CagA-induced BRCAness promotes the introduction of TP53 mutations in gastric epithelial cells. Inactivation of p53 in gastric epithelial cells, which mostly occurs only in elderly people allows survival and expansion of CagA-expressing cells with BRCAnessinduced genomic instability, from which CagA-independent cancer-precursor cells will eventually arise. Since CagA-mediated BRCAness induction may be substantially easier in cells carrying pathogenic BRCA variants, CagA and BRCA variants may act synergistically to induce gastric cancer. To answer the long-sought question of why only a small portion of H. pylori-infected individuals develop gastric cancer (even if they are infected by highly oncogenic H. pylori strains, such as in Japan), we need to consider the genetic and/or environmental factors that robustly potentiate the oncogenic potential of CagA, as demonstrated in cases of pathogenic BRCA variants. Screening for BRCA genes, such as BRCA1, BRCA2, PALB2, and ATM, should be considered immediately for identifying high-risk groups for gastric cancer in H. pylori-infected populations. Other genes that can also induce BRCAness-like cellular status, such as Fanconi Anemia genes (Kolinjivadi et al. 2022), may be additional candidates for genetic screening. Finally, in contrast to cancers carrying pathogenic BRCA mutations in the genome, such as breast, ovarian, pancreatic, and prostate cancers, PARP inhibitors, which are used for the treatment of cancers with BRCAness, may not be effective in the treatment of gastric cancer because CagAinduced BRCAness is transient and the function of BRCA1 is fully restored in established gastric cancer cells in the absence of CagA injection. Acknowledgements This work was supported by Grant-in-Aids for Scientific Research from Japan Society for the Promotion of Science (JSPS) to M.H. (16H06373 and 21H04804) and by Project for Cancer Research and Therapeutic Evolution (P-CREATE) from Japan Agency for Medical Research and Development (AMED), Japan, to M.H. (160200000291).

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Bacterial Proteases in Helicobacter pylori Infections and Gastric Disease Silja Wessler and Gernot Posselt

Abstract Helicobacter pylori (H. pylori) proteases have become a major focus of research in recent years, because they not only have an important function in bacterial physiology, but also directly alter host cell functions. In this review, we summarize recent findings on extracellular H. pylori proteases that target host-derived substrates to facilitate bacterial pathogenesis. In particular, the secreted H. pylori collagenase (Hp0169), the metalloprotease Hp1012, or the serine protease High temperature requirement A (HtrA) are of great interest. Specifically, various host cell-derived substrates were identified for HtrA that directly interfere with the gastric epithelial barrier allowing full pathogenesis. In light of increasing antibiotic resistance, the development of inhibitory compounds for extracellular proteases as potential targets is an innovative field that offers alternatives to existing therapies.

1 Introduction Helicobacter pylori (H. pylori) colonizes the gastric mucosa of more than half of the world’s population and is the most important risk factor for the development of gastric cancer (GC). It is estimated that H. pylori infection is responsible for more than 75% of GC cases worldwide (Ferlay et al. 2015). The fact that GC is the fifth most common cancer worldwide and the third leading cause of cancer-related death underlines the importance of understanding the cellular and molecular mechanisms of H. pylori pathogenesis (Fitzmaurice et al. 2017). In addition to GC, gastric and duodenal ulcers, mucosa-associated lymphoid tissue (MALT)-lymphoma, and chronic gastritis S. Wessler (B) · G. Posselt Department of Biosciences and Medical Biology, Laboratory for Microbial Infection and Cancer, Paris-Lodron University of Salzburg, Salzburg, Austria e-mail: [email protected] Cancer Cluster Salzburg and Allergy-Cancer-BioNano Research Centre, Salzburg, Austria G. Posselt e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 S. Backert (ed.), Helicobacter pylori and Gastric Cancer, Current Topics in Microbiology and Immunology 444, https://doi.org/10.1007/978-3-031-47331-9_10

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are diseases that are closely associated with H. pylori infection (Gobert and Wilson 2022). The process of H. pylori-mediated gastric tumor induction and progression is a prime example of inflammation-induced carcinogenesis that spans several decades and proceeds through defined precancerous steps commonly referred to as the Correa cascade. This cascade includes distinct sequential stages: chronic active gastritis, chronic atrophic gastritis, intestinal metaplasia, and dysplasia, ultimately leading to invasive carcinoma (Correa 2013). The second malignant entity that is strongly associated with H. pylori infection is the diffuse large B-cell lymphoma of the MALT type in the stomach (Varon et al. 2022). Still, most H. pylori infected individuals do not develop clinically relevant symptoms and even beneficial effects of H. pylori colonization in the host have been reported (Oertli and Müller 2012; Engler et al. 2015). Despite the fact, that H. pylori induces chronic gastritis in virtually all infected individuals, the immune response to the bacterium is multifaceted and the immune system has been suggested to be an important player in the manifestation of disease (Pachathundikandi et al. 2015; Blaser et al. 2019; Sato et al. 2019). Notably, H. pylori was the primary cause of more than 36% of diagnosed infection-associated cancer cases (de Martel et al. 2020). Therefore, it is of utmost importance to understand the disease-causing factors of this pathogen and their complex interaction with host cell structures and functions. In the last 15 years, novel and fascinating mechanisms of several H. pylori proteases have been discovered. After secretion in the environment, these H. pylori proteases can target host cell substrates, and thus exert a direct influence on pathogenesis. We aim to summarize the most important recent advances in this field in this review.

1.1 The Gastric Epithelium The gastric mucosa is the primary niche for H. pylori and is bounded by a simple columnar epithelium covered by a thick protective layer of mucus. At the same time, the mucosal epithelium is the first innate line of defense and barrier against infectious agents, constantly intercepting and excreting pathogens and toxins. The barrier function of a healthy gastric epithelium is strongly dependent on three characteristics: (i) an intact mucus layer, (ii) an intact apico-basolateral polarization of the cellular monolayer, and (iii) intact junctional integrity (Cone 2009; Martin-Belmonte and Perez-Moreno 2011; Backert et al. 2017) (Fig. 1a). The mucus layer is a dynamic structure that is constantly renewing itself and changing drastically in response to infection. Over the course of co-evolution, commensal bacteria have adapted to not cross the mucus layer; however, pathogenic bacteria and viruses have developed mechanisms to evade mucus trapping and resist antimicrobial activity within the mucus (Cone 2009). Moreover, during H. pylori infection mucin production is impaired and mucus flow is reduced (Navabi et al. 2013). Epithelia line the cellular boundary of the inside-world of the human gastrointestinal tract, and thus form a highly directional structure. Segregation in apical and basolateral domains allows for a vectorial organization of functional compartments and transport machineries. An

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epithelial polarity program results in polarized segregation of protein structures and membrane domains (Rodriguez-Boulan and Macara 2014). Tight junctions (TJs) can act as “membrane fences” preventing intramembrane diffusion of proteins and lipids, thereby establishing and maintaining apico-basal polarity of epithelia (Otani et al. 2019). In the cytoplasm, a PAR3–PAR6–atypical PKC (aPKC) complex localizes at TJs, which is implicated in the control of cell polarity (Martin-Belmonte and PerezMoreno 2011). Depolarization of the epithelium is a hallmark of H. pylori infections. For instance, H. pylori can directly interfere with cell polarity by targeting TJ integrity (Amieva et al. 2003; Krueger et al. 2007) and the PAR1b-dependent polarity program in epithelial cells (Lu et al. 2008). Another hallmark of intact epithelia is their regulated intercellular adhesion. In this context, TJs are significant not only for cell polarity, but also for functional intercellular adhesions. TJs consist of a variety of transmembrane proteins (claudins, occludin, JAM-A, tricellulin, etc.) and intracellular proteins of the zonula occludens (ZO-family) and form the paracellular barrier in epithelia. TJ proteins are functionally associated with signaling molecules and the cytoskeleton (Otani and Furuse 2020). Despite their name, TJs are not necessarily “tight” in the sense of sealing to the outside; in fact, tight junctions actively regulate paracellular permeability for water, ions, and macromolecular solutes (Monaco et al. 2021). In general, TJs in the stomach are considered to display low water and ion permeability, yet TJs have been described as target structures in H. pylori infection by several distinct mechanisms (Caron et al. 2015). Adherens junctions (AJs) are lateral cell–cell junctions of the cadherin type, which provide mechanical strength to the tissue. In epithelial cells, the cell adhesion molecule and transmembrane protein E-cadherin forms homotypic and calcium-dependent interactions via the extracellular domain with neighboring cells. With its intracellular domain, Ecadherin builds complexes with proteins of the catenin family, such as β-catenin and p120 catenin, that link the intercellular junctions to the actin cytoskeleton, but also regulate signaling in homeostasis and disease (Harris and Tepass 2010). Further, AJs serve as sensors of junctional integrity, and AJ proteins induce cell proliferation to facilitate epithelial homeostasis and proliferation-driven closure of micro-lesions (Garcia et al. 2018). Importantly, E-cadherin is an important tumor suppressor. Loss of E-cadherin function is associated with many epithelial tumor entities and is a critical feature in the epithelial-mesenchymal transition (EMT) and metastasis (Na et al. 2020). In addition to AJs and TJs, the gastric epithelium features desmosomes, spot-like connections that link intermediate filaments of neighboring cells in tissues exposed to high mechanical stress (Fig. 1a). Desmosomes contribute to functional intercellular adhesion in epithelia mediated by a number of transmembrane proteins of the cadherin superfamily. The main members of the family include the human desmogleins 1–4 and desmocollins 1–3. Their cytoplasmic domains recruit the armadillo proteins plakoglobin and plakophilins 1–3, which are connected to the intermediate filaments of the cytoskeleton of the cell via desmoplakin (Müller et al. 2021). Importantly, these types of intercellular adhesions have been found to be important targets in H. pylori infections and the disruption of intercellular adhesions represents an integral part of the bacterial pathogenesis.

