Fungal Extracellular Vesicles: Biological Roles (Current Topics in Microbiology and Immunology, 432) 3030833909, 9783030833909

Citation preview

Current Topics in Microbiology and Immunology

Marcio Rodrigues Guilhem Janbon   Editors

Fungal Extracellular Vesicles Biological Roles

Current Topics in Microbiology and Immunology Volume 432

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 Klaus Aktories, Faculty of Medicine, Institute of Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, Freiburg, Baden-Württemberg, Germany Arturo Casadevall, W. Harry Feinstone Department of Molecular Microbiology & Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA Richard W. Compans, Department of Microbiology and Immunology, Emory University, Atlanta, GA, USA Jorge E. Galan, Boyer Ctr. 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 Bernard Malissen, Parc Scientifique de Luminy, Centre d’Immunologie de Marseille-Luminy, Marseille, France Rino Rappuoli, GSK Vaccines, Siena, Italy

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. 2020 Impact Factor: 4.291, 5-Year Impact Factor: 5.110 2020 Eigenfaktor Score: 0.00667, Article Influence Score: 1.480 2020 Cite Score: 7.7, h5-Index: 38

More information about this series at http://www.springer.com/series/82

Marcio Rodrigues • Guilhem Janbon Editors

Fungal Extracellular Vesicles Biological Roles

Editors Marcio Rodrigues Instituto Carlos Chagas Fundação Oswaldo Cruz (Fiocruz) Curitiba, Paraná, Brazil

Guilhem Janbon Département de Mycologie Institut Pasteur Paris, France

ISSN 0070-217X ISSN 2196-9965 (electronic) Current Topics in Microbiology and Immunology ISBN 978-3-030-83390-9 ISBN 978-3-030-83391-6 (eBook) https://doi.org/10.1007/978-3-030-83391-6 © Springer Nature Switzerland AG 2021 This work is subject to copyright. All rights are reserved 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

Foreword

On the Discovery of Fungal Extracellular Vesicles and Its Resonance for Gram-Positive Bacteria Abstract This essay recounts the personal reminisces of the author involving the events leading to the discovery of fungal extracellular vesicles (exosomes) in 2005. The goal of this essay is to describe the background, thoughts, and events essential for that discovery that are not always captured by the scientific literature. With the clarity that only hindsight provides, it is apparent that a set of fortuitous events came together to create the conditions for the discovery of extracellular vesicles in fungi, which then led to the subsequent discovery of extracellular vesicles in gram-positive bacteria. Today, the study of extracellular vesicles in cell-walled microorganisms such as fungi and gram-positive bacteria is a thriving area of research, but major challenges remain for understanding this complex process, seven of which are listed here as major unsolved problems. Keywords Fungi, Gram-positive, Bacteria, Extracellular vesicles, Exosomes Today the study of function, structure, and biogenesis of microbial extracellular vesicles is a vibrant field. There is now general acceptance of the compelling evidence that cells from both unicellular and extracellular organisms produce, and use, extracellular vesicles for many functions ranging from export to intercellular communication (Fig. 1) (Brown et al. 2015; Rodrigues and Casadevall 2018; Freitas et al. 2019; Munhoz da Rocha et al. 2020). However, the road to scientific acceptance that these structures were biologically relevant and not just artifacts of culture conditions was long and hard. Scientific discovery is human endeavor, which makes any story of discovery replete with the strengths and foibles of our species, and the inevitable intervention by the hand of fortune, which is often the key element in tipping the scale. Here I describe the discovery of fungal extracellular vesicles through my personal reminisces and recount the challenges involves in gaining acceptance. Since the history of science cannot be easily gleaned from reading the literature, which often omits key details on how the process came about, my goal is to provide details of the v

vi

Foreword

Fig. 1 Watercolor representing proposed scheme for the synthesis of vesicles in fungal multivesicular body, release of vesicles at the cell membrane and transit through the cell wall. Painting was adapted from model proposed in (Casadevall et al. 2009)

discovery in light of the need to appreciate the history of any field as a necessary condition for understanding the workings of science (Casadevall and Fang 2015a).

Personal Reminiscences on the Discovery Fungal extracellular vesicles were discovered by Dr. Marcio Rodrigues in the summer of 2005 when he spent 3 months in the Casadevall laboratory doing a short sabbatical. Dr. Rodrigues had a longstanding interest in fungal lipids and had traveled to the

Foreword

vii

Albert Einstein College of Medicine to visit the Casadevall laboratory with the goal of making monoclonal antibodies (mAb) to glucosylceramide. Earlier, in his career, Dr. Rodrigues had made the fascinating observation that human antibodies to glucosylceramide inhibited the growth of Cryptococcus neoformans (Rodrigues et al. 2000). To follow up those studies he wanted to make murine mAbs, which would give him defined consistent reagents that would be more reliable than working with human polyclonal sera. His plan was to make them and return to continue his studies in Brazil. At the time, the Casadevall laboratory had a large research focus on studying mAbs to fungal antigens and was a natural place to generate those reagents. An earlier connection had occurred when Dr. Rodrigues’ mentor, Dr. Luis Travassos, had met this writer at the International Society of Human and Animal Mycoses (ISHAM) meeting in Buenos Aires in the spring of 2000 and where they discussed how antibodies to lipids could affect fungal growth. At the time, Dr. Travassos was perplexed by Dr. Rodrigues’ results, since lipids were envisioned as being part of the cell membrane, which is located inside the cell wall, and he did not believe large immunoglobulins could penetrate through the cryptococcal capsule and cell wall. This writer recalls that the conversation went on for hours for he greatly enjoyed the enormous breath of Dr. Travassos knowledge and his quick intelligence. This writer mentioned to Dr. Travassos about older papers revealing vesicles being released towards the cell wall (Takeo et al. 1973a) suggesting how lipids could reach the cell wall. In that regard, our laboratory had made mAbs to cryptococcal melanin, which is deposited in the cell wall and had shown that these would bind to intact cells and mediate fungistatic effects (Rosas et al. 2001). Dr. Travassos was surprised at the notion that the fungal cell wall contained lipids and we subsequently exchanged correspondence that helped establish a relationship between the two laboratories. In fact, that initial conversation spawned a friendship that lasted for the next two decades until Dr. Travassos death in 2020. Also cementing the connection between the Travassos and Casadevall groups was that Dr. Carlos Taborda, who had trained with Dr. Travassos, had joined the Casadevall group for a very successful postdoctoral fellowship. Although Dr. Taborda was not involved in the vesicle discovery work he was an essential link between the groups and subsequently continued to collaborate with the major groups working on vesicles. In December 2000, Dr. Rodrigues and the Travassos laboratory published a paper titled “Human antibodies against a purified glucosylceramide from Cryptococcus neoformans inhibit cell budding and fungal growth” in Infection and Immunity describing that these antibodies mediated their effects by penetrating the capsule and binding to the cell wall (Rodrigues et al. 2000). This paper is a key antecedent for the discovery of fungal extracellular vesicles for it sets the stage for the importance of lipids in the cell wall and eventually led Dr. Rodrigues to travel to New York City with the goal of making mAbs. The discovery that antibodies to the lipids in the cell wall mediated antifungal activity, the scientific connection between Drs. Travassos and Casadevall laboratories and the need for better reagents to study this phenomenon in the form of mAbs set the stage for Dr. Rodrigues’ trip to the United States in 2005. Since mAb generation in mice requires the availability of animals making an antibody response to the antigen in question, and since eliciting such antibodies by vaccination can take time,

viii

Foreword

Dr. Rodrigues had shipped cryptococcal glucosylceramide to the Casadevall laboratory in anticipation of his arrival and mice had been immunized with the compound. Antonio Nakouzi, a senior research scientist in the Casadevall laboratory, had received the lipids from Brazil and immunized the mice. When Dr. Rodrigues arrived in our laboratory, he immediately checked the mice for serum antibody to glucosylceramide but found that none of the animals had mounted a strong antibody response. This essentially precluded making mAbs since Dr. Rodrigues had only 3 months in the lab and if he chose to continue to immunize mice these would require weeks and months to make a sufficient antibody response that would insure that they had sufficient B cells for hybridoma generation. Hence, he faced the question of how to best use his time in the United States. The author remembers a conversation in his office where he suggested to Dr. Rodrigues that maybe he could “spin down the culture supernatant” and see what is there. Within days Dr. Rodrigues would discover fungal extracellular vesicles, which would change the trajectory of his career and open a vast new area of research in the fungal field. However, the road to that conversation was paved by prior observations that also require explanation. In the early 2000s the Casadevall had begun to use biophysical techniques including dynamic and static light scattering to study C. neoformans capsular polysaccharide. Dr. Diane McFadden used light scattering to show that capsular polysaccharide molecules were enormous macromolecules with mass that exceeded one million Daltons (McFadden et al. 2005). Earlier, Dr. Marta Feldmesser had used immunoelectron microscopy to show that capsular material in the fungal cell cytoplasm (Feldmesser et al. 2001), suggesting the possibility for intracellular synthesis. Since we knew that the capsular polysaccharide was synthesized intracellularly in vesicles and that for the capsule to be assembled, that the polysaccharide needed to be shipped to the extracellular space, we were befuddled by how such a large polysaccharide could get through the cell wall. Hence, Dr. Rodrigues began to discuss these problems with the author and somehow the possibility of vesicles came up. During that discussion Dr. Rodrigues brought up electron microscopic images obtained by Dr. Kildare Miranda from Rio de Janeiro showing staining of membrane-like structures at the cell wall with an antibody to glucosylceramide. Dr. Rodrigues remembers that during that discussion this writer said something to the effect of “if these membrane structures are on the cell wall, they probably go outside too—why don’t you take a look.” Since Dr. Rodrigues was an expert on fungal lipids who was now looking for a productive project that could be done in a few months, the stage was set to look for them. Dr. Rodrigues then took supernatant from a cryptococcal culture, filtered to remove cell debris, and centrifuged it hard to pellet any vesicular structures. He then analyzed the pellet for lipids and by electron microscopy and discovered vesicles. Dr. Rodrigues returned to Brazil in the fall of 2005 and continued the work of characterizing the vesicles, with an essential participation of his Ph.D. Student, Debora Oliveira. There he was joined by another very important participant in the vesicle story: Dr. Leonardo, a terrific experimentalist who brought tremendous expertise and energy to the project. Dr. Nimrichter was an expert in the analysis of membranes and for years he supposed that fungal cells could produce exosome-like vesicles. It is also noteworthy that sometime in 2005 Dr. Rodrigues had a

Foreword

ix

conversation with two other of Dr. Travassos’ pupils, Drs. Igor Almeida and Rosana Puccia, who shared with him unpublished evidence suggesting that Paracoccidioides brasiliensis produced EVs too. Although this work was developed in different direction and eventually published later (Vallejo et al. 2011) it was an independent, but simultaneous observation for vesicular-type structures outside of the cell, which provided confidence in the existence of extracellular vesicles. During his stay in the Casadevall laboratory, Marcio Rodrigues had made friendships and recruited several investigators who continued to collaborate with him on the project. Dr. Susana Frases had become an expert on light scattering and she used that technique to characterize vesicles. Also at the time that Dr. Rodrigues was visiting the Casadevall laboratory, Dr. Oscar Zaragoza was finishing his postdoctoral work and preparing to transition to an independent position in Spain and he was drafted into some of the vesicle experiments given his expertise with C. neoformans capsule induction. Dr. Zaragoza had worked out conditions for reproducible induction of the C. neoformans capsule and since the early work of Takeo et al. (ref) had associated vesicles with capsule enlargement this was an obvious avenue to purse when looking for conditions that enhanced extracellular vesicle production. Dr. Zaragoza was initially very critical and was worried that the vesicles were just sedimented cell debris, but he too became convinced of their existence when helping to show that capsular induction increased extracellular vesicle production. By 2006, we had enough data to generate a manuscript. Cognizant of the significance of the discovery we submitted the manuscript first to Cell, which was and continues to be a leading scientific journal. Unfortunately, the journal rejected our manuscript as too preliminary. In the meantime, the laboratory of Dr. Tamara Doering published a study buttressing the notion that GXM was synthesized in cytoplasmic vesicles (Yoneda and Doering 2006) and we decided to send the paper to Eukaryotic Cell, where it was published in 2007 (Rodrigues et al. 2007). In retrospect, the discovery of fungal vesicles in 2005 came about by a fortuitous combination of expertise, scientific problems, serendipity, and good fortune. Dr. Rodrigues had found himself without a project, but his expert knowledge of lipids and outstanding experimental prowess allowed him to rapidly pivot to doing other work, which led to a discovery that would prove transformative for the fungal field and set a precedent that would resonate for gram-positive bacteria.

The Discovery in the Context of Antecedent Studies Like most major scientific findings, the discovery of C. neoformans extracellular vesicles did not occur in the setting of a historical tabula rasa. In fact, there were hints for such a process in papers going back several decades. In 1970, Heath and collaborators working at Imperial College in London described that cell wall components of Saprolegnia ferax were found in vesicles and called these “wall vesicles,” but could not make a connection to Golgi synthesis and called their origin “uncertain” (Heath et al. 1971). In 1973, Takeo et al. using electron microscopy had noted accumulation of vesicles on cell membrane of C. neoformans cells with enlarged

x

Foreword

capsules and suggested that the capsular polysaccharide was synthesized and secreted in vesicle (Takeo et al. 1973a, b), drawing that remarkable insight from looking at microphotographs. Dr. Rodrigues’ own prior work demonstrating the glycosphingolipid glucosylceramide in the cell wall (Rodrigues et al. 2000) placed lipids in the outside of the cell and raised many questions about the mechanism of their delivery and function. Hence, antecedent papers provided strong hints for a connection between vesicles and cell wall synthesis but none of the prior studies suggested, proposed, or envisioned vesicle transport across the cell wall. This was understandable because the fungal cell wall was viewed as a rigid structure akin to medieval chain mail armor, whereby a dense network of crosslinked polysaccharides would preclude the transit of such large structures as a membrane-derived vesicle. Hence, the key new contribution and insight from Dr. Rodrigues’ 2007 paper was to describe extracellular vesicles (Rodrigues et al. 2007), which implied transport across the cell wall into the culture supernatant (Fig. 2). This, in turn, would require a new concept of the fungal cell wall, which would take years to develop.

Fig. 2 Photograph of Drs. Carlos Taborda (left), Josh Nosanchuk (middle), and Marcio Rodrigues (right) taken during a visit by Dr. Nosanchuk to Latin America in 2007. Dr. Taborda had trained with Dr. Travassos and then did postdoctoral training with Dr. Casadevall, thus serving a key bridge between the laboratories. Dr. Nosanchuk carried out key work to show that ascomycetes produced vesicles

Foreword

xi

Reception The initial reception to the 2007 paper (Rodrigues et al. 2007) was muted with most colleagues in the field being uncertain of what to make of the discovery. At conferences, common questions in information conversations were: why would fungi need to make vesicles? What is the advantage of vesicular transport to fungal cells? How do you know that these are not the results of cells lysing and releasing lipids that reassembled into vesicles? Others noted that if fungal vesicles existed then they wondered how come these had not been discovered in the much-studied Saccharomyces cerevisiae system. In general, there was not outright opposition to the finding but a sense of surprise coupled with difficulty in how to incorporate the notion that fungi made extracellular vesicles into the larger picture of fungal biology. Some did recognize the importance of the work as evident by the anecdote that Dr. Rodrigues went to a meeting in Europe where he met a Nobelist who asked him: why is this work not published in the journal Cell? Dr. Rodrigues responded that the paper had in fact been first submitted to Cell but not accepted. The question itself implied that the work was so important that it should have been published in one of the leading journals of science. In subsequent years, several papers helped answer some of the questions posed by the initial report of the discovery and helped gain acceptance for the notion that EVs were an important aspect of fungal physiology. Dr. Rodrigues authored another first author paper the following year detailing how C. neoformans extracellular vesicles contained features associated with virulence such as phenol oxidase and urease activities (Rodrigues et al. 2008). Dr. Rodrigues and I collaborated with the laboratory of Dr. Joshua Nosanchuk to establish that extracellular vesicles were also produced by ascomycetous yeasts, reporting them in Histoplasma capsulatum, S. cerevisiae, Candida albicans, and Sporothrix schenckii (Albuquerque et al. 2008). This together with the report from the laboratory of Dr. Rosana Puccia demonstrating extracellular vesicles in Paracoccidioides brasiliensis (Vallejo et al. 2011) helped establish them as a general property of fungal cells. Later, the finding of extracellular vesicles in Alternaria infectoria extended this phenomenon to molds (Silva et al. 2014). Extending the observation to other fungi was important for the process of general acceptance because the original discovery in C. neoformans occurred in the only encapsulated fungus, and some viewed it as perhaps an unusual adaptation in relation to capsule synthesis. However, extension to other fungal species implied a general mechanism for unconventional secretion in fungi and other investigators began to take notice. Figure 2 shows a picture of three of the principals in this story taken during a meeting in Mexico in 2007. One of the large intellectual obstacles to initial acceptance of extracellular vesicles was the requirement for “genetic proof” by many scientists in the field, which insisted on proving that the phenomenon was real by the creation of null mutants unable to release vesicles. In other words, some people refused to believe that extracellular vesicles were not some sort of artifact unless one can provide evidence that mutants existed arguing instead that one could not rule out their origin

xii

Foreword

from a small population of cells that had lysed during culture growth. The discovery that S. cerevisiae made vesicles suggested that perhaps this criterion could be met by screening the large number of mutants available for this species. Hence, Dr. Rodrigues laboratory set out to screen S. cerevisiae mutants with wellcharacterized defects in secretory pathways for vesicle production (Oliveira et al. 2010). He found quantitative differences in extracellular vesicle synthesis but all produced vesicles (Oliveira et al. 2010). Although this study did not quiet the skeptics, it did imply that extracellular vesicle production was under genetic regulation, which increased interest in these structures. Additional evidence from genetic regulation came shortly from the laboratory of Peter Williamson who implicated Sec6 on C. neoformans vesicle secretion and used the word “exosome” in referring to vesicles (Panepinto et al. 2009). To date no null mutant for extracellular vesicle production has been identified, and we have suggested that these do not exist because vesicular synthesis is an inherent property of membrane such that this criticism is akin to implying the falsity of cell membrane existence without having a cell membrane null mutant, which clearly cannot exist since that structure is essential for cell viability (Coelho and Casadevall 2019). The cell wall transit mechanism problem was another tough question to answer. How could vesicles get through the cell wall? The problem here was the general belief that the cell wall was rigid and thus provided an impenetrable barrier to such large structures as vesicles. Much of this view originated from studies that used rough chemical treatments such as alkali to recover fungal cell particles, such as zymosan, which have little relation to those found in living cells. However, it was also known that the cell wall is a dynamic structure capable of rapid morphological change as evident by the rapid emergence of germ tubes in Candida spp. and the yeast-to-hyphal cell transition. Proponents of extracellular vesicles argued for flexibility in the cell wall, or the existence of pores, that would allow vesicular transport while doubters frowned upon such explanations. In support of the transport thesis, electron microscopy showed the presence of vesicular structures inside the cell wall. However, a conversation with Dr. Neil Gow at a meeting in Brasilia in 2014 led to a collaboration that helped reframe the issue. Dr. Gow mentioned his observation that vesicles containing amphotericin B could penetrate the cell wall when added to C. albicans cultures. The Casadevall laboratory then collaborated with the Gow group and showed this effect for C. neoformans, which reinforced the notion of the living cell wall as being a fluid structure that would allow vesicular transit (Walker et al. 2018). This view of the cell wall was also consistent with reports that bacteria could penetrate the fungal cell wall to become endosymbionts (Moebius et al. 2014). Hence, the criticisms about the reality of vesicles based on the transit mechanism problem gave way to acceptance that the living fungal cell wall was not a barrier to vesicle secretion although this change in thinking did not come about from a mechanistic explanation on how this occurred. There is also the hypothesis that vesicles transit in cell wall pores, which found support in the observation that melanized cell wall contained spaces consistent with pores (Eisenman et al. 2005). At the time of this writing, the mechanism for vesicular transit across the cell wall remains unsolved, but there is widespread acceptance that the cell wall is not a barrier to vesicular transit or release.