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Fig. 1 Bacterial proteases in H. pylori infection. a H. pylori infects the gastric mucosa via the oral route. While a significant proportion of bacteria is already trapped in the outer layer of the protective mucus, H. pylori can swim freely through the inner mucus layer and attach to the apical side of gastric epithelial cells. The H. pylori metallopeptidase Hp0506 has been described to affect bacterial shape and colonization, however no host cell targets have been identified. The aminopeptidase Hp1037 has been proven to be an extracellularly active protease, yet biological targets remain to be identified. Nevertheless, both proteases, Hp0506 as well as Hp1037 have been identified in the H. pylori secretome. The junctional complexes in tight junctions have been described as a target for Hp1012/Hp0657 complexes, and the H. pylori collagenase Hp0169 has been identified to cleave triple-helical collagen in the extracellular matrix (ECM). b H. pylori HtrA targets molecules in all types of lateral junctions. The tight junction proteins occludin and claudin-8 are cleaved to enter the lateral intercellular space. E-cadherin is the most prominent target of H. pylori HtrA and E-cadherin cleavage results in opening of the adherens junctions. Finally, desmoglein-2 shedding by HtrA disintegrates desmosomal junctions. As a result, H. pylori gains access to the basolateral compartment, where it interacts with α5β1-integrins to deliver the oncoprotein CagA in a T4SS dependent manner. Created with BioRender.com

1.2 Helicobacter pylori Disease Mechanisms H. pylori is an incredibly successful bacterium colonizing more than 50% of the world’s population and it has been speculated that the genetic event of acquiring the cytotoxin-associated gene (cag) pathogenicity island (cagPAI) triggered evolutionary events that transformed H. pylori from a commensal to a pathogen (Censini et al. 1996). H. pylori is now classified as a class I (definite) carcinogen by the International Agency for Research on Cancer (IARC) (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans 1994). A significant association of the cagPAI presence in H. pylori with its carcinogenic potential has been demonstrated, however, even in gastritis H. pylori type I strains, (i.e., strains positive for cytotoxinassociated gene A (CagA)- and vacuolating cytotoxin A (VacA)), were found to cause an increase in gastritis markers as compared to strains expressing neither CagA nor VacA in a retrospective study (Liu et al. 2021). The CagA protein is injected into

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the cytoplasm of gastric epithelial cells by a specialized type IV secretion system (T4SS). CagA itself and the structural components of this T4SS are encoded within the cagPAI (Fischer et al. 2020). Once delivered into the host cytoplasm, CagA serves as a signaling adaptor protein, derailing numerous signaling cascades in tyrosine phosphorylation-dependent and -independent ways (Takahashi-Kanemitsu et al. 2020). Notably, CagA itself has the potential for oncogenic tissue transformation in the gastric epithelial- and B-cell compartment (Ohnishi et al. 2008). The second type I strain defining factor VacA is a pore-forming toxin, that interferes with the plasma membrane and the organization and trafficking of membranous cell organelles such as mitochondria (Foegeding et al. 2016). The different properties of CagA and VacA in Western- versus East Asian strains are a major determinant of the different risk potentials associated with the respective strains (Yamaoka et al. 2008). Although CagA and VacA are the most prominent protein factors in H. pylori pathogenesis, a plethora of other bacterial molecules are involved in successful colonization of the niche and persistent infection, which are prerequisites for the development of gastric disease (Posselt et al. 2013). One of the factors promoting gastric pathologies is an arsenal of intracellular and extracellular protease activities of bacterial and host origin. These proteases target crucially important molecules in bacterial colonization, mucosal inflammation, epithelial junctional integrity, tissue transformation, and metastasis (Bernegger et al. 2022b; Sokolova and Naumann 2022).

2 H. pylori Proteases with Implications in Pathogenesis Over the past three decades, research on H. pylori pathogenesis leading to inflammation-related carcinogenesis has mostly focused on the well-studied virulence factors, while proteases have played a minor role. However, the implication of extracellular H. pylori proteases in pathogenesis has been repeatedly suggested in several early reports. Piotrowski and colleagues proposed a secreted H. pylori protease that cleaves PDGF (platelet derived growth factor) and TGF-β (transforming growth factor beta) in 1997 (Piotrowski et al. 1997). A mucinase similar to the Vibrio cholerae haemagglutinin/protease (HAP) was also described in the 1990s, which showed weak activity in the digestion of gastric mucus (Smith et al. 1994). However, subsequent analyses did not find a protease in the H. pylori genome comparable to the HAP of V. cholerae, so the identity of a mucinase remains questionable (Suerbaum and Friedrich 1996; Wadström et al. 1997). Signature-tagged mutagenesis analyses identified H. pylori secreted Hp0169 as a “true” collagenase that is not only active as a gelatinase but cleaves triple-helical type I collagen (Fig. 1a). The expression of Hp0169 is essential for successful colonization of the stomach in Mongolian gerbils suggesting that the collagenolytic activity is important for the establishment of persistent infection (Kavermann et al. 2003). Accordingly, Hp0169 has been found to be prevalently expressed in approximately 98% of H. pylori isolates as implied by an Iranian study of patients suffering from gastroduodenal disorders (Gharibi et al. 2017).

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The H. pylori genome encodes several putative and factual proteases with functions in bacterial physiology. The MEROPS peptidase database lists 108 peptidases and 51 non-peptidase homologs (that are similar in amino acid sequence to peptidases but lack one or more of the expected catalytic residues) for H. pylori strain 26695 (Rawlings et al. 2018). This includes molecular chaperones of the caseinolytic protease (Clp) and Lon families, which are thought to have important functions in the elimination of misfolded or defective proteins and in the regulation of the cellular activities of critical regulatory proteins (Rath et al. 2012; Kim and Kim 2008; Loughlin et al. 2009; Luo et al. 2016; Tu et al. 2014). Clp proteins are important ATPdependent stress response factors and are represented by ClpX, ClpA, ClpB and ClpP (Rath et al. 2012). As chaperones, ClpA and ClpX are implicated in protein folding, while ClpP represents a proteolytic component. Functionally, the chaperones ClpA and ClpX flank the proteolytic ClpP and ClpAP or ClpXP complexes target misfolded or damaged proteins for degradation (Loughlin et al. 2009). An isogenic H. pylori clpB mutant showed increased sensitivity to high-temperature stress (Allan et al. 1998) and clpA deletion led to a survival defect in human macrophages (Loughlin et al. 2009). The ATP-dependent LonA and LonB proteases have similar functions in degrading misfolded proteins, but comprehensive functional studies are still rare. Pulldown experiments identified several Lon substrates (e.g., RdxA) involved in sensitivity to metronidazole (Tu et al. 2014) that is frequently used in triple and quadruple therapies against H. pylori infection. Since Clp and Lon proteases are localized in the cytoplasm of Gram-negative bacteria, they play an indirect role in H. pylori infection due to the increase in bacterial tolerance to stress conditions. Analysis of the genome of H. pylori strain 26695 in a comprehensive sequence screen predicted more than 85 putative proteases with extracellular localization and complete or at least partial conservation of the active-site region (Löwer et al. 2008). Most of these hypothetical proteases are uncharacterized, but some of them have been detected in proteomics analyses of the H. pylori´s exoproteome, including Hp0506 (Cds3, HdpA), Hp0657 (YmxG), Hp1012 (PqqE), HtrA (Hp1018/Hp1019), and Hp1037 (Bumann et al. 2002; Smith et al. 2007; Snider et al. 2015). A biochemical report demonstrated an aminopeptidase activity for Hp1037; however, a biologically significant substrate has not been identified so far (Choi et al. 2013), and therefore, it remains unknown whether Hp1037 plays an active role in bacterial physiology or pathogenesis, despite being an extracellular localized protease (Fig. 1a). The other proteases Hp0506, Hp0657, Hp1012, and HtrA were completely undescribed for a long time, but recent studies have shown that these proteases have direct effects on bacterial pathogenesis (summarized in Table 1). Hp0506 belongs to the M23B family of metallopeptidases and harbors a Zn++ binding LytM (lysostaphin/peptidase M23 domain) domain with homologies to N. meningitidis NMB0315, V. cholerae VC0503, and S. aureus LytM (An et al. 2015) representing glycyl-glycine endopeptidases that cleave peptidoglycan (Firczuk et al. 2005). It was initially described as cell shape-determining gene 3 (csd3) important for bacterial curvature and helical shape of H. pylori (Sycuro et al. 2010). Hp0506 has then been characterized as a peptidoglycan-modifying enzyme exhibiting a D,Dcarboxypeptidase activity that cleaves-off the terminal D-Ala(5) from the muramyl

Yes

Yes

Non-peptidase homologues, YmxG

Metalloprotease, PqqE

HtrA peptidase, chaperone and serine protease

Aminopeptidase

Hp0657

Hp1012

HtrA

Hp1037

Yes

N-succinyl-Ala-Ala-Ala-Pro-Phe-p-nitroanilide

E-cadherin, occludin, claudin-8, fibronectin, desmoglein-2

JAM-A?