Foreword

xiii

Resonance in Gram-Positive Bacteria The finding that fungi could produce extracellular vesicles raised the question whether other cell-walled microbes like gram-positive bacteria could do the same. Gram-negative bacteria had long been known to make extracellular vesicles, but this was considered plausible given that they have an outer cell membrane suitable for vesicle synthesis. However, gram-positive bacteria, like fungi, are encased in a tough cell wall that was perceived as too rigid to allow vesicular transit. At the time that extracellular vesicles were discovered, the Casadevall laboratory was also working with the gram-positive bacterium B. anthracis and Mycobacteria tuberculosis. In the Casadevall laboratory, Dr. Johanna Rivera and Mr. Antonio Nakouzi had developed mAbs to anthrax toxins components (Rivera et al. 2006), which were used to study toxin production by B. anthracis. When B. anthracis bacteria were studied for anthrax toxin localization using immune-gold particle labeling and electron microscopy, the gold particles were not homogenously distributed through the cell wall but rather, many were clustered into discrete spots, which in retrospect suggests concentration in vesicles (Rivera et al. 2009). Given the excitement surrounding the discovery of extracellular vesicles in fungi and the fact that the laboratory was rapidly acquiring expertise in handling vesicles through continued collaboration with Dr. Rodrigues and others, Dr. Johanna Rivera decided to see if B. anthracis produced extracellular vesicles. She applied the methods developed for C. neoformans to B. anthracis and recovered vesicles in her first experimental try (Rivera et al. 2010). Remarkably, she found that anthrax toxin was packaged in vesicles, which upended notions of how toxin was released and immediately posed several fundamental questions of how this process occurred and its implications for pathogenesis (Rivera et al. 2010). At about the same time another group independently reported extracellular vesicles from the gram-positive bacterium Staphylococcus aureus (Lee et al. 2009), providing encouragement for the notion that the phenomenon was generalizable across this group of bacteria. Dr. Rafael Prados-Rosales joined the Casadevall laboratory around that time to work on M. tuberculosis. He too applied the techniques for vesicle isolation to mycobacterial cultures and was able to isolate extracellular vesicles from several Mycobacterium spp. (Prados-Rosales et al. 2011). Dr. Prados-Rosales went on to show their role in mycobacterial iron acquisition (Prados-Rosales et al. 2014a) and their ability to elicit protective immune responses when used as experimental vaccines (Prados-Rosales et al. 2014b). Lisa Brown, a graduate student in the Casadevall laboratory, then took on Bacillus subtilis and showed that it too produced extracellular vesicles that were disrupted by the lipopeptide surfactin (Brown et al. 2014). Criticism from the B. subtilis field that these structures were artifactual led Dr. Brown to devise a clever experiment whereby bacteria were grown with cryptococcal polysaccharide followed by analysis of its location relative to vesicles (Brown et al. 2014). She showed that cryptococcal polysaccharide was outside vesicles, but when these were disrupted by sonication and allowed to reform, some of the polysaccharide was internalized thus providing strong experimental

xiv

Foreword

evidence against being the result of simple self-assembly of shed lipids (Brown et al. 2014). The discovery that B. anthracis packaged its tripartite lethal and edema toxins in vesicles posed a conundrum for many—how do these toxins get out? The then accepted was that these toxins were released by bacteria in soluble form and migrated to the host cell surface where they attached to receptors, were internalized, and then mediated their toxic effects. Placing the toxins in vesicles was not reconcilable with these views of toxin action. Although Dr. Rivera’s paper included images of what appeared to be toxin-carrying vesicles fusing with macrophage cell membranes and others in the macrophage cytosol, which implied that these toxins could reach macrophages, neither observation fitted easily within models of toxin action. Dr. Julie Wolf, a postdoctoral fellow in the Casadevall laboratory, was studying vesicle stability and she made the key observation that these structures were disrupted by serum proteins such as albumin (Wolf et al. 2012), which suggested that vesicles were short lived in tissues. The notion that serum proteins disrupted vesicles and released their contents including toxins removed a potential hurdle in acceptance of the fact that gram-positive bacteria packaged their toxin in vesicles for it allowed reconciliation with existing views of toxin action. A collaboration with the laboratories of Drs. Liise-anne Pirofski at Einstein and Manuel Rodrigues-Ortega from Spain led to the finding that Streptococcus pneumoniae also released extracellular vesicles (Olaya-Abril et al. 2014). The last bacterium to be studied by the Casadevall lab was Listeria monocytogenes, and Drs. Carolina Coelho and Lisa Brown showed that it too made vesicles and packaged listeriolysin in them (Coelho et al. 2018). One of the most interesting aspects of the journey describing extracellular vesicles in fungi and gram-positive bacteria was the differences in resistance and acceptance depending on the organism involved. If resistance to a new idea can be inferred by the difficulty of publishing a paper, then the first reports of extracellular vesicles in fungi and B. anthracis found the easiest path to publication. The report of extracellular vesicles in mycobacteria generally went smoothly, possibly because the field was already conditioned to the notion that M. tuberculosis-infected macrophages released bacterial lipids. The finding in B. subtilis encountered considerable skepticisms, which led to considerable experimentation to overcome the concern that these were simply the result of shed lipid self-assembly into vesicles. However, perhaps the most difficult paper to publish was that reporting that Listeria monocytogenes made extracellular vesicles that carried listeriolysin, which we attribute to the fact that acceptance of this notion would require considerable re-examination of the mechanisms by which this bacterium caused disease in a well-established field. In the discipline of microbiology, fields are often defined by communities of scientists who work on specific organisms and these develop norms that tend to be microbe specific (Casadevall and Fang 2015b). Hence, differences in acceptance of the existence of extracellular vesicles among the different fields probably reflected differences in epistemic approaches and how the information meshed with existing paradigms for those microbes.

Foreword

xv

Explanatory Power Despite the tepid reception given to the initial reports of the discovery that fungi produced extracellular vesicles, acceptance was hastened by the realization that their existence had great explanatory power for some nagging problems in the field. At the level of the cell wall, there had been consistent reports that it contained lipids and cytoplasmic proteins, such as Heat Shock Proteins. This had led to some acrimony and controversy when these findings were criticized as artifacts of contamination of cell wall fractions with cytoplasmic components. For example, a fragment of heat shock protein 90 was identified in the cell wall of C. albicans in the late 1980s (Matthews et al. 1988), which was highly antigenic. Antibody therapy was developed against this antigen and tested clinically, but some in the field remained perplexed about the role and function of such proteins in the cell wall. The existence of extracellular vesicles that carried cytoplasmic proteins provided a possible explanation to this finding since one could now explain how such proteins could reach the cell wall preparations. A similar criticism was commonly applied to reports of lipids in the cell wall. Vesicles also provided an explanation how many proteins and small molecules were released to the extracellular space when there was no obvious classical secretion mechanism. For example, in the M. tuberculosis field vesicles could explain siderophore release (Prados-Rosales et al. 2014a). C. albicans extracellular vesicles were shown to contain matrix components (Zarnowski et al. 2018) and that interfering with their transport by the experimental antifungal drug turbinmicin inhibited biofilm formation (Zhao et al. 2020). Antibodies to Histoplasma capsulatum were shown to modulate vesicle content (Matos Baltazar et al. 2016; Baltazar et al. 2018), implying that the fungal cell responded to antibody binding by altering vesicle metabolism, a finding that suggested new mechanisms of action for humoral immunity in host defense. This connection between immune function and vesicle release was strengthened by the finding that vesicular production was altered by growth conditions (Cleare et al. 2020), which in turn had different effects on host response (Marina et al. 2020). Hence, for many scientists the explanatory power associated with finding new roles for vesicles and how vesicles affected fungal physiology and pathogenesis were major factors leading to their acceptance.

Raison-d’etre An early question posed to us was: why do microbes need extracellular vesicles? To this writer the most satisfying answer is that vesicles allow the delivery of a concentrated punch to a point at a distance from the microbial surface without dilutional effects (Fig. 3). Alternatively, microbes would face the problem that material secreted at the cell surface would be rapidly diluted as it diffuses into the extracellular space, which would require greater production to achieve an effect at a distance. This may be of particular importance for delivering enzymatic cocktails

xvi

Foreword

Fig. 3 Unlike secretion on export components at the cell surface, which is diluted as a function of distance traveled, packaging of components into vesicles allows them to travel distances in concentrated form. Concentrated delivery may be important for the delivery of complex enzyme mixtures to substrates such as decaying vegetation where release of nutrients would require digestion of multiple biochemical forms of material

needed to digest complex materials such as plant matter. For C. neoformans, enzymatic activities such as laccase and urease are exported to the exterior in vesicles. Although both laccase and urease are important for cryptococcal virulence, it is worthwhile to remember that the overwhelming majority of cryptococci at any one time are in the environment surviving by obtaining food from organic matter and that each of these enzymes contributes to nutrient acquisition. The finding that fungal vesicles contained numerous components associated with virulence led to them being called “virulence bags” to convey the notion that these represented structures with mixed cargo that could contribute to fungal pathogenesis. However, the recent findings that vesicles also contain nucleic acids and that these are involved in cell-tocell communication imply that in different contexts they could function as “diplomatic pouches.” Hence, whether they are functioning as virulence bags or diplomatic pouches the most powerful raison-d’etre for the existence of extracellular vesicular export systems is that they allow concentration of diverse components that include proteins, lipids, and nucleic acids for action at a distance.

Continuing Challenges Well into its second decade, the study of fungal vesicles remains at its infancy. In 1900, the mathematician David Hilbert published 23 outstanding mathematical

Foreword

xvii

problems and by listing them he brought attention that led to the solution of many of them. In 2012, Dr. Maurizio del Poeta and this writer took a page from Dr. Hilbert’s example and posted a list of ten challenges in the C. neoformans field with less success (Del and Casadevall 2012), since none have been fully solved despite tremendous progress in the past decade. Nevertheless, the exercise of listing a problem can be useful, since this requires formulating in a manner that can attract attention and resources. 1. How do vesicles cross the cell wall? The mechanism by which vesicles transit the cell wall remains a black box. The finding that liposomes were able to penetrate the cell wall if they contained amphotericin b suggested the possibility of a transport system (Walker et al. 2018), but at this time this is speculation. Providing an answer to how vesicles cross the fungal cell may require a greater understanding of how this structure is assembled and maintained. 2. How many types of vesicles. And mechanisms for vesicle formation, are there? Electron microscopy shows differences in extracellular vesicle morphology, some of which have electron dense structures (Rodrigues et al. 2008). Defining the different types of vesicle populations is essential since differences between these could help understand their purpose, destination, and assembly. 3. How are vesicle components selected for export? Vesicle cargo is a myriad of components that include proteins, lipids, polysaccharides, and nucleic acids. It is likely that each component has a specific purpose and is loaded into vesicles through different, regulated mechanisms. For example, Vps27 of the endosomal sorting complex required for transport (ESCRT) pathway is essential for laccase secretion in C. neoformans (Park et al. 2020). Defining vesicle loading mechanisms could uncover new fundamental processes in eukaryotic cell biology. 4. How do vesicles know where they are going? Some vesicles function to deliver cell wall raw materials for its growth and remodeling, others like C. neoformans melanosomes are intended for cell wall insertion (Camacho et al. 2019), and those in the extracellular space are likely to have functions in microbial communication, biofilm formation, nutrient acquisition, and virulence. This suggests that vesicles are marked by a delivery code system like that used for postal services (ZIP code) to deliver mail. Deciphering this code would require correlation of components with destination, with particular emphasis on surface components that facilitate cell wall attachment or unimpeded transit. 5. How do vesicles unpack their contents? For vesicular delivery to be effective there must be a mechanism for unpacking the contents once they arrive at their intended destination. The finding that some serum proteins like albumin destabilize bacterial and fungal vesicles by interacting with their lipids suggests that specialized proteins could unpack those destined for the cell wall. Hence, part of the riddle of how some vesicles end in the cell wall while others transit to the cell exterior could depend on local unpacking mechanisms that recognize some label marking their intended destination. 6. What are the physical constraints that determine vesicle dimensions? One of the fascinating aspects of microbial vesicles is that despite all their diversity they tend

xviii

Foreword

to exist within a narrow set of dimensions, with most being in the vicinity of 100 microns. This suggests the existence of optimal dimensions for packaging, export, and release into the extracellular space. The mechanisms by which such phylogenetically diverse microbes as fungi and bacteria produce structure of similar dimensions and appearance are not understood, but these are likely to reflect a set of biophysical and biochemical constrains that converge to define optimal dimensions. 7. What are the economics of extracellular vesicles? The complexity of extracellular vesicles combined with the fact that these involve the export of biologically important molecules suggests that it must be energetically expensive. Clearly, the benefits must outweigh the costs for such a system to be universally deployed in such diverse microbial species as prokaryotes and eukaryotes. Understanding the economics of extracellular vesicle synthesis would require knowing the energetic cost of running such a complex cellular system and the benefits accrued by vesicular export. Such benefits may include unexpected functions like mycobacteria using vesicular transport to share siderophores as a form of community altruism. Accounting for the economics of this system would require knowing the answers to some of the above questions.

Perspective from Hindsight The discovery of fungal extracellular vesicles involved a fortuitous confluence of propitious events that included Dr. Rodrigues arrival in the Casadevall laboratory with experimental plans to make mAbs upended by lack of lipid immunologic responsiveness in vaccinated mice, scientific problems ripe for new approaches, the availability of suitable methods, and a willingness to abandon prevailing paradigms such as the rigidity of the cell wall. Adding to the favorable confluence set of events was the fact that Dr. Rodrigues was interested in lipids, had expertise in lipid chemistry, and had already encountered a problem that required new ways of thinking to explain the observation human antibodies to glucosylceramide bound to the C. neoformans cell wall (Rodrigues et al. 2000). The fact that the Casadevall laboratory was working with gram-positive bacteria at the time of the discovery provided an accessible rampart to another class of cell-walled microorganisms, which enormously extended the scientific scope of this vesicular transport system. Science is a human endeavor and the discovery of fungal extracellular vesicles in 2005 required an almost magical combination of the essential personal factors brought together by a hefty portion of good fortune. Department of Molecular Microbiology and Immunology, Johns Hopkins School of Public Health, Baltimore, MD, USA

Arturo Casadevall

Foreword

xix

Acknowledgments I am grateful to Drs. Oscar Zaragoza, Joshua Nosanchuk, Marcio Rodrigues, and Rosanna Puccia for comments on this manuscript and suggestions for improvement. A.C. was supported in part by NIH grants AI052733, AI15207, and HL059842. References Albuquerque PC, Nakayasu ES, Rodrigues ML, Frases S, Casadevall A, ZancopeOliveira RM, Almeida IC, Nosanchuk JD (2008) Vesicular transport in Histoplasma capsulatum: an effective mechanism for trans-cell wall transfer of proteins and lipids in ascomycetes. Cell Microbiol 10:1695–1710 Baltazar LM, Zamith-Miranda D, Burnet MC, Choi H, Nimrichter L, Nakayasu ES, Nosanchuk JD (2018) Concentration-dependent protein loading of extracellular vesicles released by Histoplasma capsulatum after antibody treatment and its modulatory action upon macrophages. Sci Rep 8:8065 Brown L, Kessler A, Cabezas-Sanchez P, Luque-Garcia JL, Casadevall A (2014) Extracellular vesicles produced by the Gram-positive bacterium Bacillus subtilis are disrupted by the lipopeptide surfactin. Mol Microbiol 93:183–198 Brown L, Wolf JM, Prados-Rosales R, Casadevall A (2015) Through the wall: extracellular vesicles in Gram-positive bacteria, mycobacteria and fungi. Nat Rev Microbiol 13:620–630 Camacho E, Vij R, Chrissian C, Prados-Rosales R, Gil D, O'Meally RN, Cordero RJB, Cole RN, McCaffery JM, Stark RE, Casadevall A (2019) The structural unit of melanin in the cell wall of the fungal pathogen Cryptococcus neoformans. J Biol Chem 294:10471–10489 Casadevall A, Fang FC (2015a) (A)Historical science. Infect Immun 83:4460–4464 Casadevall A, Fang FC (2015b) Field science--the nature and utility of scientific fields. MBio 6:e01259-01215 Casadevall A, Nosanchuk JD, Williamson P, Rodrigues ML (2009) Vesicular transport across the fungal cell wall. Trends Microbiol 17:158–162 Cleare LG, Zamith D, Heyman HM, Couvillion SP, Nimrichter L, Rodrigues ML, Nakayasu ES, Nosanchuk JD (2020) Media matters! Alterations in the loading and release of Histoplasma capsulatum extracellular vesicles in response to different nutritional milieus. Cell Microbiol 22:e13217 Coelho C, Casadevall A (2019) Answers to naysayers regarding microbial extracellular vesicles. Biochem Soc Trans 47:1005–1012 Coelho C, Brown LC, Maryam M, Vij R, Smith DF, Burnet MC, Kyle JE, Heyman HM, Ramirez J, Prados-Rosales R, Lauvau G, Nakayasu ES, Brady NR, Hamacher-Brady A, Coppens I, Casadevall A (2019) Listeria monocytogenes virulence factors, including listeriolysin O, are secreted in biologically active extracellular vesicles. J Biol Chem 294(4):1202–1217 Del PM, Casadevall A (2012) Ten challenges on Cryptococcus and cryptococcosis. Mycopathologia 173:303–310 Eisenman HC, Nosanchuk JD, Webber JB, Emerson RJ, Camesano TA, Casadevall A (2005) Microstructure of cell wall-associated melanin in the human pathogenic fungus Cryptococcus neoformans. Biochemistry 44:3683–3693

xx

Foreword

Feldmesser M, Kress Y, Casadevall A (2001) Dynamic changes in the morphology of Cryptococcus neoformans during murine pulmonary infection. Microbiology 147:2355–2365 Freitas MS, Bonato VLD, Pessoni AM, Rodrigues ML, Casadevall A, Almeida F (2019) Fungal extracellular vesicles as potential targets for immune interventions. mSphere 4(6):e00747-19 Heath I, Gay J, Greenwood A (1971) Cell wall formation in the Saprolegniales: cytoplasmic vesicles underlying developing walls. Microbiology 65:225–232 Lee EY, Choi DY, Kim DK, Kim JW, Park JO, Kim S, Kim SH, Desiderio DM, Kim YK, Kim KP, Gho YS (2009) Gram-positive bacteria produce membrane vesicles: proteomics-based characterization of Staphylococcus aureus-derived membrane vesicles. Proteomics 9:5425–5436 Marina CL, Bürgel PH, Agostinho DP, Zamith-Miranda D, Las-Casas LO, Tavares AH, Nosanchuk JD, Bocca AL (2020) Nutritional conditions modulate C. neoformans extracellular vesicles' capacity to elicit host immune response. Microorganisms 8(11):1815 Matos Baltazar L, Nakayasu ES, Sobreira TJ, Choi H, Casadevall A, Nimrichter L, Nosanchuk JD (2016) Antibody binding alters the characteristics and contents of extracellular vesicles released by Histoplasma capsulatum. mSphere 1(2): e00085-15 Matthews R, Wells C, Burnie JP (1988) Characterisation and cellular localisation of the immunodominant 47-Kda antigen of Candida albicans. J Med Microbiol 27:227–232 McFadden DC, De Jesus M, Casadevall A (2005) The physical properties of the capsular polysaccharides from Cryptococcus neoformans suggest features for capsule construction. J Biol Chem 281:1868–1875 Moebius N, Üzüm Z, Dijksterhuis J, Lackner G, Hertweck C (2014) Active invasion of bacteria into living fungal cells. elife 3:e03007 Munhoz da Rocha IF, Amatuzzi RF, Lucena ACR, Faoro H, Alves LR (2020) Crosskingdom extracellular vesicles EV-RNA communication as a mechanism for host-pathogen interaction. Front Cell Infect Microbiol 10:593160 Olaya-Abril A, Prados-Rosales R, McConnell MJ, Martin-Pena R, Gonzalez-Reyes JA, Jimenez-Munguia I, Gomez-Gascon L, Fernandez J, Luque-Garcia JL, Garcia-Lidon C, Estevez H, Pachon J, Obando I, Casadevall A, Pirofski LA, Rodriguez-Ortega MJ (2014) Characterization of protective extracellular membrane-derived vesicles produced by Streptococcus pneumoniae. J Proteome 106:46–60 Oliveira DL, Nakayasu ES, Joffe LS, Guimaraes AJ, Sobreira TJ, Nosanchuk JD, Cordero RJ, Frases S, Casadevall A, Almeida IC, Nimrichter L, Rodrigues ML (2010) Characterization of yeast extracellular vesicles: evidence for the participation of different pathways of cellular traffic in vesicle biogenesis. PLoS One 5: e11113 Panepinto J, Komperda K, Frases S, Park YD, Djordjevic JT, Casadevall A, Williamson PR (2009) Sec6-dependent sorting of fungal extracellular exosomes and laccase of Cryptococcus neoformans. Mol Microbiol 71:1165–1176