None

Peptidoglycan

Helical type I collagen

Suggested substrates

Proposed function

IVC using rec. Hp1037

Bonis et al. (2010); Sycuro et al. (2010)

Kavermann et al. (2003)

Reference

Disruption of TJs?

n.d

Choi et al. (2013)

Bernegger et al. (2021); Hoy et al. (2010); Tegtmeyer et al. (2017)

Marques et al. (2021)

Increases Marques et al. Hp1012 activity (2021)

Ko mutant, IVC using Disruption of rec. HtrA intercellular adhesion

IEX/SEC fraction containing Hp1012

–

Ko mutant, IVC using Cell shape and rec. Hp0506 colonization

Ko mutant, IVC using Important for rec. Hp0169 colonization

Experimental evidencea

IEX, ion exchange chromatography; IVC, in vitro cleavage experiment; ko, knockout mutant; n.d., not determined; rec., recombinant; SEC, size exclusion chromatography

a Abbreviations:

Yes

Metallopeptidase, HdpA

Hp0506

No

Yes

Collagenase, PrtC

Hp0169

Activity

Description

Protease

Table 1 Overview of extracellular H. pylori proteases

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pentapeptide and a D,D-endopeptidase activity that cleaves the D-Ala(4)-mDAP(3) peptide bond between cross-linked muramyl-tetrapeptides and pentapeptides. Deletion of the hp0506 gene led to alterations in H. pylori’s phenotype, which was then more compact and branched. The authors suggested that the changed bacterial phenotype has consequences for the colonization of mice (Bonis et al. 2010). Even though periplasmic function for Hp0506 has been demonstrated, it remains unclear whether it can directly interfere with host cells since it has been predicted to be an extracellular protease (Löwer et al. 2008) (Fig. 1a). Recently, a study on the Zinc-dependent metalloprotease Hp1012 was published, suggesting a role in TJ disruption (Marques et al. 2021). Marquez and colleagues showed that Hp1012 binds to Hp0657 to enhance Hp1012 protease activity. Hp0657 has previously been predicted to be a Zn++ -dependent M16 family peptidase; however, it lacks the Zn++ binding HXXEH motif, resulting in an inactive protein. A crude protein fraction obtained by combined ion exchange chromatography and size exclusion chromatography containing Hp1012/Hp0657 complexes has been shown to cleave synthetic peptides derived from the TJ protein JAM-A (Marques et al. 2021) (Fig. 1a). However, generation of a genomic hp1012 deletion mutant including a complementary H. pylori strain or in vitro cleavage experiments with recombinant Hp1012 protein did not reveal JAM-A cleavage activity (our unpublished data).

2.1 The Important Function of HtrA in Opening Lateral Junctions 2.1.1

HtrA is a Chaperone and Serine Protease

HtrA is a H. pylori factor implicated in pathogenesis, which has been intensively investigated over the last decade. A unique feature of H. pylori HtrA is the fact that it is an essential gene product and HtrA-negative isolates are not yet known (Tegtmeyer et al. 2016), underlining the importance of HtrA functions as a chaperone and serine protease (Fig. 2a). The generation of an HtrA deletion mutant in H. pylori was not possible for a long time (Salama et al. 2004; Hoy et al. 2010; Tegtmeyer et al. 2016). Exclusively, in the H. pylori N6 strain, inactivation was possible due to a coincidental mutation in the secA gene, which is part of the Sec translocon apparatus that transports proteins into the periplasm (Zawilak-Pawlik et al. 2019). Subsequently, the availability of an H. pylori HtrA knockout mutant has greatly facilitated research on the role of HtrA in pathogenesis. The structure of H. pylori HtrA is related to that of Escherichia coli (E. coli) HtrA proteases. E. coli expresses the three HtrA orthologs DegP, DegQ, and DegS. DegP and DegQ share a similar domain structure, composed of an N-terminal signal peptide, followed by a linker region and the serine protease domain containing the catalytic triad histidine (H), aspartic acid (D), and serine (S). The C-terminal region is characterized by one PDZ1- and one PDZ2 domain (postsynaptic density

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Fig. 2 H. pylori expresses the chaperone and serine protease HtrA. a Scheme of HtrA proteases in H. pylori and E. coli. H. pylori HtrA, E. coli DegP and E. coli DegQ carry a signal peptide (SP) at the N-terminus. After a linker region (LR), the protease domain is characterized by the catalytic triad histidine (H), aspartic acid (D), and serine (S) followed by the two PDZ domains 1 and 2. E. coli DegS harbors a transmembrane domain (TMD) instead of a SP and only one PDZ domain. b HtrA is a crucially important chaperone and serine protease in the bacterial periplasm. It recognizes misfolded proteins to either refold or degrade them. Via an unknown mechanism, HtrA is secreted into the bacterial environment. Created with BioRender.com

protein, Drosophila disc large tumor suppressor, and zonula occludens-1 protein (PDZ)), which are important in substrate recognition, substrate binding, and homomultimerization. The main difference between DegP and DegQ proteases is the lack of the linker region in the LA loop in DegQ. E. coli DegS has a transmembrane domain instead of the N-terminal signal peptide and contains only one PDZ domain (Fig. 2a). Homo-multimerization is important for the proteolytic activity of HtrA proteins. HtrA proteins have been shown to form inactive trimeric structures.

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Trimeric building blocks assemble to hexamer and finally to active dodeca- and 24mers upon substrate binding. Several regulatory loops (LA, LD, and L1–L3 loops) have been investigated for E. coli DegP, which share high homology with the computational model and X-ray structure of H. pylori HtrA (Perna et al. 2014). HtrA also contains an “orphan” loop (I161 -S169 ) that regulates the oligomerization of H. pylori HtrA (Bernegger et al. 2020; Perna et al. 2014). This orphan loop interferes with the active site cavity containing the catalytic S221 and the PDZ1 domain and represents an allosteric ligand binding site (Perna et al. 2014). Mutational analyses of the orphan loop revealed that mutation of S166 and D168 decreased, while K328 and S164 strongly increased the formation of active oligomeric HtrA structures (Perna et al. 2014; Bernegger et al. 2020). Recently, S171 to L171 mutations in close proximity to the orphan loop were identified in 40 clinical H. pylori isolates, which resulted in a stabilization of HtrA trimers (Zarzecka et al. 2023). These data support the regulatory function of the orphan loop, which can be targeted by highly functional small molecule compounds that act as allosteric inhibitors (Perna et al. 2014). In contrast to E. coli, H. pylori expresses only one HtrA ortholog. As in E. coli DegQ, the linker region is not present in H. pylori HtrA, suggesting that H. pylori expresses a DegQ homolog. However, H. pylori HtrA exhibits greater sequence similarity to DegP proteins (Abfalter et al. 2016). Hence, it remains unclear whether H. pylori HtrA represents a DegP or DegQ protein and whether the lack of the linker region influences the protease function. Although the secretion of HtrA has been shown in previous reports (Bumann et al. 2002; Snider et al. 2015), the proteolytic activity of H. pylori was first described in 2008 (Löwer et al. 2008). HtrA expressed from the hp1018/hp1019 gene locus of the Hp26695 strain showed caseinolytic activity. Zymography analyses, showed that secreted as well as intracellular HtrA are proteolytically active and form oligomers. H. pylori HtrA exhibits a molecular weight of approximately 52 kDa. Comparing the molecular weight of secreted and intracellular HtrAs, it became apparent that secreted HtrA has a lower molecular weight of approximately 50 kDa indicating removal of the signal peptide during secretion (Löwer et al. 2008). In addition, autocleavage of the C-terminus of N-terminal GST-tagged HtrA was observed, resulting in an active ~45 kDa protein presumably lacking the PDZ2 domain (Hoy et al. 2013; Löwer et al. 2008). For H. pylori HtrA, further N-terminal autoprocessing was detected HtrA. In a detailed study, the cleavage sites between amino acids H46 /D47 and K50 / D51 were determined by Edman sequencing. It could be shown that the N-terminal region of HtrA is important for higher order oligomerization and activity (Albrecht et al. 2018). This finding has been supported by structural analyses of H. pylori HtrA (Zhang et al. 2019; Perna et al. 2014). The crystal structure of H. pylori HtrA revealed that the N-terminal domain stabilizes the HtrA trimer, which is important for its protease activity (Zhang et al. 2019). Autoprocessing has been observed for other bacterial HtrAs as well. In contrast to H. pylori, in E. coli, Salmonella Typhimurium, or Yersinia enterocolitica, truncation led to an inactivation of DegP (Abfalter et al. 2016) suggesting that an increase of truncated HtrA reflects the need to remove active HtrA after disintegration of oligomers (Krojer et al. 2002).

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As a bifunctional protein, HtrA proteins act as a serine protease and a chaperone. These activities are crucially important for bacterial physiology as HtrA binds, refolds, or degrades misfolded proteins under environmental stress conditions (Zarzecka et al. 2019) (Fig. 2b). H. pylori HtrA is a highly active protein whose structural integrity and oligomeric activity are maintained under stress conditions (Hoy et al. 2013). Analyses of the PDZ domains revealed that the first PDZ motif is important for substrate cleavage, whereas both PDZ domains are dispensable for the chaperone activity. Moreover, the PDZ1 domain is also important for bacterial growth under stress conditions, which could be explained by the fact that the PDZ1 domain is also critical for the oligomerization of H. pylori HtrA (Zarzecka et al. 2021).