Foreword

xxi

Park YD, Chen SH, Camacho E, Casadevall A, Williamson PR (2020) Role of the ESCRT pathway in laccase trafficking and virulence of Cryptococcus neoformans. Infect Immun 88(7):e00954-19 Prados-Rosales R, Baena A, Martinez LR, Luque-Garcia J, Kalscheuer R, Veeraraghavan U, Camara C, Nosanchuk JD, Besra GS, Chen B, Jimenez J, Glatman-Freedman A, Jacobs WR Jr, Porcelli SA, Casadevall A (2011) Mycobacteria release active membrane vesicles that modulate immune responses in a TLR2-dependent manner in mice. J Clin Invest 121:1471–1483 Prados-Rosales R, Weinrick BC, Pique DG, Jacobs WR Jr, Casadevall A, Rodriguez GM (2014a) Role for Mycobacterium tuberculosis membrane vesicles in iron acquisition. J Bacteriol 196:1250–1256 Prados-Rosales R, Carreno LJ, Batista-Gonzalez A, Baena A, Venkataswamy MM, Xu J, Yu X, Wallstrom G, Magee DM, LaBaer J, Achkar JM, Jacobs WR Jr, Chan J, Porcelli SA, Casadevall A (2014b) Mycobacterial membrane vesicles administered systemically in mice induce a protective immune response to surface compartments of Mycobacterium tuberculosis. MBio 5:e01921-01914 Rivera J, Nakouzi A, Abboud N, Revskaya E, Goldman D, Collier RJ, Dadachova E, Casadevall A (2006) A monoclonal antibody to Bacillus anthracis protective antigen defines a neutralizing epitope in domain 1. Infect Immun 74:4149–4156 Rivera J, Nakouzi AS, Morgenstern A, Bruchertseifer F, Dadachova E, Casadevall A (2009) Radiolabeled antibodies to Bacillus anthracis toxins are bactericidal and partially therapeutic in experimental murine anthrax. Antimicrob. Agents Chemother 53:4860–4868 Rivera J, Cordero RJ, Nakouzi AS, Frases S, Nicola A, Casadevall A (2010) Bacillus anthracis produces membrane-derived vesicles containing biologically active toxins. Proc Natl Acad Sci U S A 107:19002–19007 Rodrigues ML, Casadevall A (2018) A two-way road: novel roles for fungal extracellular vesicles. Mol Microbiol 110:11–15 Rodrigues ML, Travassos LR, Miranda KR, Franzen AJ, Rozental S, De Souza W, Alviano CS, Barreto-Bergter E (2000) Human antibodies against a purified glucosylceramide from Cryptococcus neoformans inhibit cell budding and fungal growth. Infect Immun 68:7049–7060 Rodrigues ML, Nimrichter L, Oliveira DL, Frases S, Miranda K, Zaragoza O, Alvarez M, Nakouzi A, Feldmesser M, Casadevall A (2007) Vesicular polysaccharide export in Cryptococcus neoformans is a eukaryotic solution to the problem of fungal trans-cell wall transport. Eukaryot Cell 6:48–59 Rodrigues ML, Nakayasu ES, Oliveira DL, Nimrichter L, Nosanchuk JD, Almeida IC, Casadevall A (2008) Extracellular vesicles produced by Cryptococcus neoformans contain protein components associated with virulence. Eukaryot Cell 7:58–67 Rosas AL, Nosanchuk JD, Casadevall A (2001) Passive immunization with melaninbinding monoclonal antibodies prolongs survival in mice with lethal Cryptococcus neoformans infection. Infect Immun 69:3410–3412

xxii

Foreword

Silva BM, Prados-Rosales R, Espadas-Moreno J, Wolf JM, Luque-Garcia JL, Goncalves T, Casadevall A (2014) Characterization of Alternaria infectoria extracellular vesicles. Med Mycol 52:202–210 Takeo K, Uesaka I, Uehira K, Nishiura M (1973a) Fine structure of Cryptococcus neoformans grown in vivo as observed by freeze-etching. J Bacteriol 113:1449–1454 Takeo K, Uesaka I, Uehira K, Nishiura M (1973b) Fine structure of Cryptococcus neoformans grown in vitro as observed by freeze-etching. J Bacteriol 113:1442–1448 Vallejo MC, Matsuo AL, Ganiko L, Medeiros LC, Miranda K, Silva LS, FreymüllerHaapalainen E, Sinigaglia-Coimbra R, Almeida IC, Puccia R (2011) The pathogenic fungus Paracoccidioides brasiliensis exports extracellular vesicles containing highly immunogenic α-Galactosyl epitopes. Eukaryot Cell 10:343–351 Walker L, Sood P, Lenardon MD, Milne G, Olson J, Jensen G, Wolf J, Casadevall A, Adler-Moore J, Gow NAR (2018) The viscoelastic properties of the fungal cell wall allow traffic of ambisome as intact liposome vesicles. MBio 9(1):e02383-17 Wolf JM, Rivera J, Casadevall A (2012) Serum albumin disrupts Cryptococcus neoformans and Bacillus anthracis extracellular vesicles. Cell Microbiol 14:762–773 Yoneda A, Doering TL (2006) A eukaryotic capsular polysaccharide is synthesized intracellularly and secreted via exocytosis. Mol Biol Cell 17:5131–5140 Zarnowski R, Sanchez H, Covelli AS, Dominguez E, Jaromin A, Bernhardt J, Mitchell KF, Heiss C, Azadi P, Mitchell A, Andes DR (2018) Candida albicans biofilm-induced vesicles confer drug resistance through matrix biogenesis. PLoS Biol 16:e2006872 Zhao M, Zhang F, Zarnowski R, Barns KJ, Jones R, Fossen JL, Sanchez H, Rajski SR, Audhya A, Bugni TS, Andes DR (2021) Turbinmicin inhibits Candida biofilm growth by disrupting fungal vesicle-mediated trafficking. J Clin Invest 131(5):e145123

Preface

The study of extracellular vesicles (EVs) emerged in the last decade as an exciting and dynamic field of biological research. Simple searches in bibliographic repositories using the term “extracellular vesicles” reveal a rapidly growing area, which produced only dozens of scientific papers in the late 1970s and early 1980s, but almost 5000 documents in 2020. EVs participate in many aspects of cell biology under conditions of health and disease, impacting immunological mechanisms, tumor biology, and microbial pathogenesis, among others. EVs in fungi were first described in 2007 and proposed at that time as vehicles involved in the trans-cell wall transport of macromolecules in fungal cells. Although their existence has been initially controversial, fungal EVs are now seen as essential players of fungal physiology. So far, they were detected in at least 20 fungal species, where they mediate cell-to-cell interactions, populational communication, prion transmission, antifungal resistance, and, in the case of pathogens, interaction with host cells, delivery of virulence factors, and stimulation of immune responses resulting in vaccine potential. Although much progress has been made, there is still a lot to discover, which makes this field of research even more exciting. This said we invite the reader to visit the most important findings showing how fungal EVs dynamically affect multiple biological phenomena and how they impacted other areas of microbial biology. Curitiba, Brazil Paris, France

Marcio Rodrigues Guilhem Janbon

xxiii

About the Book

This volume focuses on an emerging and exciting area: the production of extracellular vesicles (EVs) by fungi. EVs are essential for the physiology of any cell type. They participate in numerous events, both in homeostasis and in illness. The ability of fungi to produce EVs was unknown until recently. Its discovery in 2007 opened investigations on several models, and today the perception that they impact pathogenesis mechanisms, biofilm formation, prion transmission, drug resistance, and cellular communication is clear. The vaccine potential of EVs produced by fungi is also clear. The scientific content of this volume was produced by authors from Brazil, France, USA, United Kingdom, Portugal, and Spain, which agrees with the global interest in fungal EVs. The first chapters illustrate the original findings in the field of fungal EVs, including a historical view. The production of EVs in specific fungal models is then discussed in the next chapters. Communication events that require fungal EVs, including prion transmission, biofilm formation, cell-to-cell communication, and stimulation of host responses, are then discussed. The final chapters discuss the vaccine potential of fungal EVs and the most sophisticated mechanisms for their analysis. This volume offers an overview of the multiple aspects involved in the formation and functions of fungal EVs and discusses up-to-date approaches for the study of these structures. Target Group Scientists (undergraduate and graduate students; junior and senior scientists) in the fields of Cell Biology, Mycology, Microbiology, Infectious Diseases, Biology, and Medicine.

xxv

Contents

Biogenesis of Fungal Extracellular Vesicles: What Do We Know? . . . . . Haroldo C. de Oliveira, Amanda F. Kato, Bianca A. G. Sena, Iraine Duarte, Luísa J. Jozefowicz, Rafael F. Castelli, Diogo Kuczera, Flavia C. G. Reis, Lysangela Ronalte Alves, and Marcio L. Rodrigues Lessons Learned from Studying Histoplasma capsulatum Extracellular Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Daniel Zamith-Miranda, Lysangela Ronalte Alves, Ernesto Satoshi Nakayasu, and Joshua Daniel Nosanchuk Current Status on Extracellular Vesicles from the Dimorphic Pathogenic Species of Paracoccidioides . . . . . . . . . . . . . . . . . . . . . . . . . . Rosana Puccia

1

13

19

Extracellular Vesicles from Sporothrix Yeast Cells . . . . . . . . . . . . . . . . . Marcelo Augusto Kazuo Ikeda and Karen Spadari Ferreira

35

Filamentous Fungi Extracellular Vesicles . . . . . . . . . . . . . . . . . . . . . . . . Teresa Gonçalves, Joana Oliveira, and Chantal Fernandes

45

Extracellular Vesicles and the Propagation of Yeast Prions . . . . . . . . . . Mehdi Kabani

57

Contributions of Extracellular Vesicles to Fungal Biofilm Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Marienela Heredia and David Andes Fungal Extracellular Vesicles in Interkingdom Communication . . . . . . . Maria Makarova and Robin C. May

67 81

xxvii

xxviii

Contents

Interactions of Extracellular Vesicles from Pathogenic Fungi with Innate Leukocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mateus Silveira Freitas, Andre Moreira Pessoni, Carolina Coelho, Vânia Luiza Deperon Bonato, Marcio L. Rodrigues, Arturo Casadevall, and Fausto Almeida

89

Fungal Extracellular Vesicles as a Potential Strategy for Vaccine Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 Leandro Honorato, Jhon Jhamilton Artunduaga Bonilla, Alicia C. Piffer, and Leonardo Nimrichter Current Microscopy Strategies to Image Fungal Vesicles: From the Intracellular Trafficking and Secretion to the Inner Structure of Isolated Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 Camila Wendt, Vânia Vieira, Adélia Lima, Ingrid Augusto, Fernando P. de Almeida, Ana Paula R. Gadelha, Leonardo Nimrichter, Marcio L. Rodrigues, and Kildare Miranda Proteomic Characterization of EVs in Non-pathogenic Yeast Cells . . . . . 161 Pilar Morales, Ana Mencher, Jordi Tronchoni, and Ramon Gonzalez

About the Editors1

Marcio Rodrigues is a senior investigator in the Carlos Chagas Institute (Oswaldo Cruz Foundation, Fiocruz, Brazil). His laboratory is mainly interested in the mechanisms by which fungal cells export biologically active molecules to the extracellular space. In collaboration with Drs. Arturo Casadevall, Leonardo Nimrichter, and Kildare Miranda, all authors in this volume, Marcio Rodrigues led the discovery of fungal extracellular vesicles in 2007. His laboratory has been investigating the role of extracellular vesicles in fungal physiology and pathogenesis for the last decade, mainly using the Cryptococcus model of secretion. The group is especially interested in how lipids and glycans participate in secretory processes that are essential for fungi, aiming to connect basic cell biology mechanisms with the identification of cellular pathways that could be targeted by novel antifungal agents. Guilhem Janbon is the head of the Unit RNA Biology in Fungal Pathogens and the director of the Department of Mycology of the Institut Pasteur in Paris (France). He has a very long experience working and publishing papers dealing with different aspect of pathogenic or nonpathogenic fungi. His laboratory studies different aspects of the RNA biology of some pathogenic fungi. His group is also interested in the analysis of the structure and genetics of fungal EVs. Recently, in collaboration with Drs. Marcio Rodrigues and R May, his laboratory is using the most updated technologies to revisit fungal EV structure and to study their diversity of shape, size, and structure.

1

Marcio Rodrigues and Guilhem Janbon collaborate at multiple levels, including EV research, and partnerships involving their countries (Brazil and France) and institutions (Fiocruz, Brazil; Institut Pasteur, France). xxix

Biogenesis of Fungal Extracellular Vesicles: What Do We Know? Haroldo C. de Oliveira, Amanda F. Kato, Bianca A. G. Sena, Iraine Duarte, Luísa J. Jozefowicz, Rafael F. Castelli, Diogo Kuczera, Flavia C. G. Reis, Lysangela Ronalte Alves, and Marcio L. Rodrigues

Contents 1 Eukaryotic EVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Fungal EVs: An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Intracellular Biogenesis of Fungal EVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 EV Formation at the Plasma Membrane Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Closing Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 3 3 5 7 7

Abstract So far, extracellular vesicles (EVs) have been described in 15 genera of fungi. They carry molecules that contribute to the interaction of fungal cells with the host. Although the number of studies on fungal EVs has increased, the mechanisms involved in their biogenesis are still poorly understood. The current knowledge of EV biogenesis shows us that they can originate both in the cytoplasm and at the plasma membrane. In this chapter, we will focus on these two cellular sites to review what is known about the biogenesis of fungal EVs. H. C. de Oliveira · A. F. Kato · B. A. G. Sena · I. Duarte · L. J. Jozefowicz · D. Kuczera · L. R. Alves Instituto Carlos Chagas, Fundação Oswaldo Cruz (Fiocruz), Curitiba, Brazil R. F. Castelli Instituto Carlos Chagas, Fundação Oswaldo Cruz (Fiocruz), Curitiba, Brazil Programa de Pós-Graduação em Biologia Parasitária, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil F. C. G. Reis Instituto Carlos Chagas, Fundação Oswaldo Cruz (Fiocruz), Curitiba, Brazil Centro de Desenvolvimento Tecnológico em Saúde (CDTS), Fundação Oswaldo Cruz (Fiocruz), Rio De Janeiro, Brazil M. L. Rodrigues (*) Instituto Carlos Chagas, Fundação Oswaldo Cruz (Fiocruz), Curitiba, Brazil Instituto de Microbiologia Paulo de Góes (IMPG), Universidade Federal do Rio de Janeiro, Cidade Universitária, Rio de Janeiro, Brazil e-mail: marcio.rodrigues@fiocruz.br © Springer Nature Switzerland AG 2021 M. Rodrigues, G. Janbon (eds.), Fungal Extracellular Vesicles, Current Topics in Microbiology and Immunology 432, https://doi.org/10.1007/978-3-030-83391-6_1

1

2

H. C. de Oliveira et al.

1 Eukaryotic EVs EVs are nano-sized, spherical structures composed of a phospholipid bilayered membrane and molecules of a very diverse nature (Słomka et al. 2018). They are produced by all cell types and have important roles in numerous biological processes (Joffe et al. 2016). The various functions displayed by EVs depend on the producing cell type and the environmental conditions at which these cells live (Raposo and Stoorvogel 2013; de Toledo Martins et al. 2019). In general, the main components of EVs are proteins, lipids, and RNA. Early studies described EV-like structures as “clotting factors” (Chargaff and West 1946). In 1967, similar structures were called “platelet dust” (Wolf 1967). The term exosome was used for the first time in 1981 by Trams and colleagues (Trams et al. 1981). Initially, these structures were thought to be involved in the elimination of specific molecules and their role in cell communication was only discovered later on (Raposo and Stoorvogel 2013). Cell communication through EVs can happen between cells of the same species or even between host and pathogen cells, where they can act locally or at a distance (Al-Nedawi et al. 2008; Raposo and Stoorvogel 2013; Colombo et al. 2014; Record et al. 2014; Desrochers et al. 2016; van Niel et al. 2018). It has also been shown that EVs can mediate RNA export, which can lead to protein translation by the recipient cell (Raposo and Stoorvogel 2013), drug resistance (Zarnowski et al. 2018), prion transference (Kabani and Melki 2015; Liu et al. 2016), and transport of virulence factors (Bielska et al. 2018). Some studies have also implicated EVs in the host immune response against pathogens by stimulating or increasing cytokine secretion, antibody production against an invader, and also by activating receptors, thus, contributing to the outcome of the infection (de Toledo Martins et al. 2019). Considering all these aspects, the importance of studying and comprehending the processes involving EV biogenesis is clear. According to their origin and size, EVs can be generally classified into three distinct categories: exosomes, microvesicles, and apoptotic bodies (Maas et al. 2017). In addition, it is worth mentioning that the International Society for Extracellular Vesicles (ISEV) suggests the classification of EVs according to their sedimentation properties as small EVs (sEVs), medium EVs (mEVs), and large EVs (lEVs) based upon the speed at which they sediment (Słomka et al. 2018). Since this classification was proposed only in 2018, most authors still use the former classification method based on EV origin and size. The occurrence of apoptotic bodies in fungi is controversial. Therefore, in this chapter, we will classify fungal EVs as exosome- or microvesicle-like structures. Exosomes are small membrane vesicles that derive from the endosomal pathway (Colombo et al. 2014; Maas et al. 2017). In the early endosome, cargo destined for degradation concentrates in intraluminal vesicles that accumulate within the vacuolar domain and originate the multivesicular endosomes/bodies (MVBs). MVBs have multiple functions and destinations in the eukaryotic cell, including the plasma membrane. The fusion of MVBs with the plasma membrane results in the extracellular release of intraluminal MVB vesicles, at this point, denominated exosomes.

Biogenesis of Fungal Extracellular Vesicles: What Do We Know?

3

Exosomes are EVs that range from 30–100 nm in size (Eitan et al. 2016). Exosome release is coordinated by the endosomal sorting complex required for transport (ESCRT), a sophisticated machinery composed of 4 protein complexes, namely ESCRT-0, -I, -II, and -III (Chiaruttini et al. 2015; Maas et al. 2017). Microvesicles, also called ectosomes, are usually larger than the exosomes and originate directly from the plasma membrane. These particles can range from 100 nm up to 1μm in size (Maas et al. 2017).

2 Fungal EVs: An Overview The first indirect evidence of EV production in fungi date from the 1970s when Takeo et al. (1973) used high-resolution microscopy to detect exosome-like particles in Cryptococcus neoformans. At that time, those structures were referred to as paramural bodies (Takeo et al. 1973). However, it was only in 2007, that Rodrigues et al. (2007) isolated and characterized these structures isolated from C. neoformans culture supernatants. Cryptococcal EVs were demonstrated to contain components involved in both housekeeping metabolism and pathogenicity (Rodrigues et al. 2007, 2008). Since these first reports on Cryptococcus, the field of fungal EVs has expanded considerably (Rizzo et al. 2020b; de Oliveira et al. 2020). So far, EVs have been described in 15 additional genera: Candida (Anderson et al. 1990; Albuquerque et al. 2008), Saccharomyces (Albuquerque et al. 2008), Pichia (Leone et al. 2018), Histoplasma (Albuquerque et al. 2008), Paracoccidioides (Vallejo et al. 2011), Sporothrix (Albuquerque et al. 2008; Ikeda et al. 2018), Malassezia (Gehrmann et al. 2011), Trichophytum (Bitencourt et al. 2018), Aspergillus (Souza et al. 2019), Fusarium (Bleackley et al. 2019), Alternaria (Silva et al. 2014), Rhizopus (Liu et al. 2018), Trichoderma (de Paula et al. 2019), and Exophiala (Lavrin et al. 2020). The molecular mechanisms concerning EV formation in fungi are mostly unknown. However, different studies combining multiple approaches for the study of EVs have provided some insights into how the biogenesis of EVs occurs in these organisms.