2.1.2

H. pylori HtrA Cleaves Folded Cell Adhesion Proteins to Disrupt the Epithelial Barrier

The finding that HtrA plays a crucially important role in H. pylori pathogenesis is based on the observation that HtrA specifically cleaves folded E-cadherin and its orthologues on the surface of infected gastric epithelial cells (Hoy et al. 2010; Bernegger et al. 2022a) (Fig. 1b). During infection, H. pylori sheds the extracellular domain of E-cadherin as a 90 kDa fragment in the supernatant of infected cells (Weydig et al. 2007; Schirrmeister et al. 2009). In a previous report, H. pylorimediated E-cadherin shedding has been associated with the activation of A disintegrin and metalloprotease 10 (ADAM10) (Schirrmeister et al. 2009). However, pharmacological inhibition and siRNA-mediated downregulation of ADAM10 expression revealed that activated ADAM10, matrix metalloproteinase 3 (MMP3), and MMP7 contribute to E-cadherin cleavage, but HtrA is the main protease in this phenomenon (Hoy et al. 2010; Bernegger et al. 2021). E-cadherin is the key molecule in adherence junctions, implicated in intercellular adhesion of epithelial cells. HtrA mediates ectodomain shedding of E-cadherin resulting in a local opening of adherens junctions allowing bacterial transmigration of H. pylori to the basal compartment of polarized gastric epithelial cells (Hoy et al. 2010). At the molecular level, HtrA targets signature sites containing the [VITA]-[VITA]-x-x-D-[DN] sequence pattern, which has been identified by mass spectrometry-based proteomics and Edman degradation (Schmidt et al. 2016b). These cleavage sites are located in the extracellular domain of E-cadherin, which represent calcium ion binding sites. The binding of calcium ions is a prerequisite for proper adhesive functions of E-cadherin and required to form homophilic interactions with the E-cadherin of the neighboring cell. In an in vitro cleavage assay, the addition of calcium efficiently blocks HtrA-mediated E-cadherin cleavage (Schmidt et al. 2016a). Hence, it was speculated that targeting the signature sites of E-cadherin explains the fragmentation pattern in in vitro experiments, while the formation of the stable 90 kDa fragment requires an additional cleavage site in the E-cadherin molecule that is unaffected by calcium ion binding. Accordingly, the 698 AQPV↓EA709 linker region between the EC5 and the transmembrane domain of

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E-cadherin was recently identified as an HtrA cleavage site with the valine in the P1 position, which is responsible for the release of the soluble extracellular domain (Bernegger et al. 2020). E-cadherin was the first biologically significant substrate identified for H. pylori HtrA and plays a crucial role in H. pylori pathogenesis (Hoy et al. 2010). In addition to E-cadherin, several other host cell-derived HtrA substrates have been described. Using a proteomic approach, which also confirmed the well-characterized target Ecadherin, the human desmosome component desmoglein-2 was found to be a novel extracellularly exposed H. pylori HtrA substrate (Bernegger et al. 2021). Additionally, the TJ proteins claudin-8 and occludin were identified as HtrA targets, explaining the disruption of TJs during infections (Tegtmeyer et al. 2017) (Fig. 1b). Cleavage sites have been modelled for the HtrA ortholog in Campylobacter jejuni (C. jejuni) at A58 ↓N59 in claudin-8 (Sharafutdinov et al. 2020) and A224 ↓T225 in occludin (Harrer et al. 2019), which may also be cut by H. pylori HtrA. Collectively, these data indicate that the major function of secreted H. pylori HtrA is to locally open intercellular adhesions. Previous reports have described other H. pylori factors that open cell–cell junctions, such as VacA or urease (Papini et al. 1998; Tombola et al. 2001; Lytton et al. 2005), but whether these factors operate independently of HtrA has not been addressed. Characterization of HtrA revealed that secreted HtrA targets intercellular adhesions at all three levels, including TJs, AJs, and desmosomes (Fig. 1b). This complex mechanism allows H. pylori to transmigrate across the polarized gastric epithelium, a process which can be observed not only in experimental in vitro infection experiments (Hoy et al. 2010; Tegtmeyer et al. 2016, 2017; Sharafutdinov et al. 2023) (Fig. 1b), but also in biopsies from H. pylori-infected individuals in vivo (Necchi et al. 2007, 2019). Thus, the question arose as to why H. pylori uses this elaborate mechanism as part of its cancer-associated pathogenesis? After apical colonization, a proportion of H. pylori is localized in the basolateral compartment (Hoy et al. 2010). In fact, the T4SS of H. pylori directly contacts basolateral β1-integrin receptors for efficient CagA translocation and tyrosine phosphorylation (Kwok et al. 2007). HtrA-mediated disruption of intercellular adhesions allows bacterial transmigration across polarized epithelial cells to bind β1-integrin for CagA delivery over the basolateral membranes (Tegtmeyer et al. 2017), which subsequently controls cancer-associated signaling (Fig. 1b). Interestingly, α5β1-integrins bind the tripeptide arginine-glycine-aspartic acid (RGD) in fibronectin (Ruoslahti and Pierschbacher 1987) to establish cell–matrix adhesions of gastric epithelial cells. The observation that HtrA also cleaves fibronectin (Hoy et al. 2010) (Table 1) suggests that HtrA directly facilitates CagL binding to α5β1-integrins and CagA translocation (Fig. 1b). In summary, HtrA is a fascinating factor that paves the way for H. pylori to promote full pathogenesis.

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3 Concluding Remarks Proteases of host- and bacterial origin play important roles in the onset and progression of H. pylori-related pathology (Posselt et al. 2013). In the context of a worsening antibiotic crisis, novel treatment options for H. pylori infection and intervention strategies for H. pylori-associated disease are in demand (Tshibangu-Kabamba and Yamaoka 2021). The essential gene products of H. pylori, such as the protease HtrA, may serve as attractive targets for the development of future drug options for eradication. Of note, HtrA is essential in H. pylori, but was found to be dispensable in C. jejuni (Tegtmeyer et al. 2016) and is thought to be a non-essential gene in most commensal gut microbiota. Bacteria-specific HtrA inhibitors could offer advantages over conventional antibiotics by targeting only microbials whose survival is critically dependent on HtrA activity such as H. pylori, while leaving the bulk of the gut microbiota unaffected. Moreover, extracellular proteases are ideal candidates for inhibitory compounds because they can directly block the enzymatic reaction with host cell substrates without the need to cross the bacterial cell wall. Although our understanding of bacterial proteases is still incomplete, in summary, targeting bacterial proteases holds great potential for new drug development because of their serious impact on pathogenesis. Acknowledgements The work of SW was supported by the grant I_4360 and P_31507 from the Austrian Science Fund (FWF).

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Clinical Management of Gastric Cancer Treatment Regimens Juliette Boilève, Yann Touchefeu, and Tamara Matysiak-Budnik

Abstract Gastric cancer is the fifth most common cancer and the fourth leading cause of cancer-associated death in the world. Endoscopic resection can be the treatment in selected cases of very early gastric cancer. Surgery is recommended for tumors that do not meet the criteria for endoscopic resection or for tumors with lymph node invasion but without distant metastases. Gastrectomy should include D2 lymphadenectomy without splenectomy. Perioperative or adjuvant chemotherapy improves survival and is recommended in locally advanced gastric cancer (>T1 and/or with lymph nodes positive). In locally advanced cancer with microsatellite instability (MSI), immunotherapy should be considered. Advanced unresectable or metastatic gastric cancer has a poor prognosis. The basis of the treatment is cytotoxic chemotherapy, with platinum and fluoropyrimidine doublet in the first line. Targeted therapies can be combined with chemotherapy. Trastuzumab (anti-HER2) is recommended in the first line in HER2-positive cancer. Ramucirumab (anti-VEGFR2) is recommended in the second line, in addition to paclitaxel chemotherapy. Zolbetuximab (anti-Claudine 18.2) should also be considered in the first line in Claudine 18.2-positive cancer. Immunotherapy can also be associated with chemotherapy in the first line of PD-L1-positive cancer. In HER2-positive and PD-L1-positive cancer, adjunction of trastuzumab and immunotherapy should be considered. In advanced and metastatic cancer with microsatellite instability (MSI), immunotherapy should be the first choice depending on its availability. Important progress has been made in recent years in the treatment of gastric cancer, especially due to a better understanding of molecular characteristics and heterogeneity of this disease. New targets and therapeutic approaches are being developed, which will very likely lead to changes in the management of gastric cancer. J. Boilève · Y. Touchefeu · T. Matysiak-Budnik (B) Institut Des Maladies de L’Appareil Digestif (IMAD), Nantes Université, CHU Nantes, Hépato-Gastroentérologie, Inserm CIC 1413, 44000 Nantes, France e-mail: [email protected] J. Boilève e-mail: [email protected] Y. Touchefeu e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 S. Backert (ed.), Helicobacter pylori and Gastric Cancer, Current Topics in Microbiology and Immunology 444, https://doi.org/10.1007/978-3-031-47331-9_11

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1 Introduction Gastric cancer (GC) is the fifth most commonly occurring cancer and the fourth leading cause of cancer-associated death in the world (Sung et al. 2021). GC is a disease of a globally poor prognosis, with an overall 5-year survival rate of about 30%, all stages included. This survival strictly depends on the stage of the disease at diagnosis and is about 72% for localized disease, 33% for the disease with regional extension, and 6% for advanced disease with distant metastases (Sung et al. 2021). There is an anatomical distinction between cardia and non-cardia GCs. These two entities differ in terms of molecular characteristics, risk factors, epidemiological trends, and specific treatment modalities (Colquhoun et al. 2015). Helicobacter pylori infection is the most important risk factor of non-cardia GC (see Chap. 2 of this book). Other risk factors include the Epstein–Barr virus (EBV) infection, autoimmune atrophic gastritis, family history of GC, and some ethnic origins. On the other hand, obesity and gastroesophageal reflux are associated with cardia GC (Smyth et al. 2020b). The treatment strategies depend on the disease stage at the diagnosis and the general status of the patient, including factors like age and comorbidities. Recently, the systematic treatment of localized or advanced GC has undergone significant progress, with the emergence of new molecules and biomarkers to guide prescriptions. The aim of this review is to discuss the different therapeutic options in the management of GC.

2 Treatment of Localized Disease 2.1 Endoscopic Resection of Early Gastric Cancer For localized disease without metastases, surgical or endoscopic resection remains an essential treatment with curative intent. Endoscopic resection is the initial treatment for selected cases of very early GC, i.e. T1a (tumors that invade the lamina propria or muscularis mucosae), if they are confined to the mucosa, well-differentiated, with a tumor size of less than 2 cm and non-ulcerated, but expanded endoscopic resection criteria concerning size, depth of submucosal invasion, and grade of differentiation have been also proposed (Probst et al. 2017). There are two different endoscopic resection techniques: endoscopic mucosal resection and endoscopic submucosal dissection. European guidelines recommend endoscopic submucosal dissection as the treatment of choice for most early GCs. Endoscopic mucosal resection is an acceptable option for lesions smaller than 10– 15 mm with a low probability of advanced histology (Pimentel-Nunes et al. 2015). Three meta-analyses (Park et al. 2011; Lian et al. 2012; Facciorusso 2014) have compared endoscopic mucosal resection with endoscopic submucosal dissection for the treatment of early GC. Endoscopic submucosal dissection obtained higher en

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bloc resection rates (92% vs. 52%; OR 9.69, 95% CI 7.74–12.13) and histologically complete resection rates (82% vs. 42%; OR 5.66, 95% CI 2.92–10.96), and lower recurrence frequency (1% vs. 6%; OR 0.10, 95% CI 0.06–0.18), even for lesions smaller than 10 mm. However, endoscopic submucosal dissection was associated with a longer procedure time (more than 59.4 min; 95% CI 16.8–102) and higher risk of perforation (4% vs. 1%; OR 4.67, 95% CI 2.77–7.87). This data suggest that endoscopic submucosal dissection is a treatment of choice, but with a higher risk of complications, and therefore, should be reserved for the expert centers.

2.2 Surgery Surgery remains the basis of treatment in localized GC. It is recommended for stage T1 tumors (tumors that invade lamina propria, muscularis mucosae or submucosa), which do not meet the criteria for endoscopic resection, and for tumors >T1 and/or with lymph node-positive and no metastases. The two main procedures are distal gastrectomy or total gastrectomy. In distal gastrectomy, two thirds of the stomach is resected the proximal stomach is anastomosed to the small bowel. In total gastrectomy, the anastomosis is between the esophagus and the small bowel. The optimal extent of lymphadenectomy has been the subject of debate during recent decades. A D1 dissection removes the perigastric lymph nodes and the left gastric artery lymph nodes. In D2 dissection, all D1 lymph nodes and the lymph nodes along the proper and common hepatic artery, splenic artery, and celiac axis, are removed. The current AJCC/UICC TNM (8th edition) classification recommends the excision of a minimum of 15 lymph nodes for reliable staging. In Asian countries, observational and randomized trials have demonstrated that D2 resection leads to superior outcomes compared with D1 resection (Kinoshita et al. 1993; Wu et al. 2006). In European countries, trials did not show an initial survival benefit after D2 dissection, but long-term follow-up showed less loco regional recurrences and lower gastric cancer-related mortality after D2 (Hartgrink et al. 2004; Songun et al. 2010). Currently, international consensus holds that gastrectomy for non-early GC should include D2 lymphadenectomy without splenectomy. Laparoscopic surgery has recently become an accepted surgical option for GC. A laparoscopic approach has the potential advantage of reducing postoperative morbidity and length of recovery time. Today, laparotomy is an acceptable approach to perform total or partial gastrectomy with D2 lymphadenectomy for GC. A laparoscopic approach can be offered selectively in expert hands. Robotic-assisted gastrectomy has shown similar oncologic outcomes in terms of survival and lymph node yield compared to conventional laparoscopic gastrectomy. As technology advances, GC surgery will likely become increasingly minimally invasive and will likely take advantage of rapidly developing robotic technologies.

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2.3 Adjuvant Treatments: Chemotherapy and Radiotherapy Several studies have shown that survival in GC may be improved by adding adjuvant or neoadjuvant treatment (chemotherapy ± radiotherapy). The type of adjuvant treatment currently recommended differs among different countries and continents, following the corresponding studies: in Europe, a perioperative chemotherapy has been shown to be the most effective, in Asian countries an adjuvant postoperative chemotherapy is usually proposed, while in the USA, a postoperative radiochemotherapy has been traditionally a preferred option, although more recently a tendency to a perioperative chemotherapy is observed.

2.3.1

Perioperative Chemotherapy

Perioperative chemotherapy became the standard for the management of resectable GC in Europe for patients with tumors >T1 and/or with lymph nodes positive. Two randomized studies initially demonstrated the efficacy of perioperative chemotherapy versus surgery alone. The first was the UK MAGIC trial which showed an improvement in 5-year survival rate from 23 to 36% (hazard ratio (HR) for death 0.75; 95% confidence interval (CI) 0.60–0.93; P ¼ 0.009) with six cycles (three pre- and three postoperative) of ECF protocol (epirubicin, cisplatin and 5-fluorouracil) compared with surgery alone in patients with resectable stage 2 and 3 GCs (Cunningham 2006). Another phase III French trial showed similar results with the use of perioperative cisplatin and 5-fluorouracil (5-year rate from 24 to 38%) (Ychou et al. 2011). More recently, in 2019, a phase II-III study, comparing eight cycles of perioperative 5-fluorouracil, leucovorin, oxaliplatin, and docetaxel (FLOT) versus six cycles of ECF/epirubicin, cisplatin, and capecitabine (ECX) showed a significant improvement in the primary endpoint of overall survival (hazard ratio [HR] 0·77, 95% CI 0·63– 0·94; median overall survival 50 months [38·33 to not reached] versus 35 months [27·35–46·26] (Al-Batran et al. 2019). Perioperative chemotherapy with FLOT is now the recommended standard of care for patients with locally advanced cancer (>T1 and/or with lymph nodes positive gastric), who are able to tolerate a triple cytotoxic drug regimen. In all the trials, the postoperative chemotherapy regimen was the same as the preoperative regimen. Continuation of the same chemotherapy in patients who poorly responded to the preoperatively given cycles is a matter of debate that is still unsolved. However, in the absence of a clinical trial, continuation of the same chemotherapy is recommended.

2.3.2

Postoperative Chemotherapy

Postoperative chemotherapy for patients not treated preoperatively has been shown to be effective. Several Asian studies have shown improvements in overall survival using adjuvant chemotherapy, with capecitabine and S-1 (a combination of tegafur,

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gimeracil, and oteracil) (Shinichi et al. 2007; Bang et al. 2012). Thus, the current Japanese guidelines recommend postoperative adjuvant chemotherapy with S-1 monotherapy or oxaliplatin and capecitabine (Park et al. 2015). A large patientlevel meta-analysis of adjuvant chemotherapy in GC has confirmed a 6% absolute benefit in 5-year overall survival for 5-fluorouracil-based chemotherapy compared with surgery alone (HR 0.82, 95% CI 0.76–0.90, p < 0.001) in all subgroups tested, including Western patients (Dikken et al. 2010). However, adjuvant chemotherapy is less well tolerated than neoadjuvant chemotherapy and neoadjuvant chemotherapy leads to tumor downsizing; therefore, a perioperative approach is recommended if possible, so that more patients can benefit from systemic treatment even if the postoperative chemotherapy cannot be delivered.

2.3.3

Chemoradiotherapy

The postoperative chemoradiotherapy after surgery for GC was for the first time evaluated in a randomized study performed in the USA in 2001 and became since then a standard of care in the USA (Macdonald et al. 2001). The CRITICS study, a randomized phase III trial, concluded that postoperative chemoradiotherapy did not improve overall survival compared with postoperative chemotherapy in patients with resectable GC treated with adequate preoperative chemotherapy and surgery (Cats et al. 2018). In the per-protocol analysis, adjuvant chemotherapy was even superior to chemoradiation after D2 surgery for GC (de Steur et al. 2021). The ARTIST and ARTIST II trials showed that the addition of radiotherapy to adjuvant chemotherapy did not reduce the rate of recurrence after gastrectomy with D2 lymphadenectomy, even with lymph-node positive disease (Lee et al. 2012; Park et al. 2015, 2021). Therefore, chemoradiotherapy is not recommended for patients who have received complete surgery with appropriate lymphadenectomy. However, for patients who have not received preoperative chemotherapy and have undergone an incomplete D2 lymphadenectomy, adjuvant chemoradiotherapy can be considered (Macdonald et al. 2001; Dikken et al. 2010). For patients who have undergone surgery with involved margins (R1), adjuvant radiotherapy or radiochemotherapy might be considered, but is not a standard (Ho et al. 2017). Preoperative chemoradiotherapy has only been evaluated in phase III trials for tumors of the esogastric junction. For gastric tumors, it did not show statistically significant efficacy in a randomized trial that was closed early due to poor accrual (Stahl et al. 2009). The ongoing TOPGEAR trial compares perioperative epirubicin, cisplatin, 5-FU (ECF) chemotherapy with perioperative ECF chemotherapy combined with a preoperative chemoradiotherapy. The results of the interim analysis showed that preoperative chemoradiation can be safely delivered to the vast majority of patients without a significant increase in treatment toxicity or surgical morbidity (Leong et al. 2017).