3 Intracellular Biogenesis of Fungal EVs The ESCRT complex is formed of four complexes of high-molecular-weight cytoplasmic proteins called ESCRT-0, -I, -II, and -III (Hurley and Emr 2006). Studies in S. cerevisiae revealed key roles for a class of proteins called VPS (vacuolar protein sorting) in the functionality of the ESCRT complex. VPS components play key roles at different stages of protein traffic between the Golgi and the vacuole (Katzmann et al. 2001; Hurley and Emr 2006). In yeast, more than 60 genes are involved in the vacuolar protein sorting (Bryant et al. 1998; Iwaki et al. 2007). However, a special

4

H. C. de Oliveira et al.

class of VPS protein, the E VPS class, is essential to the MVB sorting pathway (Piper et al. 1995; Bryant et al. 1998; Odorizzi et al. 1998) which can directly impact the EV biogenesis. In S. cerevisiae, the deletion of different genes encoding VPS proteins affected EV release, morphology, and cargo (Oliveira et al. 2010; Zhao et al. 2019). The mutant strains vps23Δ, vps36Δ, vps2Δ, and hseIΔ displayed a significant reduction in EV release as compared to the wild-type strains. Besides, the vps23Δ and vps36Δ mutants had enrichment of an EV population with increased size (Zhao et al. 2019). Disruption of VPS genes in S. cerevisiae also modulated the EV protein cargo. Oliveira et al. (2010) observed that the protein composition of EVs isolated from the vps23Δ and snf7Δ mutant strains was significantly distinct from that observed in the parental strains (Oliveira et al. 2010). Important differences in EV protein cargo were also observed by Zhao et al. (2019) for vps2Δ, vps23Δ, and vps36Δ mutant strains, in comparison to parental cells (Zhao et al. 2019). These results indicate that the ESCRT complexes are required for EV formation in fungal cells. In C. neoformans, disruption of the VPS27 gene led to a defect in EV export that resulted in the accumulation of MVBs in the cytosol (Park et al. 2020). Moreover, the mutant strains, vps34Δ and hseIΔ, showed defective traffic of laccase, an important cryptococcal virulence factor that is exported in EVs (Park et al. 2020). Indeed, in Cryptococcus there is an evident link between the ESCRT pathway, EV formation, and virulence factors. Glucuronoxylomannan (GXM), the most abundant component of the cryptococcal capsule, is transported in EVs and was one of the first components identified in cryptococcal EVs (Rodrigues et al. 2007, 2008). Disruption of distinct genes of the ESCRT pathway, including VPS34, HSE1, VPS23, VPS22, VPS25, VPS20, and SNF7 (Hu et al. 2013, 2015; Godinho et al. 2014; Park et al. 2020), had an important impact on the capsule size of mutant cells. This effect may be related to a defect in EV formation, as observed for the vps27Δ mutant strain, which also showed a significant decrease in capsule size (Park et al. 2020). Besides laccase and GXM, urease, which is another important virulence factor to Cryptococcus, is exported in EVs, and it was also affected by VPS27 disruption (Park et al. 2020). Recently, the importance of the ESCRT pathway in EV formation was demonstrated in C. albicans. Sixteen mutant strains in which different ESCRT genes were disrupted (VPS27, HSE1, VPS23, VPS28, MVB12, SRN2, VPS22, VPS25, VPS36, VPS2, VPS20, SNF7, VPS24, BRO1, DOA4, VPS4) showed a significant decrease in EV production, consequently influencing the export of components of the biofilm extracellular matrix and their resistance to antifungals (Zarnowski et al. 2018). The decrease in EV production may be linked with previous observations of Bruckmann et al. (2001) in a C. albicans vps34Δ mutant strain, which displayed an abnormal accumulation of vesicles in the cytoplasm with a defect in vesicle-mediated protein sorting (Bruckmann et al. 2001). Other regulators of vesicle formation are required for EV export in fungi. In S. cerevisiae, lack of expression of the Golgi reassembly and stacking protein (GRASP), which connects the early endosomal compartments and the MVB pathway (Duran et al. 2010), resulted in a significant reduction in the release of EVs

Biogenesis of Fungal Extracellular Vesicles: What Do We Know?

5

(Oliveira et al. 2010). In Cryptococcus, deletion of the GRASP gene resulted in the formation of EVs with abnormal dimensions (Peres da Silva et al. 2018). In this model, EVs produced by the graspΔ strain had an altered RNA content when compared to wild-type cells (Peres da Silva et al. 2018). Other secretory pathways apparently affect the formation of EVs in fungi. Seminal studies by the Schekman laboratory with S. cerevisiae mutants lacking SEC genes, which compose the post-Golgi secretory pathway, revealed that these cells displayed an accumulation of vesicles in the cytoplasm (Novick et al. 1980; Schekman 2002). The connections between the post-Golgi secretion pathway and the formation of EVs in fungi are unknown, but Oliveira et al. (2010) demonstrated that a sec4Δ S. cerevisiae strain produced vesicles with an altered diameter. In these cells, particles corresponding to EVs were distributed in two populations of 80–120 nm and 400–550 nm, while the in the parental strain EV diameter ranged from 100 to 200 nm (Oliveira et al. 2010). The participation of SEC genes in EV formation was also suggested in Cryptococcus, where disruption of SEC6 resulted in the lack of detection of EVs (Panepinto et al. 2009). These are puzzling results. Since post-Golgi vesicles are supposed to fuse with the plasma membrane (Schekman 2002; Lee et al. 2004), it is unknown how the deletion of SEC genes would affect EV formation in fungi. The identification of EV biomarkers in fungi could be an extremely important tool to elucidate the mechanisms of EV biogenesis. In a recent study (Dawson et al. 2020), the utilization of a proteomic approach allowed the identification of different proteins as potential EV biomarkers in C. albicans. One major candidate was Sso2, an integral membrane protein involved in the fusion of secretory vesicles with the plasma membrane. The enrichment of this protein in C. albicans EVs suggests an endocytic origin (Dawson et al. 2020). It is well-known that autophagy is involved with EV formation in different eukaryotes, including fungi (Zheng et al. 2019). Autophagy is a conserved protein degradation process functioning in different cell types that culminates with cargo delivery to lysosomes (Amaya et al. 2015; Zhang et al. 2018). One known regulator of autophagy is the Atg7 protein, a ubiquitin-activating enzyme (E1) that is involved with EV cargo selection in Cryptococcus. Vesicles from a C. neoformans atg7Δ strain displayed a different RNA content in comparison with its parental strain (Peres da Silva et al. 2018).

4 EV Formation at the Plasma Membrane Level One major difficulty in the analysis of microvesicle-like EV formation at the plasma membrane level of fungi is the presence of a thick cell wall. In 1998, Osumi (1998) observed structures resembling microvesicles in protoplasts of Schizosaccharomyces pombe (Osumi 1998). Very recently, Rizzo et al. (2020a) used A. fumigatus protoplasts to study the release of EVs from the plasma membrane. In this study, the combined use of fluorescence, super-resolution scanning

6

H. C. de Oliveira et al.

electron microscopy (SEM), and transmission electron microscopy (TEM) revealed plasma membrane projections and the release of particles resembling fungal EVs (Rizzo et al. 2020a). These results were consistent with early studies by Gibson and Peberdy (1972) demonstrating that protoplasts of A. nidulans also display membrane projections resembling EVs. In A. fumigatus, a significant increase in the release of EVs occurred when the protoplasts regenerated their cell walls (Rizzo et al. 2020a). Proteomic analysis of these EVs revealed the presence of plasma membrane enzymes required for cell wall synthesis, including β1,3 glucan synthase, Fks1, α1,3 glucan synthase, chitin synthase, and mannosyltransferases. These findings strongly support the notion that A. fumigatus EVs originate at the plasma membrane and participate in the synthesis of the cell wall (Rizzo et al. 2020a). The connections between EVs and cell wall assembling were also suggested in S. cerevisiae. Knockout strains lacking ESCRT components produced EVs enriched with cell wall remodeling enzymes, including Fks1, Chs1, and Chs3 (Zhao et al. 2019). In this model, depletion of β1,3glucan led to an increase of EV release, suggesting that the kinetics of EV release is negatively affected by the presence of the cell wall (Zhao et al. 2019). The notion that fungal EVs can be formed at the plasma membrane level is supported by early studies that were not focused on the analysis of EVs. In Cryptococcus, plasma membrane projections forming vesicle-like structures that were in contact with the cell wall were observed two decades ago (Rodrigues et al. 2000). In S. cerevisiae, plasma membrane invaginations resulting in cytoplasmic subtractions that were delivered into the periplasmic space (Rodrigues et al. 2013). This observation is compatible with the abundant detection of cytoplasmic proteins in S. cerevisiae EVs (Oliveira et al. 2010). However, in this and all other studies discussed in this section, the molecular mechanisms required for EV formation at the plasma membrane level remain unknown. Eukaryotic components required for the architecture of the plasma membrane are also required for EV formation in fungi. In C. albicans, genes involved with phospholipid biosynthesis impacted EV morphology and cargo (Wolf et al. 2015). In Cryptococcus, the lipid-translocating enzymes flippase and scramblase impacted EV formation. In this model, the deletion of ATP1, a gene encoding the Apt1 flippase, resulted in EVs with a lower GXM content (Rizzo et al. 2014, 2018). Similarly, the deletion of AIM25, a gene encoding a putative scramblase, affected the formation of cryptococcal EVs (Reis et al. 2019). An aim25Δ mutant strain produced EVs that differed from those obtained from parental cells in physical-chemical properties and cargo. Indeed, EVs obtained from the aim25Δ mutant were more efficiently used by cryptococci as a source of capsular polysaccharides. Altogether, these results strongly support the hypothesis that the plasma membrane is a fundamental cellular site operating in the formation of fungal EVs. Mechanisms of the formation of fungal exosome-like vesicles and microvesicles are illustrated in Fig. 1.

Biogenesis of Fungal Extracellular Vesicles: What Do We Know?

7 S. cerevisiae S. pombae C. neoformans C. albicans A. fumigatus A. nidulans

S. cerevisiae C. neoformans C. albicans Cell wall 30 – 100 nm

Plasma membrane

A

Exosomes

100 nm – 1 μm

B Microvesicles

Cytosol MVB

Fig. 1 Major mechanisms of biogenesis of eukaryotic EVs adapted to the knowledge available for fungal cells. A. MVBs can fuse with the plasma membrane to release exosomes to the periplasmic space, for further passage through the cell wall by still unknown mechanisms. Processes related to exosome formation were described for S. cerevisiae, C. neoformans, and C. albicans, as concluded from the microscopic observation of MVBs fusing with the plasma membrane and/or deletion of MVB-related genes resulting in altered EVs. B. Plasma membrane projections can form microvesicles. Processes related to microvesicle formation were microscopically observed in S. cerevisiae, S. pombae, C. neoformans, C. albicans, A. fumigatus, and A. nidulans. For the original references, see details in the text

5 Closing Remarks Our current knowledge of fungal EVs supports the notion that several cellular regulators and processes work simultaneously to form and export EVs. This idea is supported by the fact that, so far, we have never been able to shut down EV formation in fungi through the deletion of single genes. As mentioned before, several mutants lacking regulators of the secretory pathway displayed alterations in EVs, including the number of vesicles, morphology, and/or cargo. Nonetheless, EVs were always produced by these cells. This may be explained by compensatory mechanisms or simply by the fact that until now, we have not identified an essential regulator of EV formation in fungi. Simplified protocols for fungal EV isolation have been recently made available (Reis et al. 2019, 2021). This experimental tool, together with advances in omics studies (Rodrigues et al. 2014), and with highresolution microscopic approaches, will likely advance our knowledge of the biogenesis of fungal EVs.

References Albuquerque PC, Nakayasu ES, Rodrigues ML et al (2008) Vesicular transport in Histoplasma capsulatum: an effective mechanism for trans-cell wall transfer of proteins and lipids in ascomycetes. Cell Microbiol 10:1695–1710. https://doi.org/10.1111/j.1462-5822.2008.01160.x

8

H. C. de Oliveira et al.

Al-Nedawi K, Meehan B, Micallef J et al (2008) Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells. Nat Cell Biol 10:619–624. https://doi. org/10.1038/ncb1725 Amaya C, Fader CM, Colombo MI (2015) Autophagy and proteins involved in vesicular trafficking. FEBS Lett 589:3343–3353. https://doi.org/10.1016/j.febslet.2015.09.021 Anderson J, Mihalik R, Soll DR (1990) Ultrastructure and antigenicity of the unique cell wall pimple of the Candida opaque phenotype. J Bacteriol 172:224–235. https://doi.org/10.1128/jb. 172.1.224-235.1990 Bielska E, Sisquella MA, Aldeieg M et al (2018) Pathogen-derived extracellular vesicles mediate virulence in the fatal human pathogen Cryptococcus gattii. Nat Commun 9:1556. https://doi. org/10.1038/s41467-018-03991-6 Bitencourt TA, Rezende CP, Quaresemin NR et al (2018) Extracellular vesicles from the dermatophyte Trichophyton interdigitale modulate macrophage and keratinocyte functions. Front Immunol 9:2343. https://doi.org/10.3389/fimmu.2018.02343 Bleackley MR, Samuel M, Garcia-Ceron D et al (2019) Extracellular vesicles from the cotton pathogen Fusarium oxysporum f. sp. vasinfectum induce a phytotoxic response in plants. Front Plant Sci 10:1610. https://doi.org/10.3389/fpls.2019.01610 Bruckmann A, Kunkel W, Augsten K et al (2001) The deletion of CaVPS34 in the human pathogenic yeast Candida albicans causes defects in vesicle-mediated protein sorting and nuclear segregation. Yeast 18:343–353. https://doi.org/10.1002/1097-0061(20010315) 18:43.0.CO;2-Z Bryant NJ, Piper RC, Gerrard SR, Stevens TH (1998) Traffic into the prevacuolar/endosomal compartment of Saccharomyces cerevisiae: a VPS45-dependent intracellular route and a VPS45-independent, endocytic route. Eur J Cell Biol 76:43–52. https://doi.org/10.1016/ S0171-9335(98)80016-2 Chargaff E, West R (1946) The biological significance of the thromboplastic protein of blood. J Biol Chem 166:189–197 Chiaruttini N, Redondo-Morata L, Colom A et al (2015) Relaxation of loaded ESCRT-III spiral springs drives membrane deformation. Cell 163:866–879. https://doi.org/10.1016/j.cell.2015. 10.017 Colombo M, Raposo G, Thery C (2014) Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol 30:255–289. https://doi.org/ 10.1146/annurev-cellbio-101512-122326 Dawson CS, Garcia-Ceron D, Rajapaksha H et al (2020) Protein markers for Candida albicans EVs include claudin-like Sur7 family proteins. J Extracell Vesicles 9:1750810. https://doi.org/10. 1080/20013078.2020.1750810 de Oliveira HC, Castelli RF, Reis FCG et al (2020) Pathogenic delivery: the biological roles of cryptococcal extracellular vesicles. Pathogens 9:754. https://doi.org/10.3390/ pathogens9090754 de Paula RG, Antoniêto ACC, Nogueira KMV et al (2019) Extracellular vesicles carry cellulases in the industrial fungus Trichoderma reesei. Biotechnol Biofuels 12:146. https://doi.org/10.1186/ s13068-019-1487-7 de Toledo Martins S, Szwarc P, Goldenberg S, Alves LR (2019) Extracellular vesicles in fungi: composition and functions. Curr Top Microbiol Immunol 422:45–59. https://doi.org/10.1007/ 82_2018_141 Desrochers LM, Bordeleau F, Reinhart-King CA et al (2016) Microvesicles provide a mechanism for intercellular communication by embryonic stem cells during embryo implantation. Nat Commun 7:11958. https://doi.org/10.1038/ncomms11958 Duran JM, Anjard C, Stefan C et al (2010) Unconventional secretion of Acb1 is mediated by autophagosomes. J Cell Biol 188:527–536. https://doi.org/10.1083/jcb.200911154 Eitan E, Suire C, Zhang S, Mattson MP (2016) Impact of lysosome status on extracellular vesicle content and release. Ageing Res Rev 32:65–74. https://doi.org/10.1016/j.arr.2016.05.001

Biogenesis of Fungal Extracellular Vesicles: What Do We Know?

9

Gehrmann U, Qazi KR, Johansson C et al (2011) Nanovesicles from Malassezia sympodialis and host exosomes induce cytokine responses--novel mechanisms for host-microbe interactions in atopic eczema. PLoS One 6:e21480. https://doi.org/10.1371/journal.pone.0021480 Gibson RK, Peberdy JF (1972) Fine structure of protoplasts of Aspergillus nidulans. J Gen Microbiol 72:529–538. https://doi.org/10.1099/00221287-72-3-529 Godinho RM, Crestani J, Kmetzsch L et al (2014) The vacuolar-sorting protein Snf 7 is required for export of virulence determinants in members of the Cryptococcus neoformans complex. Sci Rep 4:6198. https://doi.org/10.1038/srep06198 Hu G, Caza M, Cadieux B et al (2013) Cryptococcus neoformans requires the ESCRT protein Vps23 for iron acquisition from heme, for capsule formation, and for virulence. Infect Immun 81:292–302. https://doi.org/10.1128/IAI.01037-12 Hu G, Caza M, Cadieux B et al (2015) The endosomal sorting complex required for transport machinery influences haem uptake and capsule elaboration in Cryptococcus neoformans. Mol Microbiol 96:973–992. https://doi.org/10.1111/mmi.12985 Hurley JH, Emr SD (2006) The escrt complexes: structure and mechanism of a membranetrafficking network. Annu Rev Biophys Biomol Struct 35:277–298. https://doi.org/10.1146/ annurev.biophys.35.040405.102126 Ikeda MAK, de Almeida JRF, Jannuzzi GP et al (2018) Extracellular vesicles from Sporothrix brasiliensis are an important virulence factor that induce an increase in fungal burden in experimental sporotrichosis. Front Microbiol 9:2286. https://doi.org/10.3389/fmicb.2018. 02286 Iwaki T, Onishi M, Ikeuchi M et al (2007) Essential roles of class E Vps proteins for sorting into multivesicular bodies in Schizosaccharomyces pombe. Microbiology 153:2753–2764. https:// doi.org/10.1099/mic.0.2007/006072-0 Joffe LS, Nimrichter L, Rodrigues ML, Del Poeta M (2016) Potential roles of fungal extracellular vesicles during infection. mSphere 1(4). https://doi.org/10.1128/mSphere.00099-16 Kabani M, Melki R (2015) Sup35p in its soluble and prion states is packaged inside extracellular vesicles. MBio 6. https://doi.org/10.1128/mBio.01017-15 Katzmann DJ, Babst M, Emr SD (2001) Ubiquitin-dependent sorting into the multivesicular body pathway requires the function of a conserved endosomal protein sorting complex, ESCRT-I. Cell 106:145–155. https://doi.org/10.1016/S0092-8674(01)00434-2 Lavrin T, Konte T, Kostanjšek R et al (2020) The neurotropic black yeast Exophiala dermatitidis induces neurocytotoxicity in neuroblastoma cells and progressive cell death. Cell. https://doi. org/10.3390/cells9040963 Lee MCS, Miller EA, Goldberg J et al (2004) BI-directional protein transport between the ER and Golgi. Annu Rev Cell Dev Biol 20:87–123. https://doi.org/10.1146/annurev.cellbio.20.010403. 105307 Leone F, Bellani L, Muccifora S et al (2018) Analysis of extracellular vesicles produced in the biofilm by the dimorphic yeast Pichia fermentans. J Cell Physiol 233:2759–2767. https://doi. org/10.1002/jcp.25885 Liu S, Hossinger A, Hofmann JP et al (2016) Horizontal transmission of cytosolic Sup35 prions by extracellular vesicles. MBio 7. https://doi.org/10.1128/mBio.00915-16 Liu M, Bruni GO, Taylor CM et al (2018) Comparative genome-wide analysis of extracellular small RNAs from the mucormycosis pathogen Rhizopus delemar. Sci Rep 8:5243. https://doi.org/10. 1038/s41598-018-23611-z Maas SLN, Breakefield XO, Weaver AM (2017) Extracellular vesicles: unique intercellular delivery vehicles. Trends Cell Biol 27:172–188. https://doi.org/10.1016/j.tcb.2016.11.003 Novick P, Field C, Schekman R (1980) Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell 21:205–215. https://doi.org/10. 1016/0092-8674(80)90128-2 Odorizzi G, Babst M, Emr SD (1998) Fab1p PtdIns(3)P 5-kinase function essential for protein sorting in the multivesicular body. Cell 95:847–858. https://doi.org/10.1016/S0092-8674(00) 81707-9

10

H. C. de Oliveira et al.

Oliveira DL, Nakayasu ES, Joffe LS et al (2010) Characterization of yeast extracellular vesicles: evidence for the participation of different pathways of cellular traffic in vesicle biogenesis. PLoS One 5:e11113. https://doi.org/10.1371/journal.pone.0011113 Osumi M (1998) The ultrastructure of yeast: cell wall structure and formation. Micron 29:207–233. https://doi.org/10.1016/S0968-4328(97)00072-3 Panepinto J, Komperda K, Frases S et al (2009) Sec6-dependent sorting of fungal extracellular exosomes and laccase of Cryptococcus neoformans. Mol Microbiol 71:1165–1176. https://doi. org/10.1111/j.1365-2958.2008.06588.x Park YD, Chen SH, Camacho E et al (2020) Role of the ESCRT pathway in laccase trafficking and virulence of Cryptococcus neoformans. Infect Immun. https://doi.org/10.1128/IAI.00954-19 Peres da Silva R, Martins ST, Rizzo J et al (2018) Golgi reassembly and stacking protein (GRASP) participates in vesicle-mediated RNA export in Cryptococcus neoformans. Genes (Basel) 9. https://doi.org/10.3390/genes9080400 Piper RC, Cooper AA, Yang H, Stevens TH (1995) VPS27 controls vacuolar and endocytic traffic through a prevacuolar compartment in Saccharomyces cerevisiae. J Cell Biol 131:603–617. https://doi.org/10.1083/jcb.131.3.603 Raposo G, Stoorvogel W (2013) Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol 200(4):373–383 Record M, Carayon K, Poirot M, Silvente-Poirot S (2014) Exosomes as new vesicular lipid transporters involved in cell–cell communication and various pathophysiologies. Biochim Biophys Acta Mol Cell Biol Lipids 1841:108–120. https://doi.org/10.1016/j.bbalip.2013.10. 004 Reis FCG, Borges BS, Jozefowicz LJ et al (2019) A novel protocol for the isolation of fungal extracellular vesicles reveals the participation of a putative scramblase in polysaccharide export and capsule construction in Cryptococcus gattii. mSphere 4:e00080-19. https://doi.org/10.1128/ mSphere.00080-19 Reis FCG, Gimenez B, Jozefowicz LJ, Castelli RF, Martins ST, Alves LR, de Oliveira HC, Rodrigues ML (2021) Analysis of cryptococcal extracellular vesicles: experimental approaches for studying their diversity among multiple isolates, kinetics of production, methods of separation, and detection in cultures of titan cells. Microbiol Spectr 4:e0012521. https://doi.org/10. 1128/Spectrum.00125-21. Epub ahead of print. PMID: 34346749 Rizzo J, Oliveira DL, Joffe LS et al (2014) Role of the Apt1 protein in polysaccharide secretion by Cryptococcus neoformans. Eukaryot Cell 13:715–726. https://doi.org/10.1128/EC.00273-13 Rizzo J, Colombo AC, Zamith-Miranda D et al (2018) The putative flippase Apt1 is required for intracellular membrane architecture and biosynthesis of polysaccharide and lipids in Cryptococcus neoformans. Biochim Biophys Acta, Mol Cell Res 1865:532–541. https://doi.org/10. 1016/j.bbamcr.2017.12.007 Rizzo J, Chaze T, Miranda K et al (2020a) Characterization of extracellular vesicles produced by Aspergillus fumigatus protoplasts. mSphere 5(4). https://doi.org/10.1128/mSphere.00476-20 Rizzo J, Rodrigues ML, Janbon G (2020b) Extracellular vesicles in fungi: past, present, and future perspectives. Front Cell Infect Microbiol 10. https://doi.org/10.3389/fcimb.2020.00346 Rodrigues ML, Travassos LR, Miranda KR et al (2000) Human antibodies against a purified glucosylceramide from Cryptococcus neoformans inhibit cell budding and fungal growth. Infect Immun 68:7049–7060. https://doi.org/10.1128/iai.68.12.7049-7060.2000 Rodrigues ML, Nimrichter L, Oliveira DL et al (2007) Vesicular polysaccharide export in Cryptococcus neoformans is a eukaryotic solution to the problem of fungal trans-cell wall transport. Eukaryot Cell 6:48–59. https://doi.org/10.1128/EC.00318-06 Rodrigues ML, Nakayasu ES, Oliveira DL et al (2008) Extracellular vesicles produced by Cryptococcus neoformans contain protein components associated with virulence. Eukaryot Cell 7:58–67. https://doi.org/10.1128/EC.00370-07 Rodrigues ML, Franzen AJ, Nimrichter L, Miranda K (2013) Vesicular mechanisms of traffic of fungal molecules to the extracellular space. Curr Opin Microbiol 16:414–420. https://doi.org/ 10.1016/j.mib.2013.04.002

Biogenesis of Fungal Extracellular Vesicles: What Do We Know?

11

Rodrigues ML, Nakayasu ES, Almeida IC, Nimrichter L (2014) The impact of proteomics on the understanding of functions and biogenesis of fungal extracellular vesicles. J Proteome 97:177–186. https://doi.org/10.1016/j.jprot.2013.04.001 Schekman R (2002) Lasker basic medical research award. SEC mutants and the secretory apparatus. Nat Med 8:1055–1058. https://doi.org/10.1038/nm769 Silva BM, Prados-Rosales R, Espadas-Moreno J et al (2014) Characterization of Alternaria infectoria extracellular vesicles. Med Mycol 52:202–210. https://doi.org/10.1093/mmy/myt003 Słomka A, Urban SK, Lukacs-Kornek V et al (2018) Large extracellular vesicles: have we found the holy grail of inflammation? Front Immunol 9:2723. https://doi.org/10.3389/fimmu.2018.02723 Souza JAM, Baltazar LM, Carregal VM et al (2019) Characterization of Aspergillus fumigatus extracellular vesicles and their effects on macrophages and neutrophils functions. Front Microbiol 10:2008. https://doi.org/10.3389/fmicb.2019.02008 Takeo K, Uesaka I, Uehira K, Nishiura M (1973) Fine structure of Cryptococcus neoformans grown in vitro as observed by freeze-etching. J Bacteriol 113:1442–1448 Trams EG, Lauter CJ, Salem N, Heine U (1981) Exfoliation of membrane ecto-enzymes in the form of micro-vesicles. Biochim Biophys Acta 645:63–70. https://doi.org/10.1016/0005-2736(81) 90512-5 Vallejo MC, Matsuo AL, Ganiko L et al (2011) The pathogenic fungus Paracoccidioides brasiliensis exports extracellular vesicles containing highly immunogenic α-Galactosyl epitopes. Eukaryot Cell 10:343–351. https://doi.org/10.1128/EC.00227-10 van Niel G, D’Angelo G, Raposo G (2018) Shedding light on the cell biology of extracellular vesicles. Nat Rev Mol Cell Biol 19:213–228. https://doi.org/10.1038/nrm.2017.125 Wolf P (1967) The nature and significance of platelet products in human plasma. Br J Haematol 13:269–288. https://doi.org/10.1111/j.1365-2141.1967.tb08741.x Wolf JM, Espadas J, Luque-Garcia J et al (2015) Lipid biosynthetic genes affect Candida albicans extracellular vesicle morphology, cargo, and immunostimulatory properties. Eukaryot Cell 14:745–754. https://doi.org/10.1128/EC.00054-15 Zarnowski R, Sanchez H, Covelli AS et al (2018) Candida albicans biofilm–induced vesicles confer drug resistance through matrix biogenesis. PLoS Biol 16:e2006872. https://doi.org/10. 1371/journal.pbio.2006872 Zhang N, Yang X, Yuan F et al (2018) Autophagy-deficient tumor cells rely on extracellular amino acids to survive upon glutamine deprivation. Autophagy 14:1652–1653. https://doi.org/10. 1080/15548627.2018.1493314 Zhao K, Bleackley M, Chisanga D et al (2019) Extracellular vesicles secreted by Saccharomyces cerevisiae are involved in cell wall remodelling. Commun Biol 2:305. https://doi.org/10.1038/ s42003-019-0538-8 Zheng J, Tan J, Miao Y-Y, Zhang Q (2019) Extracellular vesicles degradation pathway based autophagy lysosome pathway. Am J Transl Res 11:1170–1183

Lessons Learned from Studying Histoplasma capsulatum Extracellular Vesicles Daniel Zamith-Miranda, Lysangela Ronalte Alves, Ernesto Satoshi Nakayasu, and Joshua Daniel Nosanchuk

Contents 1 2 3 4

Histoplasma capsulatum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discovery of EV in H. capsulatum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. capsulatum EV Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. capsulatum EV Cargo Loading and Release Is a Highly Dynamic Regulated Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Monoclonal Antibody Binding to Fungal Surfaces Impacts the Characteristics and Payloads of EV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Growth Medium Significantly Regulates EV Loading and Secretion in H. capsulatum 5 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

14 14 14 15 15 17 17 18

Abstract Histoplasma capsulatum is a major endemic mycosis. Our laboratories have demonstrated that H. capsulatum produces extracellular vesicles (EV) that are loaded with diverse compounds that influence virulence. We have further shown that H. capsulatum dynamically regulates the loading and release of fungal EV in response to stimuli and growth conditions. This chapter details the current knowledge of EV biology in H. capsulatum and the impact of this information on our understanding of this important process that is closely linked to pathogenesis.

D. Zamith-Miranda · J. D. Nosanchuk (*) Albert Einstein College of Medicine, Bronx, NY, USA e-mail: [email protected] L. R. Alves Instituto Carlos Chagas, Fundação Oswaldo Cruz (Fiocruz), Curitiba, Brazil E. S. Nakayasu Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA © Springer Nature Switzerland AG 2021 M. Rodrigues, G. Janbon (eds.), Fungal Extracellular Vesicles, Current Topics in Microbiology and Immunology 432, https://doi.org/10.1007/978-3-030-83391-6_2

13

14

D. Zamith-Miranda et al.

1 Histoplasma capsulatum Histoplasma capsulatum is a major global fungal pathogen (Azar et al. 2020; Rakislova et al. 2021). It is remarkable for its thermodimorphism, existing as a filamentous form in the environment that undergoes rapid changes to yeast during human infection. Infection in endemic regions is common, but clinical disease is relatively infrequent. Pulmonary disease is most common, but H. capsulatum can widely disseminate. During infection, yeast cells survive intracellularly within phagosomes of macrophages, which protects the fungus from diverse host effector responses (Shen and Rappleye 2020).

2 Discovery of EV in H. capsulatum Following the identification of extracellular vesicles (EV) in C. neoformans, a basidiomycete fungus, by Dr. Marcio Rodrigues in 2007 (Rodrigues et al. 2007), we sought to determine if fungi in the phylum Ascomycota similarly produced EV. In 2008, we demonstrated that H. capsulatum along with the ascomycetes Candida parapsilosis, C. albicans, Sporothrix schenckii, and Saccharomyces cerevisiae produced large amounts of EV (Albuquerque et al. 2008). Transmission electron microscopy (TEM) revealed that the H. capsulatum EV was similar to that previously characterized in C. neoformans with the presence of bilayered vesicles of different sizes carrying molecules of variable electron density. Additionally, analyses of TEM photomicrographs identified vesicles internal to the cell, transitioning the cell wall, and exiting the cell wall as well as EV that appeared to be recently released from cells (Albuquerque et al. 2008). These findings solidly formed the foundation for EV being viewed as a remarkably conserved and important mechanism in fungal biology.

3 H. capsulatum EV Contents Even though our methodologies for analyzing the proteome of fungal EV were nescient in 2008, we identified 283 proteins in H. capsulatum EV (Albuquerque et al. 2008). Included in the molecules were proteins that have been well established as virulence factors in histoplasmosis, such as catalase, superoxide dismutase, and a variety of heat shock proteins. Overall, there were numerous proteins associated with chaperone functions and regulators of cell wall architecture, cell signaling, cytoskeleton, antioxidant responses, cell cycle, lipid metabolism, and sugar metabolism. There were also diverse proteins involved with functions in the nucleus, ribosome, and proteasome. Although there were similarities to many proteins identified in C. neoformans EV (Rodrigues et al. 2007), a significant number were distinct to

Lessons Learned from Studying Histoplasma capsulatum Extracellular Vesicles

15

H. capsulatum (Albuquerque et al. 2008), which is consistent with the difference in the pathobiology of the two species. Another major finding was that sera from individuals with histoplasmosis were immunoreactive with many of the proteins contained within the H. capsulatum EV (Albuquerque et al. 2008), and binding to heat shock protein and histone 2B were confirmed using monoclonal antibodies specific to these two important virulence determinants. Membrane phospholipids were also highly diverse in EV, with 17 major lipid species identified, including phosphatidylethanolamine (PE), phosphatidylcholine (PC), and phosphatidylserine (PS) (Albuquerque et al. 2008). A recent analysis showed that EV is enriched in PE and monohexosylceramide, but depleted in triacylglycerol (TG), inositolphosphoceramide (PI_Cer), cardiolipin (CL), phosphatidylglycerol (PG), phosphatidylinositol (PI), and PS. This is consistent with the lipids required to build the EV bilayered membrane. For instance, the depletion of the energy storage lipid, TG, and the mitochondrial marker, CL, is evidence of a specialized mechanism for sorting lipids to build EV membranes. More recently, we also demonstrated a rich presence of RNAs as cargo within H. capsulatum EV (Alves et al. 2019). Using two commonly studied H. capsulatum strains, we identified 124 mRNAs. However, the mRNAs were differentially enriched in the two strains, which suggests that there are key differences among strains. This is not surprising as there are distinct differences between these two strains as the G217B lacks an alpha-glucan outer wall layer. We also identified a distinct class of ncRNAs with fragments sizes varying from 20 to 40 nt in length that aligned complementary to specific mRNAs, indicating a role in gene expression regulation.

4 H. capsulatum EV Cargo Loading and Release Is a Highly Dynamic Regulated Process Investigators in the area of fungal EV biology were asking questions about the regulation of EV biogenesis and active discussions were presented during the BrazilUS Colloquium on Fungal Extracellular Vesicles held in 2016 at the Albert Einstein College of Medicine, Bronx, NY. Work on H. capsulatum was the first to provide specific insights into the nuanced regulation of EV loading by fungi.

4.1

Monoclonal Antibody Binding to Fungal Surfaces Impacts the Characteristics and Payloads of EV

In 2016, we demonstrated that the binding of either protective or non-protective monoclonal antibodies (mAb) to cell surface-displayed heat shock protein 60 (hsp60) significantly altered the biology of H. capsulatum EV (Matos Baltazar

16

D. Zamith-Miranda et al.

et al. 2016). The size of both small and large EV populations increased significantly with mAb binding. Interestingly, although the sterol content of the EVs remained similar, yeast interacting with mAb, produced EV with significantly greater amounts of protein compared to what was quantified in EV from untreated yeast. Certain virulence factors were assayed and mAb binding reduced phosphatase and laccase activity in EV compared to EV from untreated yeast cells. The non-protective mAb 7B6 also reduced the amount of catalase in EV. Demonstrating the advances in our capacity to perform proteomic analyses in EV compared to our 2008 paper (Albuquerque et al. 2008), we identified 1125 proteins and 250 were differentially present in EV from yeast cells cultivated with or without mAb (Matos Baltazar et al. 2016). Overall, the most plentiful proteins were associated with amino acid, protein, sugar, and lipid metabolism as well as nuclear processes. Both mAbs enhanced and downregulated large numbers of proteins. For the protective mAb 6B7, 46% increased and 47% decreased. Comparisons were also made for EV from yeast cells cultivated with mAb to EV from other fungal species to determine whether a pattern of changes in abundance could be associated with alterations in virulence. Both mAbs caused alterations in abundance relative to orthologous proteins in EV from Paracoccidioides brasiliensis, S. cerevisiae, and C. neoformans. The conclusion was that the mAbs, although with different capacities to modify histoplasmosis, nevertheless, both differentially regulated pathways that were conserved in closely and distantly related fungi. This work also demonstrated a new role for mAb action, specifically that mAb from the host can regulate fungal secretion, by acting as an agonist for a signaling molecule on the surface of the fungal cell. We next, in 2018, discovered that EV biogenesis was differentially regulated by antibodies as variations in mAb led to concentration dependent and independent changes (Baltazar et al. 2018). Comparing 6 and 20 μg of mAb, the most alterations by KEGG Pathway analysis were glycolysis/gluconeogenesis (12 fold enrichment) and phagosome (11 fold enrichment) with proteins associated with the endoplasmic reticulum, ribosomes, and oxidative phosphorylation, among others, also markedly altered. This work also advanced the study of H. capsulatum EV into mechanisms of pathogenesis. Bone marrow-derived murine macrophages were treated with EV derived from control or mAb-treated yeast cells and then challenged with yeast cells. Although control EV inhibited phagocytosis by 35%, EV from mAb-treated yeast suppressed phagocytosis by as much as 60%. Interestingly, the 6 μg mAb concentration condition was more suppressive than the 20 μg of mAb. However, EV from both control and mAb-treated H. capsulatum increased the survival of yeast cells that were phagocytosed, and EV from yeast cells bound by 6 μg of mAb 7B6 had the highest survival rates, consistent with the non-protective nature of this antibody. Human macrophages were also less able to kill H. capsulatum yeast cells when they were pretreated with EV from yeast cells incubated with mAb 7B6. Finally, EV derived from mAb-treated yeast cells significantly reduced reactive oxygen species generation compared to both untreated and control EV-treated murine macrophages. Altogether, these findings further supported the remarkably responsive and dynamic fine-tuning of the biogenesis of H. capsulatum EV.

Lessons Learned from Studying Histoplasma capsulatum Extracellular Vesicles

4.2

17

Growth Medium Significantly Regulates EV Loading and Secretion in H. capsulatum

In 2020, we studied whether growth medium affected EV production and release, and found that “media matters!” (Cleare et al. 2020). Our capacity to perform proteomics continued to advance with our ongoing experience in EV research in H. capsulatum and other fungi such that we were able to identify nearly 2000 proteins in H. capsulatum EV and determined that 270 were significantly differentially regulated in the different growth medium conditions. Notably, the rich media, particularly Ham’s medium followed by Brain Heart Infusion (BHI) medium. Of the regulated proteins in rich medium, proteins linked to carbohydrate and polysaccharide metabolism, amino acid and protein transport, and phospholipid translocation were upregulated. For Ham’s, proteins associated with DNA repair, mRNA splicing, endocytosis, and lipid metabolism were also upregulated. In the restrictive medium, RPMI, the upregulated proteins were primarily associated with transcription and protein folding. Although we only identified 17 lipids in H. capsulatum EV in 2008 (Albuquerque et al. 2008), our advances in lipidomics facilitated the detection of 100 lipids in the EV derived from the different growth media (32406582) and 44 were differentially altered. Similar to the proteomics, culture in Ham’s medium led to the greatest variety of lipids in EV, and these were mostly PC and PE species. Interestingly, lipids abundant in EV from yeast grown in BHI were also present in EV from the other culture conditions with the exception of sphingomyelins that were in the BHI-derived EV. The EV from BHI contained several sphingomyelin species as well as ceramide and a PI_ceramide. The EV from RPMI cultures had the most abundant of lysophospholipids, particularly lysophophatidylcholine. Hence, the nutritional state of H. capsulatum is an important factor in EV loading and secretion, which has significant implications as the availability of nutrients varies significantly in different tissues, and during the intracellular phase of the fungus, within the host cells.

5 Summary Since their description in 2007 (Rodrigues et al. 2007), remarkable insights into the processes of generating fungal EV and their biological actions. The work in H. capsulatum was fundamental in extending the finding of EV biogenesis in a fungus in the phylum Basidiomycota to ascomycete fungi. The work on H. capsulatum also led to innovations in omic techniques to improve our capacity to identify diverse compounds, including proteins, lipids, metabolites, and RNAs. H. capsulatum has also served as a model system to demonstrate the fine specificity of the regulation of EV biogenesis using mAbs and nutritional conditions to explore this area, and these advances were crucial to demonstrating that cargo sorting in EV

18

D. Zamith-Miranda et al.

is an active and highly regulated event. Finally, EVs from the fungus were used to assess how these structures can affect host-pathogen processes. In sum, H. capsulatum is a model organism for the study of EV cargo and its function in the host-pathogen interface. Acknowledgement J.D.N., E.S.N., and D.Z-M were supported in part by NIH R21AI124797. LRA received financial support from Inova Fiocruz/Fundação Oswaldo Cruz [Grant number VPPCB-07-FIO-18-2-52] and CNPq [Grant number 442317/2019-0]. L.R.A is a research fellow awardee from CNPq.