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Other Adjuvant Treatments

The interest in targeted therapies associated with adjuvant chemotherapy is the subject of several ongoing studies. The amplification of HER2 (also known as ERBB2) and overexpression of the HER2 protein occur in approximately 15% of patients with GCs (The Cancer Genome Atlas Research Network 2014). HER2-targeted therapies are being evaluated in combination with perioperative chemotherapy for HER2-positive tumors. In particular, the INNOVATION study compares the efficacy of perioperative chemotherapy alone or in combination with trastuzumab ± pertuzumab (Wagner et al. 2019). Immunotherapy could also be an interesting approach in an adjuvant setting as shown in CheckMate 577 (Kelly et al. 2021) study evaluating nivolumab in resected esophageal or gastroesophageal junction cancer. In this randomized phase III trial, adjuvant nivolumab was studied in patients with esophageal or gastroesophageal junction cancer resected after neoadjuvant chemoradiotherapy and with residual pathologic disease. The disease-free survival was significantly longer among patients who received adjuvant nivolumab (22.4 months vs 11.0 months; HR: 0.69; 96.4%CI, 0.56 to 0.86; p < 0.001). The VESTIGE trial, a randomized phase II, is currently in progress. The study compares adjuvant immunotherapy in patients with resected esophageal, gastroesophageal junction, and GC following preoperative chemotherapy with high risk for recurrence (Smyth et al. 2020a, b).

2.3.5

A Special Case of Microsatellite Instability (MSI) Tumors

Microsatellites are repeated sequences of the genome. Microsatellite instability (MSI) status is the consequence of a deficiency in the DNA mismatch repair system (MMR). The prognosis of patients with MSI GC who have undergone resection is better than the non-MSI GC. The addition of postoperative chemotherapy does not seem to have a beneficial effect and adjuvant chemotherapy should be avoided, according to retrospective data (Pietrantonio et al. 2019). For perioperative treatment of MSI GC, data from randomized trials did not show a benefit of perioperative chemotherapy for patients with MSI GC, but this was before the use of taxanes including regimens such as FLOT (association of 5-fluorouracil, leucovorin, oxaliplatin, and docetaxel). Data from MSI patients treated with FLOT showed a better response rate (Al-Batran et al. 2021). Therefore, if a downstaging before surgery is desired, FLOT is recommended. In the future, immunotherapy will probably be an alternative to MSI GC (André et al. 2023). Indeed, the phase II NEONIPIGA study evaluated the benefit of preoperative immunotherapy with nivolumab [anti-PD-1 (Programmed Death-Ligand 1) antibody] and ipilimumab (anti-CTLA-4) and postoperative immunotherapy, with nivolumab alone, in patient with GC or esophagogastric MSI phenotype cancer: the study showed in the 29 patients analyzed, a histological complete response rate of 58.6%. The DANTE study (Al-Batran et al. 2022), a phase IIb, showed beneficial effects of atezolizumab (anti-PD-L1) combined with FLOT versus FLOT alone on

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pathological stage and regression. A randomized phase III trial, MATTERHORN, investigating the efficacy and safety of neoadjuvant-adjuvant durvalumab (anti-PDL1) and FLOT chemotherapy followed by adjuvant durvalumab monotherapy in patients with resectable gastric/gastroesophageal junction cancer is ongoing (Janjigian et al., 2022b). Early results confirm that immunotherapy, combined with chemotherapy and surgery, represents a new therapeutic approach for improving outcomes.

3 Treatment of Advanced and Metastatic Disease Locally advanced unresectable or metastatic GC has a poor prognosis. The overall survival of patients with GC treated with chemotherapy is 1 year (Glimelius et al. 1997). Chemotherapy improves survival and quality of life for patients with locally advanced unresectable or metastatic GC. The management of advanced GC has evolved considerably in recent years. Prior to the 1980s, the best supportive care was the usual strategy in advanced GC. Then, cytotoxic chemotherapies started to be introduced, first as monotherapy and then as bi- or tri-therapy. Subsequently, targeted therapies have been developed including trastuzumab (anti-HER2) and ramucirumab (antiangiogenic). Finally, immunotherapy has made its appearance in the treatment of GC, with nivolumab or pembrolizumab (anti-PD-1). In the near future, new approaches will allow the use of combo therapies, such as the combination of antiHER2 and immunotherapy, and new targets will be used, such as zolbetuximab (anti-claudin-18.2).

3.1 Cytotoxic Chemotherapies Cytotoxic chemotherapy improves survival in comparison to the best supportive care, and a combination of chemotherapy improves survival compared to mono chemotherapy with 5-fluorouracil (Wagner et al. 2017). Standard first-line chemotherapy for GC is a platinum-fluoropyrimidine doublet. The platinum drugs the most commonly used are oxaliplatin and cisplatin. Fluoropyrimidines may be administered as an infusion (5-fluorouracil (5-FU)) or as oral treatment (capecitabine or S-1 which includes tegafur, gimeracil, and oteracil). S-1 is used in Asian patients and is less well tolerated. Cisplatin and oxaliplatin are equivalent in terms of efficacy (David et al. 2008; Al-Batran et al. 2008), but they are different in terms of side effects. In older patients (aged > 65 years), oxaliplatin has a superior safety profile and is more effective (Al-Batran et al. 2008). The phase III GO-2 trial showed that for elderly or frail patients, reduced-dose oxaliplatin-based chemotherapy is less toxic with comparable survival outcomes (Hall et al. 2021). The efficacy of irinotecan with 5-FU has also been evaluated. A phase III randomized study comparing irinotecan combined with 5-FU to cisplatin combined with

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5-FU in patients with advanced gastric or esophageal GC did not show a significant time to treatment failure and overall survival superiority, but the results of noninferiority were borderline (Dank et al. 2008). Another phase III study comparing epirubicin, cisplatin, and capecitabine (ECX) with irinotecan and 5-FU demonstrated a longer time to treatment failure with irinotecan and 5-FU than ECX (Guimbaud et al. 2014). Irinotecan and 5-FU can be an alternative option for patients who do not tolerate platinum chemotherapy. Triplet chemotherapy with the addition of docetaxel to a doublet chemotherapy with cisplatin and 5-FU improved overall survival and response rate in advanced GC patients in a phase III randomized trial but were associated with a higher toxicity (Van Cutsem et al. 2006). However, in a phase III, Japanese randomized trial (Yamada et al. 2019), in which patients with advanced GC were randomly assigned to receive docetaxel plus cisplatin and S-1 or cisplatin and S-1, the addition of docetaxel did not improve overall survival. Finally, GASTFOX Study, a phase III trial, showed that addition of docetaxel to 5-fluorouracil and oxaliplatin significantly improved progression-free survival and overall survival in first-line treatment of advanced HER2 negative gastric or gastro-esophageal junction adenocarcinomas (Zaanan et al. 2023). mFLOT/TFOX can be considered as a new therapeutic option for patients eligible for a triplet chemotherapy. In second-line treatment and beyond, the chemotherapy options are paclitaxel, docetaxel, and irinotecan, with equivalent efficacy but different toxicity profiles (Hironaka et al. 2013; Wagner et al. 2017). More recently, the phase III TAGS trial showed that trifluridine and tipiracil (an oral nucleoside analog plus thymidine phosphorylase inhibitor) improved overall survival compared with placebo and was well tolerated (Shitara et al. 2018a).

3.2 Targeted Therapies Targeted therapy is a drug treatment that targets specific genes and proteins of cancer cells to stop the cancer from growing and spreading. In the randomized phase III TOGA trial (Bang et al. 2010), the addition of trastuzumab (a monoclonal antibody against HER2) to cisplatin and fluoropyrimidine chemotherapy was compared with chemotherapy alone, in patients with HER2 overexpressing (HER2 immunohistochemistry score 3+ of HER2 immunohistochemistry score 2+ and fluorescent in situ hybridization positive). The addition of trastuzumab improved median overall survival with 13.8 months for trastuzumab and chemotherapy patients and 11.1 months in patients with chemotherapy alone (HR 0.74, 95% CI 0.60–0.91, p = 0,0046). Thus, trastuzumab in addition to first-line chemotherapy is recommended in patients with HER2 overexpressing GC. In the phase III RAINBOW trial, the addition of ramucirumab, an anti-vascular endothelial growth factor receptor 2 (VEGFR2) antibody, to paclitaxel improved the overall survival in patients previously treated for advanced GC, compared with placebo plus paclitaxel (median 9.6 months [95% CI 8.5–10.8] vs 7.4 months [95%