References Albuquerque PC, Nakayasu ES, Rodrigues ML, Frases S, Casadevall A, Zancope-Oliveira RM et al (2008) Vesicular transport in Histoplasma capsulatum: an effective mechanism for trans-cell wall transfer of proteins and lipids in ascomycetes. Cell Microbiol 10(8):1695–1710 Alves LR, Peres da Silva R, Sanchez DA, Zamith-Miranda D, Rodrigues ML, Goldenberg S et al (2019) Extracellular vesicle-mediated RNA release in Histoplasma capsulatum. mSphere 4(2): e00176-19 Azar MM, Loyd JL, Relich RF, Wheat LJ, Hage CA (2020) Current concepts in the epidemiology, diagnosis, and management of histoplasmosis syndromes. Semin Respir Crit Care Med 41 (1):13–30 Baltazar LM, Zamith-Miranda D, Burnet MC, Choi H, Nimrichter L, Nakayasu ES et al (2018) Concentration-dependent protein loading of extracellular vesicles released by Histoplasma capsulatum after antibody treatment and its modulatory action upon macrophages. Sci Rep 8 (1):8065 Cleare LG, Zamith D, Heyman HM, Couvillion SP, Nimrichter L, Rodrigues ML et al (2020) Media matters! Alterations in the loading and release of Histoplasma capsulatum extracellular vesicles in response to different nutritional milieus. Cell Microbiol 22(9):e13217 Matos Baltazar L, Nakayasu ES, Sobreira TJ, Choi H, Casadevall A, Nimrichter L et al (2016) Antibody binding alters the characteristics and contents of extracellular vesicles released by Histoplasma capsulatum. mSphere 1(2) Rakislova N, Hurtado JC, Palhares AEM, Ferreira L, Freire M, Lacerda M et al (2021) High prevalence and mortality due to Histoplasma capsulatum in the Brazilian Amazon: an autopsy study. PLoS Negl Trop Dis 15(4):e0009286 Rodrigues ML, Nimrichter L, Oliveira DL, Frases S, Miranda K, Zaragoza O et al (2007) Vesicular polysaccharide export in Cryptococcus neoformans is a eukaryotic solution to the problem of fungal trans-cell wall transport. Eukaryot Cell 6(1):48–59 Shen Q, Rappleye CA (2020) Living within the macrophage: dimorphic fungal pathogen intracellular metabolism. Front Cell Infect Microbiol 10:592259

Current Status on Extracellular Vesicles from the Dimorphic Pathogenic Species of Paracoccidioides Rosana Puccia

Contents 1 Paracoccidioides spp. and Paracoccidioidomycosis: General Aspects . . . . . . . . . . . . . . . . . . . . . . 2 Characterization of Paracoccidioides EVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Paracoccidioides EV Proteome in Comparison with the Cell Wall . . . . . . . . . . . . . . . . . . . . . . . . . 4 Carbohydrate Delivery by Paracoccidioides EVs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Lipidome of Paracoccidioides EVs in Comparison with the Cell Wall . . . . . . . . . . . . . . . . . . . . 6 Paracoccidioides EV Transcriptome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Paracoccidioides EVs: Cell Communication and Immunomodulation . . . . . . . . . . . . . . . . . . . . . 8 Closing Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

19 21 22 23 25 26 28 29 29

Abstract Species of the Paracoccidioides brasiliensis complex and P. lutzii cause human paracoccidioidomycosis (PCM). Early interest in Paracoccidioides extracellular vesicles (EVs) resulted in a series of publications that unraveled the EVs protein, carbohydrate, lipid, and RNA cargo from isolates of different phylogenetic groups and distinct virulence degrees. EV and cell wall components were compared. These works are discussed in parallel with more recent data on the role of Paracoccidioides EVs in immunomodulation.

1 Paracoccidioides spp. and Paracoccidioidomycosis: General Aspects Human paracoccidioidomycosis (PCM) was described by Adolpho Lutz in 1908 (Lutz 1908). Until recently, the PCM etiological agent has been considered to be a unique species that was classified as Paracoccidioides brasiliensis in 1930 (Almeida R. Puccia (*) Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de MedicinaUniversidade Federal de São Paulo (EPM-UNIFESP), São Paulo, SP, Brazil © Springer Nature Switzerland AG 2021 M. Rodrigues, G. Janbon (eds.), Fungal Extracellular Vesicles, Current Topics in Microbiology and Immunology 432, https://doi.org/10.1007/978-3-030-83391-6_3

19

20

R. Puccia

1930). With the advances in nucleic acid sequencing technology and genetic divergence analysis following the multilocus sequence typing strategy, the phylogenetic species concept provided enough support to split the P. brasiliensis isolates into five distinct taxonomical species (Turissini et al. 2017). Therefore, PCM can currently be defined as a systemic mycosis caused by the P. brasiliensis complex (Matute et al. 2006; Teixeira et al. 2014), specifically, P. brasiliensis sensu strictu (S1 main group), P. americana (PS2 group), P. restrepiensis (PS3 group), P. venezuelensis (PS4 group), and by Paracoccidioides lutzii (formerly “Pb01-like” group). The P. lutzii group is genetically well separated and comprises isolates from CentralWestern and Northern Brazil (Teixeira et al. 2009, 2014; Desjardins et al. 2011). The P. lutzii genome is 32.9 Mb in size, while it varies between 29 and 30 Mb in the P. brasiliensis complex, with 94.8% identity between them. Genomic identity within the P. brasiliensis complex is over 98.5% (Desjardins et al. 2011; Muñoz et al. 2014). The Paracoccidioides genome information can be accessed through the link http://fungi.ensembl.org. Unfortunately, genetic manipulation is still an issue in Paracoccidioides studies due to the lack of efficient molecular techniques for targeted gene inactivation (Chaves et al. 2021). Therefore, in order to understand the role of individual molecules in the fungal biology and virulence, A. tumefaciensmediated transformation (ATMT) has been used in conjunction with the antisense RNA (aRNA) technology to obtain mutants with low expression of target genes (Menino et al. 2012; Sturme et al. 2011). The Paracoccidioides spp. are temperature-dependent dimorphic ascomycetes belonging to the Onygenales order and the Ajellomycetaceae family (Teixeira et al. 2014). At mild temperatures up to 28
C, Paracoccidioides spp. grow as mycelia constituted of thin septate hyphae. Conidia produced by the environmental mycelia are inhaled by the host and germinate as multi-budding and multinucleated yeasts in the alveolar spaces or at incubation temperatures around 37
C. Dimorphism is essential for active PCM to occur, considering that conidia and hyphae are promptly destroyed by the host immune defenses (McEwen et al. 1987). PCM is a granulomatous mycosis that is mainly reported in Latin American countries, especially in Brazil, which contributes to 80% of the reported cases. The clinical manifestations involve mainly the lungs, but lymphatic acute/subacute forms also occur in up to 20% of the cases (Prado et al. 2009; Martinez 2017). The Th1-driven pro-inflammatory immune response provides protection against PCM and other fungal infections (Gow et al. 2017; Burger 2021; Fernández-García and Cuellar-Rodríguez 2021). Dendritic cells make the bridge between the innate and acquired immunity, and the IL-12 cytokine they express is essential to stimulate an effective Th1 response. Active macrophages and neutrophils are key to prevent disease progression as well. Interferon γ (IFNγ) activates macrophages, which then express tumor necrosis factor-α (TNF-α) that is necessary for granuloma persistence. Essential effector mechanisms involve the production of reactive oxygen and nitrogen species of the respiratory burst by the immune cells (Gow et al. 2017; Burger 2021; Fernández-García and Cuellar-Rodríguez 2021). In PCM patients, high serum antibody levels reflect the severity of the disease: the specific antigen for the P. brasiliensis complex is the secreted glycoprotein gp43

Current Status on Extracellular Vesicles from the Dimorphic Pathogenic. . .

21

(Puccia and Travassos 1991; Pinheiro et al. 2020), which is unfortunately not diagnostic for P. lutzii infections (Leitão et al. 2014). The gp43 mediates yeast cell adherence to ECM components, probably contributing to virulence (Torres et al. 2013), but it is also a vaccine candidate due to its P10 T-cell epitope (Taborda et al. 1998; Travassos and Taborda 2012). Since their full characterization in C. neoformans (Rodrigues et al. 2007), extracellular vesicles (EVs) have been recognized as important fungal components involved in the delivery of all types of molecules to the cell wall and extracellular environment, thus highly contributing to cell communication and virulence. These topics are discussed in other chapters of this book. In addition, there are excellent reviews on fungal EVs (Rodrigues et al. 2011, 2013, 2014, 2015; Joffe et al. 2016; Nimrichter et al. 2016; Zamith-Miranda et al. 2018, 2021; Piffer et al. 2021). Early interest in studying Paracoccidioides EVs resulted in a series of publications that unraveled the EVs protein, carbohydrate, lipid, and RNA cargo and provided a detailed comparison with cell wall components. The aim of this chapter is to critically review the solid and relevant literature data related to Paracoccidioides EVs and their role in immunomodulation. We will use the old nomenclature of P. brasiliensis, when relating to the P. brasiliensis complex, and P. lutzii throughout the text.

2 Characterization of Paracoccidioides EVs Most publications on Paracoccidioides EVs were the result of projects developed in collaboration with experts in different areas, especially with Dr. Igor C. Almeida, who suggested to look for EVs in Paracoccidioides early in the 2000s. The assays were performed with EVs obtained from fungal cultures of the pathogenic yeast phase that had recently been recovered from mice organs to keep the virulence traits hopefully expressed. The original characterization of Paracoccidioides EVs was published in 2011 (Vallejo et al. 2011). The culture medium of choice for EV purification was the Ham’s F-12 defined medium (Gibco) supplemented with 1.5% glucose (F12/glc). Although Paracoccidioides grows faster and better in rich media such as yeastpeptone-dextrose YPD, cell culture-defined media should provide a more host-like environment and are free of complex molecules that might bind to the EV surface, eventually leading to artifactual results. For EV preparation, dense 500-mL cultures of logarithmic growing pre-inoculum cells were cultivated at 36
C for 2 days under shanking. EVs were isolated from culture supernatants using filtration and differential centrifugation, as standardized for C. neoformans EVs (Rodrigues et al. 2007; Vallejo et al. 2011). In order to avoid membrane contamination resulting from cell lysis in the processed supernatants, cultures had to be over 95% viable. The Paracoccidiodes projects developed by our group have been carried out using P. brasiliensis Pb18, which is highly virulent and represents phylogenetic group S1, Pb3 representing PS2, which is the most distant phylogenetic group within

22

R. Puccia

the P. brasiliensis complex, and P. lutzii Pb01. We have previously observed that P. brasiliensis isolates from phylogenetic group S1 were more virulent in the mice than those from PS2, thus, suggesting a correlation between genetic groups and virulence (Carvalho et al. 2005). Comparative results between Pb18 and Pb3 might hopefully lead to the finding of potential virulence traits. In the work by Vallejo et al. (2011), EVs could be observed at the surface of the cell wall and in 100,000 g final precipitates (1 h) by transmission electron microscopy (TEM). The EV population had different sizes between 20 and 200 nm, as estimated by TEM. Later work in the lab showed that the P. brasiliensis Pb18 EV size estimated by nanoparticle tracking analysis (NTA) peaks at 45–63 nm, 85 nm, while only trace amounts are between 100–200 nm. Nitrosative and oxidative stresses, apparently, stimulated EV export and the EVs tended to be bigger (Leitao 2017). We have lately adapted to Paracoccidioides the protocol of EV isolation from stationary cultures (Reis et al. 2019), by growing the yeasts in petri dishes containing F12/glc (0.5% glucose)-agar. Under these conditions, the EV size profile showed a sharp peak of about 48 nm and trace amounts of EVs between 100 and 200 nm (Octaviano et al. unpublished). The small sizes estimated for Paracoccidiodes EVs suggest that the preparations might be enriched with exosomes (Théry et al. 2018). In general, however, P. brasiliensis EV sizes seem to be smaller than those for most other fungal EVs (Zamith-Miranda et al. 2021).

3 Paracoccidioides EV Proteome in Comparison with the Cell Wall On the basis of peptide sequencing obtained by liquid chromatography coupled with tandem mass spectrometry, Vallejo et al. (2012b) carried out a thorough secretome characterization from the yeast phase of P. brasiliensis Pb18 that included a comparison between the 100,000 g (1 h) EV and “EV-free” fractions. Out of 205 total EV proteins, 89 were not detected in the “EV-free” sample and tended to be transmembrane or associated with the membrane, such as signaling, cell division, and transport proteins. A very useful aspect of the publication was a detailed comparison of the EV proteome from Pb18 with others described until then for C. neoformans, Histoplasma capsulatum, Candida albicans, and Saccharomyces cerevisiae, showing a 63% overlap. Seventy-two orthologs were detected in EVs of at least three species and 26 were common to all systems (Vallejo et al. 2012b). These data suggested that EVs might be the preferential means of transportation of those proteins outside the fungal cell, considering that most of them do not bear a signal sequence. Among the common EV proteins are superoxide dismutase (SOD), thioredoxin, and peroxiredoxin Prx1, which are enzymes involved in the pathogen defense against oxidative stress. Paracoccidioides PbPrx1 has recently been characterized as a 1-Cys peroxiredoxin that can efficiently decompose hydrogen

Current Status on Extracellular Vesicles from the Dimorphic Pathogenic. . .

23

peroxide and organic hydroperoxides. It localizes to the cell wall and mitochondria of yeast cells (Longo et al. 2020). Longo et al. (2014) compared non-covalently-linked cell wall proteins from the mycelial and yeast phases of P. brasiliensis Pb3 and Pb18. The proteins were extracted from live cells with 2 mM DTT for 1 h at 4
C, in a way to preserve plasma membrane integrity and cell viability. Among 132 proteins detected exclusively in the yeast pathogenic phase, 92 were only detected in the Pb18 isolate. About 60% of the total cell wall-associated proteins detected in this work had already been described in fungal EVs, thus reinforcing the notion that the EV cargo highly contributes to components of the cell wall structure (Puccia et al. 2011, 2016; Nimrichter et al. 2016). Gp43 was described both in EVs and at the cell wall, as well as enolase, glyceraldehyde-3-phosphate dehydrogenase, fructose 1,6-bisphosphate aldolase, and triosephosphate isomerase, which are glycolytic enzymes, and cellular signaling 14-3-3. All of these proteins also display adhesive properties and could have a role on the cell wall of helping fungal dissemination, with the help of proteases such as a subtilase-type proteinase psp3 that has been identified at both EV and the cell wall (Vallejo et al. 2012b; Longo et al. 2014). Heat shock proteins and enzymes required for cell wall remodeling have been identified in P. brasiliensis EVs, such as cell wall α1,3-glucan synthase mok13, endo-β1,3-glucanase, glucan β1,3-glucosidase, and α-mannosidase (Vallejo et al. 2012b), and have systematically been found in fungal EVs, suggesting that they could be involved in cell wall biosynthesis/remodeling (Nimrichter et al. 2016). There is an enormous impact fungal cell wall remodeling can have in the interaction with the host and immunomodulation. In this sense, the incredible collection of enzymes that seem to possibly interfere with the cell wall structure deserves attention and further study. Modulation of fungal EV proteome has been described in H. capsulatum by the use of different culture media and by the addition of anti-hsp60 monoclonal antibodies, since hsp60 has been described both at the cell wall and in EVs (Vallejo et al. 2012b; Matos Baltazar et al. 2016; Cleare et al. 2020). Data from our group revealed that the proteome of EVs from P. brasiliensis Pb18 yeasts cultivated under mild oxidative and nitrosative stress suffered a considerable increase in the number of proteins, showing that stress conditions can modulate the EV protein cargo in P. brasiliensis (Leitao 2017).

4 Carbohydrate Delivery by Paracoccidioides EVs Polysaccharides and carbohydrate epitopes (glycotopes) play key roles in microorganism pathogenesis because they can be highly antigenic, display adhesive properties, and interact with the innate immune system (Gow et al. 2017). Most of the fungal pathogen-associated molecular patterns (PAMPs) recognized by pathogen recognition receptors (PRRs) exposed on the cells of the innate immune system are carbohydrate in nature (Erwig and Gow 2016). Cell wall β-1,3-glucan is a

24

R. Puccia

well-studied PAMP mainly recognized by dectin-1, which can interact with toll-like receptor 2 (TLR2) to activate pro-inflammatory pathways (Gow et al. 2017). In the P. brasiliensis cell wall, α-1,3-glucan is prevalent and probably masks β-1,3-glucan recognition to evade the immune system, as demonstrated in H. capsulatum mutants (Rappleye et al. 2007; Gow et al. 2017; Puccia et al. 2016). The pioneer characterization of fungal EVs showed that C. neoformans EVs carry capsular glucuronoxylomannan (GXM), which is a major fungal immunomodulator and virulence factor of high molecular mass (Rodrigues et al. 2007). That was a breakthrough observation because it could explain how GXM and other fungal polysaccharides having bulky structures could eventually be found in the extracellular environment (Latge 2009). In addition, it paved the way to explaining how EVs could be involved in both capsule synthesis and fungal pathogenicity (Rodrigues et al. 2007). The fungal EV carbohydrate content, however, has seldom been addressed in the fungal literature. The work by Peres da Silva et al. (2015a) was entirely dedicated to evaluating and comparing the carbohydrate EV content from P. brasiliensis isolates Pb18, Pb3, and P. lutzii. The work included the analysis of total carbohydrate and also EV surface glycotopes and lectins using glycomics microarrays. Total ethanol-precipitated carbohydrates were analyzed from lipid- and proteinfree EV preparations by chemical degradation, gas chromatography-mass spectrometry, nuclear magnetic resonance, and size exclusion chromatography (Peres da Silva et al. 2015a). The main detected residue was glucose, followed by smaller amounts of mannose and galactose, which are likely to be part of: (a) a high molecular mass 4,6-α-glucan compatible with storage glycogen; (b) a galactofuranosylmannan polymer bearing a 2-α-Manp core and end units of β-Galf (1,3) and α-Manp (1,6); (c) smaller amounts of (1 ! 3)- and (1 ! 6)-glucan characteristic of cell wall components; (d) small amounts of a (1 ! 6) Manp polymer. Subtle differences between Pb18 and Pb01 EVs have been found in the linkage analysis. Therefore, EVs might contribute to the carbohydrate structure of the cell wall and also deliver their components to the extracellular environment. Recently, biofilm EVs have been shown to probably participate in matrix biofilm synthesis by transporting matrix polysaccharides (Zarnowski et al. 2018). Glycomic microarray profiles for Pb18, Pb3, and Pb01 EV surface components binding to plant and mammalian lectins showed higher intensity with Man-binding plant lectins, but there was also some reactivity with lectins specific for galactose, lactose, GlcNAc, and fucose (Peres da Silva et al. 2015a). The finding of glycotopes at the EV surface is extremely relevant because they can intermediate the interaction with the host and other microorganisms. Glycomic microarray with mammalian lectins showed binding to the dendritic cell DC-SIGN and DC-SIGNR receptors. The reaction was inhibited by Man and GlcNAc, which have been identified on the EV surface by plant lectins (Peres da Silva et al. 2015a). Hatanaka et al. (2019) observed that galectin-3 disturbs Paracoccidioides growth and evokes EV disruption at experimentally high concentrations. The same effect has previously been found in C. neoformans cells and EVs (Almeida et al. 2017). The EV lysis mechanism has not been solved, and the authors suggested it could be related to lipid

Current Status on Extracellular Vesicles from the Dimorphic Pathogenic. . .

25

binding. Galectin-3 is a β-galactoside-binding receptor and glycoarray data showed that Paracoccidioides EV glycosyl residues, including galactose, can be part of Nlinked oligosaccharides (Peres da Silva et al. 2015a). Therefore, EV membrane glycoproteins could be involved in the galectin-3 lysis mechanism. Glycan microarray showed that P. brasiliensis EVs are also bound to glycans and therefore expose surface lectin(s). Considering that the binding could be inhibited by GlcNAc, the lectin could be paracoccin, which is a well-characterized extracellular GlcNAc-binding molecule involved in P. brasiliensis morphogenesis and virulence, polarization to M1 protective macrophages, and antifungal therapy (Ganiko et al. 2007; Alegre-Maller et al. 2014; Freitas et al. 2016; Fernandes et al. 2017). That possibility, however, needs further experimental proof, since paracoccin has not been detected in P. brasiliensis EV proteomics so far (Vallejo et al. 2012b). Vallejo et al. (2011) characterized the presence of antigenic terminal alphagalactosyl (α-Gal) epitopes, of the type Gal-α-(1,3)-Gal, in EVs isolated from both Pb18 and Pb3. This glycotope was detected in assays using the Marasmius oreades (MOA) lectin and anti-α-Gal antibodies purified from plasma of Chagas’ disease and PCM patients. The α-Gal epitopes were detected inside and near the EV surface and seemed to be at least partially localized to O-linked oligosaccharides from EV glycoproteins in both Pb18 and Pb3. In Pb18 yeasts, α-Gal epitope labeling was observed by TEM at the cell wall and in clusters inside vacuoles that resembling multivesicular bodies. Although the biological role of EV α-Gal epitopes remains obscure, it could have immunomodulatory properties other than eliciting anti-α-Gal antibodies (Galili 2013). Of note, recent results from our group using anti-α-Gal antibodies suggest the presence of α-Gal epitopes in H. capsulatum, C. neoformans, C. albicans, and A. fumigatus, both at the cell surface and in the cytoplasm (Gegembauer 2019). The results mentioned above show that P. brasiliensis EVs can transport high levels of high molecular storage polysaccharides, Man oligomers, cell wall glucans, and can expose PAMPs and lectin at the surface. All of these components could potentially interact with host components and have a role in the course of infection.

5 Lipidome of Paracoccidioides EVs in Comparison with the Cell Wall The work by Vallejo et al. (2012a) focused on the comparative lipidome of EVs from P. brasiliensis Pb18 and Pb3. EV and total cell lipids were fractionated in silica-60 columns and analyzed by electrospray ionization and gas chromatography-mass spectrometry. Detailed information was obtained for phospholipid, fatty acid, sterol, and neutral glycolipids composition. Similar data were obtained in parallel for the isolated cell wall fraction of the same isolates (Longo et al. 2013b) and qualitative results with EV lipids could be performed.

26

R. Puccia

In general, similar results between EV and cell wall lipid composition suggested that part of the cell wall lipids derive from EVs that remain in that compartment. For both EVs and cell wall, most phospholipid species identified were of the phosphatidylcholine (predominant in the cell wall) and phosphatidylethanolamine types, with a few species of phosphatidic acid, phosphatidylserine, phosphatidylglycerol, and phosphatidylinositol (PI) also represented (Vallejo et al. 2012a; Longo et al. 2013b). The alkyl-C16:0-acyl-C18:2 or –C18:1-PI species were unique to Pb18 EVs, and their possible role as immunomodulators still deserves further investigation: in Trypanosoma cruzi, an alkylacylglycerol unsaturated fatty acid present in glycosylphosphatidylinositol anchors is essential to induce proinflammatory cytokines (Almeida et al. 2000). As to the fatty acid portion of phospholipids, C18:1 (most probably oleic acid) predominated in Pb3 EVs and slightly in the cell wall, while C18:2 (most probably linoleic acid) was highly abundant in both Pb18 EVs and the cell wall. In Pb3 and Pb18 whole cells, C18:2 tended to be slightly more abundant (Vallejo et al. 2012a; Longo et al. 2013b). Brassicasterol was the major sterol detected in Pb18 and Pb3 EVs, cell walls, and cells (Vallejo et al. 2012a; Longo et al. 2013b), confirming previous findings for the P. brasiliensis yeast phase (San-Bias et al. 1997). Ergosterol and lanosterol had detectable amounts in both Pb18 and Pb3 EVs, however, the amount was higher in Pb18 than Pb3 EVs. In whole cells and the cell wall, however, ergosterol and lanosterol were either undetected or detected in trace amounts. Glucosylceramide (GlcCer) was the most probable glycosphingolipid with the structure Hex-C18:0 (or C18:1)-OH/d19:2. It was detected in both Pb18 and Pb3 EV samples and their cell wall (Vallejo et al. 2012a; Longo et al. 2013a, b), and previously in Pb18 yeast and hyphae cells (Toledo et al. 1999) and C. neoformans EVs (Rodrigues et al. 2007). Although GlcCer has important roles in the biology and virulence of other pathogenic fungi (Del Poeta et al. 2014), it remains understudied in Paracoccidioides.

6 Paracoccidioides EV Transcriptome The Paracoccidioides EV transcriptome has initially been characterized by RNA-seq in comparison with other fungal EVs by Peres da Silva et al. (2015b), specifically showing the features of the EV small RNA (sRNA) fraction from P. brasiliensis Pb18, C. neoformans, C. albicans, and Saccharomyces cerevisiae. The non-coding (nc)RNA sequences represented in all species were the small nucleolar (sno)RNA and tRNA, in higher proportion, followed by rRNA, small nuclear (sn)RNA, and long (nc)RNA. In addition, a copurified population of messenger mRNA and of sequences that matched those of the micro (mi)RNAs database have also been analyzed. The publication made it clear that the amount and nature of the fungal EV RNA sequences could vary with the species (Peres da Silva et al. 2015b).

Current Status on Extracellular Vesicles from the Dimorphic Pathogenic. . .

27

Further analysis of the Paracoccidioides EV transcriptome from Pb18, Pb3, and Pb01 yeast cells (Peres da Silva et al. 2019) revealed that the nature of the sRNA species and mRNA sequences may also considerably vary with isolates representing species within the same gender, as observed for the EV RNA population from two H. capsulatum strains analyzed in parallel (Alves et al. 2019). Both the small RNA population (200 nt) fractionated from Paracoccidioides EVs were analyzed and stringent parameters were applied for mRNA sequence validation (Peres da Silva et al. 2019). The results showed the presence of 14, 5, and 18 EV mRNA sequences, respectively for Pb18, Pb3, and Pb01. It was interesting to note that only the sequences coding for polyubiquitin, histone H3, calmodulin, elongation factor 1-alpha, and histone H2a have been detected in more than one isolate. Sequences coding for transport and oxidation-reduction were described only in Pb01. Importantly, the presence of full-length functional mRNA has been confirmed by in vitro translation assay, suggesting that virulence-related active mRNA could be EV-transferred and translated by other fungal cells. Among the 71 total sequences of EV (nc)RNA, the (sno)RNA species was the most representative, followed by tRNA, rRNA, long ncRNA, and (sn)RNA (Peres da Silva et al. 2019). It is noteworthy the number of exclusive sequences found in Pb01 (20, mostly tRNA) and Pb3 (13, mostly snoRNA) versus only six in Pb18 (mostly snoRNA). The presence of 145 micro (mi)RNA sequences in Pb18 EVs has previously been suggested by the alignment with sequences of the mirbase miRNA database (Peres da Silva et al. 2015b), but only 11 seem to match the structural criteria for miRNA-like in the Pb18 genome (de Curcio et al. 2019). Partial secondary miRNA structures have later suggested the presence of 42 EV miRNA-like sequences differentially represented in Pb18, Pb3, and Pb18; 50% of them were complementary to the RNA strand, thus showing potential to modulate RNA processing through RNA interference (Peres da Silva et al. 2019). Maybe the most interesting result featured in the analysis of the Paracoccidioides transcriptome (Peres da Silva et al. 2019) was the finding of 130 short (about 25 nt) RNA sequences that aligned at a unique region of the corresponding mRNA exon. They were highly represented in Pb18 EVs (104 sequences, 47% in the reverse position, 89 exclusives), but also found in Pb3 (19 sequences, 68% in the reverse position, 3 exclusives), and Pb01 (27 sequences, 56% in the reverse position, 21 exclusives). The target sequences were related to metabolism, translation, oxidation-reduction, or signaling, however, the majority have unknown functions. They have been called exonic RNA-like in reference to fungal exonic short interfering RNAs (ex-siRNA), which can regulate translation of the origin gene via double-stranded RNA generated by both sense and antisense sequences (Nicolás and Ruiz-Vázquez 2013; Nicolás et al. 2015; Son et al. 2017). Further investigation will be necessary to confirm the exonic siRNA-like mechanism in Paracoccidioides, and if the transfer of exonic RNA-like sequences via EVs to other fungal cells can regulate the expression of virulence genes.

28

R. Puccia

7 Paracoccidioides EVs: Cell Communication and Immunomodulation The role of fungal EVs in cell-cell communication, immunomodulation, and consequently in the course of infection has been addressed in several fungal models, as nicely reviewed by Piffer et al. (2021). The conclusion of whether fungal EVs tend to enhance infection or protect the host is not consensus and seems to vary with the fungal model and experimental conditions. In vitro, co-incubation of fungal EVs and cells of the innate immune system (macrophages and dendritic cells), however, seem to generally induce pro-inflammatory mediators (recent reviews by Zamith-MIranda et al. 2018; Freitas et al. 2019; Piffer et al. 2021). In Paracoccidioides, Peres da Silva et al. (2015a) showed that EVs isolated from Pb18 stimulated the expression, in murine peritoneal macrophages, of proinflammatory mediators, specifically, NO, IL-12p40, IL-12p70, IL-6, TNF-α, IL-1α, and IL-1β. The proinflammatory profile was maintained upon stimulation of culture macrophages of the J774A.1 lineage, with the expression of IL-6, TNF-α, and IL-12. EVs were stimulatory in a dose-response manner. Not coincidentally, the authors experimentally demonstrated that P. brasiliensis EVs from Pb18 prompted peritoneal macrophage polarization to the M1 phenotype, which is related to the activation of a protective cellular immune response. Moreover, the EVs were able to revert the macrophage phenotype from M2, which is associated with non-protective Th2 response and antibody production, to M1. Importantly, EVs also stimulated high fungicidal activity after coincubation with peritoneal macrophages for 48 h. Indirect coculture of P. brasiliensis Pb18 with MoDC (monocyte-derived CD11c + cells) using a transwell system resulted in changes in the cell transcription pattern in a more physiological environment, suggesting that extracellular components can modulate gene expression. Considering that under the assay conditions fungal EVs were both released and uptaken by MoDC, they are probably responsible for the stimulus, which could occur via sRNA and/or EV surface ligands, like those binding to DC-SIGNR via mannose mentioned earlier (Peres da Silva et al. 2015a). In our laboratory, we compared the effect of EVs isolated from Pb18 (vEVs) and its attenuated variant (aEVs) upon co-incubation with lineage and bone marrow macrophages. The stimulus with aEVs resulted in significantly increased amounts of pro-inflammatory mediators. However, the previous inoculation of mice with both EV samples enhanced the infection in our experimental conditions (Octaviano et al., unpublished). Enhancement of infection due to EVs also occurred with other dimorphs, specifically, Sporothrix schenckii (Ikeda et al. 2018) and H. capsulatum, while protection was achieved for C. albicans (Vargas et al. 2020). As mentioned before, the effect of fungal EVs in the interaction with the host has not been consensus and probably depends on the fungal system and experimental conditions. Since the interest in studying fungal EVs has increased in recent years due to the undeniable importance of these components in fungal biology and virulence, many answered issues will soon be better understood.

Current Status on Extracellular Vesicles from the Dimorphic Pathogenic. . .

29

8 Closing Remarks The interest in fungal extracellular vesicles by different research groups has suddenly increased within the last few years due to the solid data published by a few initial groups that recognized the essential roles that these components could have in the interaction with the host, fungal cells, and other microorganisms. The work on Paracoccidioides EVs has been important to understand fungal EV cargo and its nuances in different isolates and fungal species. Unrevealing the EV cargo and how it may be modulated is the first step to explore its role. We are starting to rapidly understand the fungal EV roles and the work on Paracoccidioides will certainly continue to add valuable information to the field.

References Alegre-Maller AC, Mendonça FC, da Silva TA et al (2014) Therapeutic administration of recombinant paracoccin confers protection against Paracoccidioides brasiliensis infection: involvement of TLRs. PLoS Negl Trop Dis 8:e3317. https://doi.org/10.1371/journal.pntd.0003317 Almeida F (1930) Estudos comparativos do granuloma coccidióidico nos Estados Unidos e no Brasil. Novo gênero para o parasito brasileiro. An da Fac Med São Paulo 5:125–141 Almeida IC, Camargo MM, Procópio DO et al (2000) Highly purified glycosylphosphatidylinositols from Trypanosoma cruzi are potent proinflammatory agents. EMBO J 19:1476–1485. https://doi.org/10.1093/emboj/19.7.1476 Almeida F, Wolf JM, da Silva TA et al (2017) Galectin-3 impacts Cryptococcus neoformans infection through direct antifungal effects. Nat Commun 8:1968. https://doi.org/10.1038/ s41467-017-02126-7 Alves LR, Peres da Silva R, Sanchez DA et al (2019) Extracellular vesicle-mediated RNA release in Histoplasma capsulatum. mSphere 4:e00176-19. https://doi.org/10.1128/mSphere.00176-19 Burger E (2021) Paracoccidioidomycosis protective immunity. J Fungi 7:1–26. https://doi.org/10. 3390/jof7020137 Carvalho KC, Ganiko L, Batista WL et al (2005) Virulence of Paracoccidioides brasiliensis and gp43 expression in isolates bearing known PbGP43 genotype. Microbes Infect 7:55–65. https:// doi.org/10.1016/j.micinf.2004.09.008 Chaves AFA, Navarro MV, de Barros YN et al (2021) Updates in Paracoccidioides biology and genetic advances in fungus manipulation. J Fungi 7:1–28. https://doi.org/10.3390/jof7020116 Cleare LG, Zamith D, Heyman HM et al (2020) Media matters! Alterations in the loading and release of Histoplasma capsulatum extracellular vesicles in response to different nutritional milieus. Cell Microbiol 22:e13217. https://doi.org/10.1111/cmi.13217 de Curcio JS, Batista MP, Paccez JD et al (2019) In silico characterization of microRNAs-like sequences in the genome of Paracoccidioides brasiliensis. Genet Mol Biol 42:95–107. https:// doi.org/10.1590/1678-4685-GMB-2018-0014 Del Poeta M, Nimrichter L, Rodrigues ML, Luberto C (2014) Synthesis and biological properties of fungal glucosylceramide. PLoS Pathog 10:e1003832. https://doi.org/10.1371/journal.ppat. 1003832

30

R. Puccia

Desjardins CA, Champion MD, Holder JW et al (2011) Comparative genomic analysis of human fungal pathogens causing paracoccidioidomycosis. PLoS Genet 7:e1002345. https://doi.org/10. 1371/journal.pgen.1002345 Erwig LP, Gow NARR (2016) Interactions of fungal pathogens with phagocytes. Nat Rev Microbiol 14:163–176. https://doi.org/10.1038/nrmicro.2015.21 Fernandes FF, Oliveira AF, Landgraf TN et al (2017) Impact of paracoccin gene silencing on Paracoccidioides brasiliensis virulence. MBio 8:e00537-17. https://doi.org/10.1128/mBio. 00537-17 Fernández-García OA, Cuellar-Rodríguez JM (2021) Immunology of fungal infections. Infect Dis Clin N Am 35:373–388. https://doi.org/10.1016/j.idc.2021.03.006 Freitas MS, Oliveira AF, da Silva TA et al (2016) Paracoccin Induces M1 polarization of macrophages via interaction with TLR4. Front Microbiol 7:1003. https://doi.org/10.3389/ fmicb.2016.01003 Freitas MS, Bonato VLD, Pessoni AM et al (2019) Fungal extracellular vesicles as potential targets for immune interventions. mSphere 4:e00747-19. https://doi.org/10.1128/mSphere.00747-19 Galili U (2013) Anti-Gal: an abundant human natural antibody of multiple pathogeneses and clinical benefits. Immunology 140:1–11. https://doi.org/10.1111/imm.12110 Ganiko L, Puccia R, Mariano V et al (2007) Paracoccin, an N-acetyl-glucosamine-binding lectin of Paracoccidioides brasiliensis, is involved in fungal growth. Microbes Infect 9:695–703. https:// doi.org/10.1016/j.micinf.2007.02.012 Gegembauer G (2019) Characterization of alpha-galactopyranosyl epitopes in Paracoccidioides and other pathogenic fungal species. Federal University of São Paulo Gow NAR, Latge J-P, Munro CA (2017) The fungal cell wall: structure, biosynthesis, and function. Microbiol Spectr 5:1–25. https://doi.org/10.1128/microbiolspec.FUNK-0035-2016 Hatanaka O, Rezende CP, Moreno P et al (2019) Galectin-3 inhibits Paracoccidioides brasiliensis growth and impacts paracoccidioidomycosis through multiple mechanisms. mSphere 4:e0020919. https://doi.org/10.1128/mSphere.00209-19 Ikeda MAK, de Almeida JRF, Jannuzzi GP et al (2018) Extracellular vesicles from Sporothrix brasiliensis are an important virulence factor that induce an increase in fungal burden in experimental sporotrichosis. Front Microbiol 9:2286. https://doi.org/10.3389/fmicb.2018. 02286 Joffe LS, Nimrichter L, Rodrigues ML, Del Poeta M (2016) Potential roles of fungal extracellular vesicles during infection. mSphere 1:e00099-16. https://doi.org/10.1128/mSphere.00099-16 Latge J-P (2009) Galactofuranose containing molecules in Aspergillus fumigatus. Med Mycol 47: S104–S109. https://doi.org/10.1080/13693780802258832 Leitao NP (2017) Characterization of extracellular vesicles isolated from pathogenic fungi cultivated under stress and their role in cell communication. Federal University of São Paulo Leitão NP, Vallejo MC, Conceição PM et al (2014) Paracoccidioides lutzii Plp43 is an active glucanase with partial antigenic identity with P. brasiliensis gp43. PLoS Negl Trop Dis 8:e3111. https://doi.org/10.1371/journal.pntd.0003111 Longo LVG, Nakayasu ES, Matsuo AL et al (2013a) Identification of human plasma proteins associated with the cell wall of the pathogenic fungus Paracoccidioides brasiliensis. FEMS Microbiol Lett 341:87–95. https://doi.org/10.1111/1574-6968.12097 Longo LVG, Nakayasu ES, Gazos-Lopes F et al (2013b) Characterization of cell wall lipids from the pathogenic phase of Paracoccidioides brasiliensis cultivated in the presence or absence of human plasma. PLoS One 8:e63372. https://doi.org/10.1371/journal.pone.0063372 Longo LVG, da Cunha JPC, Sobreira TJP, Puccia R (2014) Proteome of cell wall-extracts from pathogenic Paracoccidioides brasiliensis: comparison among morphological phases, isolates, and reported fungal extracellular vesicle proteins. EuPA Open Proteom 3:216–228. https://doi. org/10.1016/j.euprot.2014.03.003 Longo LVG, Breyer CA, Novaes GM et al (2020) The human pathogen Paracoccidioides brasiliensis has a unique 1-cys peroxiredoxin that localizes both intracellularly and at the cell surface. Front Cell Infect Microbiol 10:394. https://doi.org/10.3389/fcimb.2020.00394

Current Status on Extracellular Vesicles from the Dimorphic Pathogenic. . .