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CI 6.3–8.4], hazard ratio 0.807 [95% CI 0.678–0.962]; p = 0.017) (Wilke et al. 2014). In the phase III REGARD trial, ramucirumab monotherapy compared with placebo demonstrated improved overall survival (5.2 months to 3.8 months, hazard ratio 0.776, 95% CI 0.603–0.998; p = 0.047), but with limited response rates (Fuchs et al. 2014). Trials evaluating second-line lapatinib and trastuzumab emtansine in patients with HER2-positive GC who have progressed on trastuzumab have been negative (ThussPatience et al. 2017). Trastuzumab-deruxtecan is an antibody–drug conjugate in which trastuzumab is linked with deruxtecan, an anti-cancer drug that interrupts DNA replication in cancer cells. An Asian randomized phase II trial, DESTINYGastric 01, evaluated trastuzumab-deruxtecan, compared with chemotherapy in HER2-positive pre-treated GC in the third line. Trastuzumab-deruxtecan led to significant improvements in objective response rate (51% vs. 14%; P < 0.001) and overall survival (median 12.5 vs. 8.4 months; hazard ratio for death, 0.59; 95% confidence interval, 0.39 to 0.88; P = 0.01) (Shitara et al. 2020a). DESTINY-Gastric 02 (Daiichi Sankyo, Inc. 2022), a phase II with Western patients, demonstrated a confirmed objective response rate with trastuzumab-deruxtecan of 38.0% (95% CI, 27.3–49.6) with an acceptable safety profile, in patients with HER-positive gastric or oesophageal-GC who progressed on or after trastuzumab-containing regimen. DESTINY-Gastric 03 (Janjigian et al. 2022a) is a phase Ib/II in progress, investigating the safety, tolerability, pharmacokinetics, immunogenicity, and preliminary antitumor activity of trastuzumab-deruxtecan alone or in combination with chemotherapy and/or immunotherapy in HER2-positive advanced/metastatic gastric/ gastroesophageal junction and esophageal adenocarcinoma patients. Finally, a phase III study is in progress, DESTINY-Gastric 04 (Daiichi Sankyo, Inc. 2023), comparing trastuzumab-deruxtecan in patients with HER2 positive metastatic or unresectable gastric or gastroesophageal junction adenocarcinoma, who have progressed on or after a trastuzumab-containing regimen.

3.3 Immunotherapies Immune checkpoint inhibitors are a type of immunotherapy that blocks checkpoint proteins in T cells and some cancer cells. Some immune checkpoint inhibitors act against checkpoint proteins such as PD-1, PD-L1 or CTLA-4. PD-L1 expression on tumor cells and tumor-associated immune cells (combined positive score (CPS)) has shown better enrichment for the efficacy of immune checkpoint inhibitors than tumor cell PD-L1 expression in advanced GC (Kulangara et al. 2018). The Asian randomized phase III ATTRACTION-4 showed that nivolumab combined with oxaliplatin-based chemotherapy significantly improved progressionfree survival, but not overall survival, in Asian patients with untreated, HER2negative, unresectable advanced or recurrent gastric or gastroesophageal junction cancer (regardless of PD-L1 expression) (Kang et al. 2022). The phase III CheckMate 649 trial (Janjigian et al. 2021b) evaluated the addition of nivolumab (anti-PD-1) to

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the first-line chemotherapy (capecitabine and oxaliplatin or 5-FU and oxaliplatin) in gastric, esophagogastric junction and esophageal adenocarcinoma. Nivolumab plus chemotherapy resulted in significant improvement in overall survival (hazard ratio [HR] 0.71 [98.4% CI 0.59–0.86]; p < 0.0001) and progression-free survival (HR 0.68 [98% CI 0.56–0.81]; p < 0.0001) as compared to chemotherapy in PD-L1 CPS ≥ 5 patients (minimum follow-up, 12.1 months), with an acceptable safety profile. In the phase III KEYNOTE-062 trial (Shitara et al. 2020b), pembrolizumab monotherapy was non-inferior to cisplatin and fluoropyrimidine chemotherapy for overall survival in patients with PD-L1 CPS ≥ 1 with gastric or esophagogastric junction, but was not superior for overall survival and progression-free survival. The KEYNOTE-590 trial (Kato et al. 2019) compared pembrolizumab plus chemotherapy to placebo plus chemotherapy and showed improvement in overall survival and progression-free survival in patients with locally advanced of metastatic esophageal cancer or gastroesophageal junction cancers with expression PD-L1 CPS ≥ 10, treated with pembrolizumab plus chemotherapy. However, KEYNOTE-590 included relatively few patients with adenocarcinoma. More recently, the interim analysis of KEYNOTE-589 trial confirmed the overall survival benefit of first-line immunotherapy with pembrolizumab plus chemotherapy (fluoropyrimidine and platinum chemotherapy) in advanced GC, with any degree of expression of PD-L1 (Helwick 2023a). Furthermore, patients with MSI-H GC have high response rates and excellent long-term outcomes when treated with anti-PD1 monotherapy (Chao et al. 2021). The phase II KEYNOTE-158 trial, in patients with previously treated unresectable or metastatic MSI-H GC, demonstrated the clinical benefit of pembrolizumab monotherapy, with an objective response rate of 45.8% and a median progression-free survival of 11 months, with median overall and median duration of response not yet reached. In the phase III KEYNOTE-061 trial, pembrolizumab monotherapy did not significantly improve overall survival compared with paclitaxel as second-line therapy for advanced gastric or gastroesophageal junction cancer with PD-L1 CPS of 1 or higher, but an exploratory subgroup analysis suggested a benefit in advanced MSI-H GC (Shitara et al. 2018b). In 3rd or 4th-line treatment, the Asian trial ATTRACTION-02 (Boku et al. 2021), compared nivolumab to a placebo in patients with advanced gastric or gastroesophageal junction cancer who had been previously treated with two or more chemotherapy regimens, regardless of MSI status and PD-L1 expression. Median overall survival was 5.26 months in the nivolumab group and 4.14 months in the placebo group (hazard ratio 0·63, 95% CI 0·51–0·78; p < 0·0001).

3.4 Surgery for Metastatic GC Classically, metastatic GC is considered a non-operable disease. However, some data, initially coming from a retrospective series of patients, have suggested that in oligometastatic disease (not more than one metastatic site present) the surgery may bring a benefit in terms of overall survival (Mariette et al. 2013). More recently,

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prospective studies have addressed this issue. The randomized phase III REGATTA trial demonstrated that gastrectomy followed by chemotherapy did not show any survival benefit compared with chemotherapy alone in advanced oligometastatic GC (Fujitani et al. 2016). The non-randomized phase II AIO- FLOT3 trial reported favorable survival in patients with oligometastatic disease who received FLOT neoadjuvant chemotherapy followed by gastrectomy and resection of the metastatic site (Al-Batran et al. 2017). The results of two phases III studies, RENAISSANCE and SURGIGAST, are expected to explore the potential benefit of surgery in oligometastatic GC. The addition of hyperthermic intraperitoneal chemotherapy (HIPEC) to cytoreduction, in cases of limited peritoneal carcinosis, has been shown to be safe and may be associated with improved oncologic outcomes (Bonnot et al. 2019; Rau et al. 2021; Bonnot et al. 2021). The prognosis of GC patients with positive peritoneal cytology without gross peritoneal metastases is poor, and the survival benefit of gastrectomy for these patients has not been established. Perioperative chemotherapy may be a strategy for these patients (Higaki et al. 2017; Valletti et al. 2021; Kobayashi et al. 2022). Another technique has been developed to allow homogeneous application of intraperitoneal chemotherapy during laparoscopy, pressurized intraperitoneal aerosol chemotherapy (PIPAC), which could be an alternative for patients with unresectable peritoneal disease. Results of the PIPAC EstoK 01 trial, which is a randomized, controlled, multicenter, and phase II evaluated PIPAC, are pending (Eveno et al. 2018).

3.5 Supportive Care Supportive care is important for the well-being of patients with GC. Early integration of palliative care in oncology is a valuable approach because it also improves the quality of life at the end of life, not just shortly after palliative care is initiated (Vanbutsele et al. 2020). A randomized phase III trial demonstrated an increase in overall survival for patients who received multidisciplinary supportive care integrated into standard oncologic care compared with those who received standard care (median OS 14.8 months vs. 11.9%; hazard ratio 0.68; 95% CI, 0.51 to 0.9; P = 0.021) (Lu et al. 2021). Supportive care includes nutritional support and palliation of symptoms. Nutritional advice should be provided to minimize weight loss, which is multifactorial. A post hoc analysis demonstrated that weight loss of ≥3% during the first cycle of treatment was a negative prognostic factor for overall survival in patients with advanced GC (Mansoor et al. 2021). Hemorrhagic tumors can be treated by endoscopic coagulation, tranexamic acid, radiotherapy, embolization or, more rarely, by surgery (Kawabata et al. 2019). Dysphagia due to proximal gastric tumors may be improved by radiotherapy or endoscopic stent placement. Stenting is proposed for patients with severe dysphagia, especially those with short life expectancy, because the effect on swallowing is immediate, whereas radiotherapy (brachytherapy or

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external beam) takes several weeks to improve dysphagia (Bergquist et al. 2005; Yang et al. 2014).

3.6 Novel Targets and New Therapeutic Approaches Recent advances in molecular biology have led to the better identification of the tumor molecular characteristics, tumor microenvironment, and oncogenic signaling pathways. One of the challenges is to translate these recent findings into effective treatment for patients with GC. The phase III SPOTLIGHT trial (Laarhoven and Derks 2023), investigated the effect of claudin-18.2 targeting, using the monoclonal antibody zolbetuximab plus modified folinic acid, fluorouracil, and oxaliplatin regimen, in patients with claudin-18.2 positive, HER2-negative, untreated, locally advanced unresectable or metastatic gastric or gastroesophageal junction adenocarcinoma. The study showed an improvement in the progression-free survival (10.61 months in the zolbetuximab group vs. 8.67 months in the placebo group; hazard ratio [HR] 0.75, 95% CI 0.60–0.94; p = 0.0066). In the same way, the GLOW trial showed an improvement in overall survival in patients with claudin-18.2 positive/HER2-negative locally advanced or metastatic gastric adenocarcinoma, treated with zolbetuximab in combination with capecitabine and oxaliplatin (CAPOX). Median progression-free survival was 8.2 months in the zolbetuximab group compared to 6.8 months in the placebo group (hazard ratio [HR] = 0.687, P = 0.0007) and median overall survival was 14.4 vs 12.2 months (HR = 0.771, P = 0.0118), respectively (Helwick 2023b). A phase II (Wainberg et al. 2022) study investigated efficacy and safety of a fucosylated, humanized IgG1 anti-fibroblast growth factor receptor 2 isoform IIb (FGFR2b) monoclonal antibody bemarituzumab with modified 5-fluorouracil, leucovorin, and oxaliplatin (mFOLFOX6) in patients with FGFR2b-selected gastric or gastroesophageal junction adenocarcinoma, and showed promising clinical efficacy with bemarituzumab. In this exploratory phase 2 study, despite no statistically significant improvement in progression-free survival, treatment with bemarituzumab showed promising clinical efficacy. A phase III trial of bemarituzumab in combination with mFOLFOX6, is being investigated in patients with advanced FGFR2b overexpressing gastric or gastroesophageal junction adenocarcinoma. Other targets are promising, such as NTRK (Neurotrophic tyrosine receptor kinase) fusion or c-MET (another tyrosine kinase receptor) pathway. TRK (tropomycin receptor kinase) inhibitor therapies have been developed for patients with NTRK fusion-positive cancers, such as larotrectinib or entrectinib, and were associated with high response rates (Drilon et al. 2017, 2018; Cocco et al. 2018; Demetri et al. 2022). In the same way, c-MET pathway inhibitors have been studied, but the results have been disappointing, and further investigation is needed (Kiyozumi et al. 2018; El Darsa et al. 2020). A new personalized approach has also been developed. The VIKTORY trial (Lee et al. 2019) was designed to classify patients with metastatic GC based on

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clinical sequencing and focused on eight different biomarker groups (RAS aberration, TP53 mutation, PIK3CA mutation/amplification, MET amplification, MET overexpression, all negative, TSC2 deficient, or RICTOR amplification) to assign patients to one of the 10 associated clinical trials in second-line treatment. 14.7% of patients received biomarker-assigned drug treatment. The biomarker-assigned treatment cohort had encouraging response rates and survival when compared with conventional second-line chemotherapy. Another approach is chimeric antigen receptor T cells (CAR-T) therapy (Jiang et al. 2019; Olnes and Martinson 2021). Several studies with Claudin 18.2-spectifc T cells showed promising results. A phase I study by Zhan and co-workers (Zhan et al. 2019) showed a total objective response rate of 33.3%, with a median progressionfree survival of 130 days, without serious adverse events, treatment-related death or severe neurotoxicity in the study. A phase I trial by Qi and colleagues (Qi et al. 2022) showed in all patients a grade 3 or higher hematologic toxicity, cytokine release syndrome (CRS) grade 1 or 2 in 94.6% of patients, but no grade 3 or higher CRS or neurotoxicities and treatment-related deaths. In GC, the overall response rate and disease control rate reached 57.1% and 75.0%, respectively. Combinations of antiangiogenic and immune checkpoint blocking therapies are also being studied, with a combination of regorafenib and nivolumab in a phase Ib and ramucirumab and pembrolizumab in a phase Ia/b (Herbst et al. 2019; Fukuoka et al. 2020). Finally, the randomized phase III KEYNOTE-811 trial compares the combinations of trastuzumab plus pembrolizumab and chemotherapy for unresectable or metastatic, HER2-positive gastric or gastroesophageal junction adenocarcinoma. Results of the - first interim analysis showed that adding pembrolizumab to standard therapy with trastuzumab and chemotherapy results in a statistically significant, clinically meaningful improvement in progression-free survival compared with trastuzumab and chemotherapy alone (Janjigian et al. 2023) (Tables 1, 2 and Figs. 1, 2).

4 Concluding Remarks GC is a frequent and deadly disease that represents a global healthcare challenge. Treatment strategy depends on the stage of the disease at the time of diagnosis and it remains challenging. Given the limits of current treatments, primary prevention of GC by eradication of Helicobacter pylori appears as a valuable strategy and is being currently evaluated in several European projects. Many advances have been made in recent years in the treatment of GC, especially thanks to a better understanding of the molecular characteristics and heterogeneity of GC. The identification of new targets has led to the development of targeted therapies. So far, two of them, trastuzumab (anti-HER2) and ramucirumab (anti-VEGFR2) have been approved but several others have already shown their efficacy, like zolbetuximab (anti-Claudin 18.2) or are being currently tested. Immunotherapy is also being developed in GC and is now recommended in combination with chemotherapy for PD-L1-positive

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Table 1 Main positive phase III clinical trials in patients with advanced or metastatic gastric cancer Clinical trial

Regimen

Line Overall survival Progression-free (months) survival (months)

GASTFOX-PRODIGE 51

mFLOT/TFOX vs FOLFOX

1st line

15,08; 12.65 (HR 0.82, P = 0.048)

7.59; 5.98 (P = 0.007)

TOGA

Chemotherapy + trastuzumab vs chemotherapy

1st line

13.8; 11.1 (HR 0.74, P < 0.01)

6.7; 5.5 (HR 0.71, P < 0.01)

DESTINY-Gastric01

Trastuzumab-deruxtecan vs chemotherapy

2nd line

12.5; 8.4 (HR 0.59, P = 0.01)

5.6; 3.5 (HR 0.47, P NR)

HER2

VEGF REGARD

Ramucirumab vs placebo 1st line

5.2; 3.8 (HR 0.77, P = 0.047)

2.1; 1.3 (HR 0.48, P < 0.01)

RAINBOW

Paclitaxel + ramucirumab vs paclitaxel

1st line

9.6; 7.4 (HR 0.80, P = 0.017)

4.4; 2.9 (HR 0.63. P < 0.01)

ATTRACTION-04

Nivolumab + chemotherapy vs chemotherapy

1st line

17.5; 17.2 (HR 0.90, P = 0.257)

0.5; 8.3 (HR 0.68, P = 0.0007)

Checkmate-649

Nivolumab + chemotherapy vs chemotherapy

1st line

14.4; 11.1 (HR 7.7; 6.0 (HR 0.68, 0.71, P < 0.001) P < 0.001)

KEYNOTE-062

Pembrolizumab + chemotherapy vs chemotherapy

1st line

10.6; 11.1 (HR 0.91, non-inferiority)

2.1; 6.4 (HR 1.66, P NR)

KEYNOTE-590

Pembrolizumab + chemotherapy vs chemotherapy

1st line

13.5; 9.4 (HR 0.62, P = 0.001)

7.5; 5.5 (HR 0.51, P < 0.001)

KEYNOTE-509

Pembrolizumab + chemotherapy vs chemotherapy

1st line

12.9; 11.5 (HR 0.78, P < 0.0001

6.9; 5.6 (HR = 0.76, P < 0.0001)

KEYNOTE-061

Pembrolizumab vs paclitaxel

2nd line

9.1; 8.3 (HR 0.82, P = 0.042)

1.5; 4.1 (HR 1.27, P NR)

ATTRACTION-02

Nivolumab vs placebo

2nd line

5.3; 4.1 (HR 1.6; 1.45 (HR 0.63, P < 0.001) 0.60, P < 0.001)

KEYNOTE-811

Pembrolizumab + trastuzumab + chemotherapy

1st line

20.0; 16.9 (HR 0.87, P = 0.084)

10.0; 8.1 (HR 0.73)

SPOTLIGHT

Zolbetuximab + chemotherapy vs chemotherapy

1st line

18.23; 15.54 (HR 0.75, P = 0.0053)

10.61; 8.67 (HR 0.75, P = 0.0066)

GLOW

Zolbetuximab + chemotherapy vs chemotherapy

1st line

14.4; 12.2 (HR 0.771, P = 0.0118)

8.2; 6.8 (HR 0.687, P = 0.0007)

Immunotherapy

Claudin 18.2

Clinical Management of Gastric Cancer Treatment Regimens Table 2 Targets, frequency, and therapies in gastric cancer (Selim et al. 2019)

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Targets

Frequency (%)

HER2

15–20

Therapies Trastuzumab

MSI

10–30

Immunotherapy

PD L1 expression

40

Immunotherapy

Claudin 18.2

50–70

Zolbetuximab

Fig. 1 Algorithm for treatment of localized gastric cancer. This figure proposes an algorithm for the treatment of localized gastric cancer. In the case of stage IA, management by endoscopic resection or surgery may be proposed. For stages IB to III, perioperative or adjuvant treatment is recommended

cancers. New therapies are also being studied, as are new therapeutic approaches, notably by combining different targeted treatments. The results of several of these promising studies are expected in the coming years and will help to change the way GC is managed.

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Fig. 2 Algorithm for treatment of advanced and metastatic gastric cancer. This figure proposes an algorithm for the treatment of advanced or metastatic gastric cancer, based on the recommendations of expert societies, as well as therapeutic proposals based on phase III studies (*). Firstline treatment includes chemotherapy, which may be combined with trastuzumab in HER2 positive cancer, immunotherapy in PD-L1 positive cancer, and zolbetuximab in Claudin 18.2 positive cancer. In second- and third-line treatment, chemotherapy is recommended. Ramucirumab can be combined with paclitacel in second-line treatment

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