31

Lutz A (1908) Uma micose pseudo-coccídica localizada na boca e observada no Brasil: Contribução ao conhecimento das hiphoblastomicoses americanas. Brasil Med 22:121–124 Martinez R (2017) New trends in paracoccidioidomycosis epidemiology. J Fungi (Basel, Switzerland) 3:1. https://doi.org/10.3390/jof3010001 Matos Baltazar L, Nakayasu ES, Sobreira TJP et al (2016) Antibody binding alters the characteristics and contents of extracellular vesicles released by Histoplasma capsulatum. mSphere 1: e00085-15. https://doi.org/10.1128/mSphere.00085-15 Matute DR, McEwen JG, Puccia R et al (2006) Cryptic speciation and recombination in the fungus Paracoccidioides brasiliensis as revealed by gene genealogies. Mol Biol Evol 23:65–73. https:// doi.org/10.1093/molbev/msj008 McEwen JG, Bedoya V, Patiño MM et al (1987) Experimental murine paracoccidiodomycosis induced by the inhalation of conidia. J Med Vet Mycol 25:165–175. https://doi.org/10.1080/ 02681218780000231 Menino JF, Almeida AJ, Rodrigues F (2012) Gene knockdown in Paracoccidioides brasiliensis using antisense RNA. Methods Mol Biol (Clifton NJ) 845:187–198 Muñoz JF, Gallo JE, Misas E et al (2014) Genome update of the dimorphic human pathogenic fungi causing paracoccidioidomycosis. PLoS Negl Trop Dis 8:e3348. https://doi.org/10.1371/journal. pntd.0003348 Nicolás F, Ruiz-Vázquez R (2013) Functional diversity of RNAi-associated sRNAs in fungi. Int J Mol Sci 14:15348–15360. https://doi.org/10.3390/ijms140815348 Nicolás FE, Vila A, Moxon S et al (2015) The RNAi machinery controls distinct responses to environmental signals in the basal fungus Mucor circinelloides. BMC Genomics 16:237. https:// doi.org/10.1186/s12864-015-1443-2 Nimrichter L, de Souza MM, Del Poeta M et al (2016) Extracellular vesicle-associated transitory cell wall components and their impact on the interaction of fungi with host cells. Front Microbiol 7:1034. https://doi.org/10.3389/fmicb.2016.01034 Peres da Silva R, Heiss C, Black I et al (2015a) Extracellular vesicles from Paracoccidioides pathogenic species transport polysaccharide and expose ligands for DC-SIGN receptors. Sci Rep 5:14213. https://doi.org/10.1038/srep14213 Peres da Silva R, Puccia R, Rodrigues ML et al (2015b) Extracellular vesicle-mediated export of fungal RNA. Sci Rep 5:7763. https://doi.org/10.1038/srep07763 Peres da Silva R, Longo LGV, da Cunha JPC et al (2019) Comparison of the RNA content of extracellular vesicles derived from Paracoccidioides brasiliensis and Paracoccidioides lutzii. Cell 8:765. https://doi.org/10.3390/cells8070765 Piffer AC, Kuczera D, Rodrigues ML, Nimrichter L (2021) The paradoxical and still obscure properties of fungal extracellular vesicles. Mol Immunol 135:137–146. https://doi.org/10.1016/ j.molimm.2021.04.009 Pinheiro BG, Hahn RC, de Camargo ZP, Rodrigues AM (2020) Molecular tools for detection and identification of Paracoccidioides species: current status and future perspectives. J Fungi 6:1– 24. https://doi.org/10.3390/jof6040293 Prado M, da Silva MB, Laurenti R et al (2009) Mortality due to systemic mycoses as a primary cause of death or in association with AIDS in Brazil: a review from 1996 to 2006. Mem Inst Oswaldo Cruz 104:513–521. https://doi.org/10.1590/S0074-02762009000300019 Puccia R, Travassos LR (1991) 43-kilodalton glycoprotein from Paracoccidioides brasiliensis: immunochemical reactions with sera from patients with paracoccidioidomycosis, histoplasmosis, or Jorge Lobo’s disease. J Clin Microbiol 29:1610–1615. https://doi.org/10.1128/JCM.29.8. 1610-1615.1991 Puccia R, Vallejo MC, Matsuo AL, Longo LVG (2011) The Paracoccidioides cell wall: past and present layers toward understanding interaction with the host. Front Microbiol 2:1–7. https:// doi.org/10.3389/fmicb.2011.00257 Puccia R, Vallejo MC, Longo LVG (2016) The cell wall-associated proteins in the dimorphic pathogenic species of Paracoccidioides. Curr Protein Pept Sci 18:1074–1089. https://doi.org/ 10.2174/1389203717666160812233437

32

R. Puccia

Rappleye CA, Eissenberg LG, Goldman WE (2007) Histoplasma capsulatum alpha-(1,3)-glucan blocks innate immune recognition by the beta-glucan receptor. Proc Natl Acad Sci 104:1366– 1370. https://doi.org/10.1073/pnas.0609848104 Reis FCG, Borges BS, Jozefowicz LJ et al (2019) A novel protocol for the isolation of fungal extracellular vesicles reveals the participation of a putative scramblase in polysaccharide export and capsule construction in Cryptococcus gattii. mSphere 4:e00080-19. https://doi.org/10.1128/ mSphere.00080-19 Rodrigues ML, Nimrichter L, Oliveira DL et al (2007) Vesicular polysaccharide export in Cryptococcus neoformans is a eukaryotic solution to the problem of fungal trans-cell wall transport. Eukaryot Cell 6:48–59. https://doi.org/10.1128/EC.00318-06 Rodrigues ML, Nosanchuk JD, Schrank A et al (2011) Vesicular transport systems in fungi. Future Microbiol 6:1371–1381. https://doi.org/10.2217/fmb.11.112 Rodrigues ML, Franzen AJ, Nimrichter L, Miranda K (2013) Vesicular mechanisms of traffic of fungal molecules to the extracellular space. Curr Opin Microbiol 16:414–420. https://doi.org/ 10.1016/j.mib.2013.04.002 Rodrigues ML, Nakayasu ES, Almeida IC, Nimrichter L (2014) The impact of proteomics on the understanding of functions and biogenesis of fungal extracellular vesicles. J Proteome 97:177– 186. https://doi.org/10.1016/j.jprot.2013.04.001 Rodrigues ML, Godinho RMC, Zamith-Miranda D, Nimrichter L (2015) Traveling into outer space: unanswered questions about fungal extracellular vesicles. PLoS Pathog 11:e1005240. https:// doi.org/10.1371/journal.ppat.1005240 San-Bias G, Urbina JA, Marchán E et al (1997) Inhibition of Paracoccidioides brasiliensis by ajoene is associated with blockade of phosphatidylcholine biosynthesis. Microbiology 143 (Pt 5):1583–1586. https://doi.org/10.1099/00221287-143-5-1583 Son H, Park AR, Lim JY et al (2017) Genome-wide exonic small interference RNA-mediated gene silencing regulates sexual reproduction in the homothallic fungus Fusarium graminearum. PLoS Genet 13:e1006595. https://doi.org/10.1371/journal.pgen.1006595 Sturme MHJ, Puccia R, Goldman GH, Rodrigues F (2011) Molecular biology of the dimorphic fungi Paracoccidioides spp. Fungal Biol Rev 25:89–97. https://doi.org/10.1016/j.fbr.2011.04. 002 Taborda CP, Juliano MA, Puccia R et al (1998) Mapping of the T-cell epitope in the major 43-kilodalton glycoprotein of Paracoccidioides brasiliensis which induces a Th-1 response protective against fungal infection in BALB/c mice. Infect Immun 66:786–793. https://doi.org/ 10.1128/IAI.66.2.786-793.1998 Teixeira MM, Theodoro RC, de Carvalho MJA et al (2009) Phylogenetic analysis reveals a high level of speciation in the Paracoccidioides genus. Mol Phylogenet Evol 52:273–283. https://doi. org/10.1016/j.ympev.2009.04.005 Teixeira MM, Theodoro RC, Nino-Vega G et al (2014) Paracoccidioides species complex: ecology, phylogeny, sexual reproduction, and virulence. PLoS Pathog 10:4–7. https://doi.org/ 10.1371/journal.ppat.1004397 Théry C, Witwer KW, Aikawa E et al (2018) Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles 7:1535750. https://doi. org/10.1080/20013078.2018.1535750 Toledo MS, Levery SB, Straus AH et al (1999) Characterization of sphingolipids from mycopathogens: factors correlating with expression of 2-hydroxy fatty acyl (E)-Δ3-unsaturation in cerebrosides of Paracoccidioides brasiliensis and Aspergillus fumigatus. Biochemistry 38: 7294–7306. https://doi.org/10.1021/bi982898z Torres I, Hernandez O, Tamayo D et al (2013) Inhibition of PbGP43 expression may suggest that gp43 is a virulence factor in Paracoccidioides brasiliensis. PLoS One 8:e68434. https://doi.org/ 10.1371/journal.pone.0068434 Travassos LR, Taborda CP (2012) Paracoccidioidomycosis vaccine. Hum Vaccin Immunother 8: 1450–1453. https://doi.org/10.4161/hv.21283

Current Status on Extracellular Vesicles from the Dimorphic Pathogenic. . .

33

Turissini DA, Gomez OM, Teixeira MM et al (2017) Species boundaries in the human pathogen Paracoccidioides. Fungal Genet Biol 106:9–25. https://doi.org/10.1016/j.fgb.2017.05.007 Vallejo MC, Matsuo AL, Ganiko L et al (2011) The pathogenic fungus Paracoccidioides brasiliensis exports extracellular vesicles containing highly immunogenic α-galactosyl epitopes. Eukaryot Cell 10:343–351. https://doi.org/10.1128/EC.00227-10 Vallejo MC, Nakayasu ES, Longo LVG et al (2012a) Lipidomic analysis of extracellular vesicles from the pathogenic phase of Paracoccidioides brasiliensis. PLoS One 7:e39463. https://doi. org/10.1371/journal.pone.0039463 Vallejo MC, Nakayasu ES, Matsuo AL et al (2012b) Vesicle and vesicle-free extracellular proteome of Paracoccidioides brasiliensis: comparative analysis with other pathogenic fungi. J Proteome Res 11(3):1676–1685 Vargas G, Honorato L, Guimarães AJ et al (2020) Protective effect of fungal extracellular vesicles against murine candidiasis. Cell Microbiol 22:e13238. https://doi.org/10.1111/cmi.13238 Zamith-Miranda D, Nimrichter L, Rodrigues ML, Nosanchuk JD (2018) Fungal extracellular vesicles: modulating host-pathogen interactions by both the fungus and the host. Microbes Infect 20:501–504. https://doi.org/10.1016/j.micinf.2018.01.011 Zamith-Miranda D, Peres da Silva R, Couvillion SP et al (2021) Omics approaches for understanding biogenesis, composition and functions of fungal extracellular vesicles. Front Genet 12: 648524. https://doi.org/10.3389/fgene.2021.648524 Zarnowski R, Sanchez H, Covelli AS et al (2018) Candida albicans biofilm–induced vesicles confer drug resistance through matrix biogenesis. PLoS Biol 16:e2006872. https://doi.org/10. 1371/journal.pbio.2006872

Extracellular Vesicles from Sporothrix Yeast Cells Marcelo Augusto Kazuo Ikeda and Karen Spadari Ferreira

Contents 1 Sporotrichosis: A Zoonotic Health Issue in Brazil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Sporothrix EVs Isolation and Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

35 39 42 42

Abstract Sporotrichosis is an emerging deep mycosis and a public health concern in Brazil. This mycosis is caused by the traumatic inoculation of several species of Sporothrix from nature. However, when cats transmit the disease to humans as zoonotic transmission, severe extracutaneous manifestations are observed. Therefore, effective control of the disease requires the engagement of host receptors by pathogen-derived molecules to stimulate the immune response. In this context, extracellular vesicles from Sporothrix sp contribute to intracellular communication with host cells. In addition, however, extracellular vesicles may contribute to the spread of this fungus via delivering molecules such as proteins, nucleic acids, and lipids. Thus, understanding mechanisms behind extracellular vesicles related to Sporothrix may provide us with a way to understand and identify its capacity to manipulate the host immune system and spread the infection.

1 Sporotrichosis: A Zoonotic Health Issue in Brazil Sporotrichosis was described for the first time in 1898 as chronic infection with a worldwide distribution (Hektoen 1900; Schenck 1898). In Brazil, this mycose is endemic, and it has been considered a neglected and emerging zoonotic fungal infection (De Beer et al. 2016). The disease affects both humans and animals and M. A. K. Ikeda · K. S. Ferreira (*) Departamento de Ciências Farmacêuticas do Instituto de Ciências Ambientais, Químicas e Farmacêuticas da Universidade Federal de São Paulo, Diadema, Brazil e-mail: [email protected] © Springer Nature Switzerland AG 2021 M. Rodrigues, G. Janbon (eds.), Fungal Extracellular Vesicles, Current Topics in Microbiology and Immunology 432, https://doi.org/10.1007/978-3-030-83391-6_4

35

36

M. A. K. Ikeda and K. S. Ferreira

Fig. 1 Role of the cat in the epidemiology of sporotrichosis. Free-roaming infected cats have lesions containing yeasts from Sporothrix brasiliensis that can colonize the oral cavity and nails (red circles). This cat can spread the fungus by causing injuries to people such as veterinarians (blue circle), or other animals, from colonies or shelters (orange circle) and owned cats with outdoor access (yellow circle), which can take home the fungus. Environmental contamination may occur by contaminated feces and secretions or by infected dead animals (green circle)

induces a typically cutaneous form in most cases. Still, it is classified as well pulmonary and disseminated form, mainly in immunocompromised patients (Chakrabarti et al. 2015; Barros et al. 2011; Schubach et al. 2008; Moreira et al. 2015). Sporotrichosis is caused by the traumatic inoculation of several species of Sporothrix. The fungi are found in the environment like soil, rose garden, and some decaying wood (Chakrabarti et al. 2015; Barros et al. 2011; Rodrigues et al. 2013). S. schenckii, for a long time, was the only known species to infect the host. In Europe and North America, the “Rose Gardener’s Disease” is caused by this species (Rodrigues et al. 2013). However, around the early 2000s, other species were described from genetic analyses, like S. Mexicana, S. lurei, S. globosa, and S. brasiliensis (Rossow et al. 2020; Marimon et al. 2007, 2008). In South America, more specifically, in Brazil, S. brasiliensis is the principal etiological agent of human and animal mycosis (Dib et al. 2017; Silva et al. 2012). This species led to outbreaks of sporotrichosis amongst humans and felines. It is responsible for the hyperendemic growth in Rio de Janeiro (Barros et al. 2011; Schubach et al. 2008; Silva et al. 2012; Almeida-Paes et al. 2014; Pereira et al. 2014). It is essential to highlight that cats have a crucial role in S. brasiliensis epidemiology in Brazil (Fig. 1). In the mid-90s, few feline and zoonotic (cat-to-human) transmission cases were reported in Brazil’s southeast region. Over the years, there

Extracellular Vesicles from Sporothrix Yeast Cells

37

was an expansion to the south and northeast regions of the country, reaching an epidemic situation with thousands of cases per year and spreading to neighboring countries like Argentina (Rossow et al. 2020; Gremião et al. 2020). Cats are very susceptible to S. brasiliensis infection and have a high fungal load in the lesions, but it is unclear which mechanisms of host-pathogen interaction are involved in this susceptibility (Miranda et al. 2018; Gremião et al. 2021). Cat’s clinical signals can range from localized skin nodules to widespread lesions. Several tools are available to diagnose feline with sporotrichosis, such as fungal culture, cytopathological, histopathological examination, serological and molecular tests. However, a great challenge is the treatment of these animals, which is long and can be refractory to the few therapeutic options available. No prophylactic treatment is available yet (Gremião et al. 2021). Several factors can explain the easy spread of the disease in the feline population. There are behavioral issues, such as the territorial dispute that leads to fights with scratches and bites that allow the inoculation of fungi present in the oral cavity and nails. Besides, cats that share the same environment, in colonies or shelters, can be contaminated by licking or sharing a feeding place. (MacêdoSales et al. 2018). Also, the environment can become contaminated with feces and secretions of infected cats and with sick animals that died and did not have an appropriate disposal (Chaves et al. 2013; Montenegro 2014). One of the reasons the S. brasiliensis strain is so important in Brazil is that it has a high virulence profile compared to other species (Almeida-Paes et al. 2015; Della Terra et al. 2017) and can induce an inflammatory immune response (BatistaDuharte et al. 2018; Arrillaga-Moncrieff et al. 2009). After traumatic inoculation with these fungi, the host develops subcutaneous mycosis, but it may spread to other tissues (Barros et al. 2011; Ramos-e-Silva et al. 2007). The dissemination depends on the fungi’ virulence and the host’s immune response (Almeida 2012). However, the immunological mechanisms involved in the control of sporotrichosis are not well understood. In 2018, Almeida and coworkers (De Almeida et al. 2018) identified the ZR8 peptide from GP70 protein, the main antigen of the Sporothrix. ZR8 peptide induced CD4+T cells and higher levels of IFN-γ, IL-17A, and IL-1β. The peptide induced a strong cellular immune response associated with fungus clearance. Additional studies have been shown other potential targets as a vaccine against sporotrichosis (Della Terra et al. 2017; De Almeida et al. 2018; Lopes-Bezerra 2011). However, at present, there is no effective vaccine available to prevent sporotrichosis. On the other hand, some studies have been shown that the fungal components are essential to invade and survive in the host. Since fungal EVs were first described (Rodrigues et al. 2007, 2008), they are associated as crucial molecules related to virulence. EVs have a vital role in the regulation of immune response. Moreover, the fungal EVs are associated with delivering essential proteins, lipids, nucleic acids, and some immunoreactive components, making them a key player in intracellular communication (Rizzo et al. 2017; Joffe et al. 2016). As Evs induces interaction between the cells, they can alter the cellular signaling in response to a different stimulus, and these properties make them excellent candidates for therapeutics.

38

M. A. K. Ikeda and K. S. Ferreira

Furthermore, their small size enables them to cross major biological membranes (El Andaloussi et al. 2013; Vickers and Remaley 2012) and present their cargo to cells from the immune system. In sporotrichosis, the therapeutic potential of S. brasiliensis EVs was analyzed (Ikeda et al. 2018). After subcutaneous yeast cells infection, BALB/c mice received two doses of different concentrations of EVs. Surprisingly, when the mice were treated with a high concentration of EVs, the infection got worse. Moreover, the mice’s immune system couldn’t respond efficiently against the fungi (Ikeda et al. 2018). Many proteins and molecules are essential for forming EVs, and others are important during fungal infection. In 2008, Albuquerque and coworkers recovered EVs from S. schenckii after ultracentrifugation assay and showed similarity with Cryptococcus EVs (Albuquerque et al. 2008). Our group conducted a proteomic study comparing the content of a virulent strain of S. brasiliensis and a less virulent S. schenckii strain to understand better the role of vesicles in the immune response sporotrichosis infection (Ikeda et al. 2018). In the comparative analysis, we found few proteins in common between the two species, which are related to the transport of molecules. These results are showing the role of EVs as transport structures, or proteins involved in the processes of transcription, translation, and catabolism of proteins, which can correlate with the function of EVs being evolved in active processes when interacting with host cells during the infection (Rodrigues et al. 2015). In the EVs of the two species, we found the Heat Shock Protein of 70 kDa (HSP70), a highly conserved chaperone found in several living organisms. HSP70 is involved in stress response processes; in pathogenic fungi, we have studies showing their influence on host response (Tiwari and Shankar 2018). In Cryptococcus neoformans, it was demonstrated that a recombinant HSP70 was able to decrease the fungicidal activity in macrophages (Silveira et al. 2013). In Candida albicans HSP70, it was shown to be important in the invasion process in the experimental infection and the fungicide activity of the host cells (Gong et al. 2017). Besides, in the thermodimorphic fungi Histoplasma capsulatum and Paracoccidioides brasiliensis, HSP70 is recognized by the serum of infected patients (Cleare et al. 2017), being a potential candidate as a target for immunotherapy. Regarding the characterized proteins found exclusively in the EVs of each species, we have in S. schenckii proteins related to the processes of regulation of transcription and translation and the transport of molecules. In S. brasiliensis, in addition to the proteins associated with these same processes, several enzymes were found. We have enzymes related to the cell wall’s structural modifications, such as Extracellular cell wall glucanase and Alpha-mannosyltransferase; they may represent a mechanism of remodeling the cell wall to facilitate EVs release. Depending on these enzymes’ levels, structural changes in the fungal cell wall may occur, which can alter the recognition by host cells (Nimrichter et al. 2016). We have the enzyme 4-hydroxyphenylpyruvate dioxygenase, which is essential in the production of the pyomelanin derived from L-tyrosine. This melanin production has been described in S. brasiliensis and S. schenckii and can play a critical role in protecting the fungus

Extracellular Vesicles from Sporothrix Yeast Cells

39

Fig. 2 Western blot of the EVs from Sporothrix brasiliensis and Sporothrix schenckii with serum from infected mice. We can observe immunoreactive components in S. brasiliensis with a double band at approximately 100 kDa and single bands at about 50 kDa and 45 kDa. In comparison, in S. schenckii, we observed a single band at around 100 kDa

against the host’s immune response and antifungals (Romero-Martinez et al. 2000; Almeida-Paes et al. 2012, 2016). We also have some enzymes involved in metabolic processes like NADH: ubiquinone oxidoreductase. In Candida albicans, they showed an essential role in respiration and cell wall synthesis, directly impacting virulence and being a possible target for antifungal drugs (She et al. 2015). We also found kinases that may be related to the response to stress and can promote regulations in the fungus that increase its pathogenicity (Day and Quinn 2019). What surprised us was the large number of uncharacterized proteins found in EVs. In our work, we performed a comparative analysis (unpublished data) by Western Blot of the EVs from the two species against serum from infected mice (Fig. 2). We observed that in the EVs of S. brasiliensis, we have more immunoreactive components than in S. schenckii, which could explain the greater virulence of S. brasiliensis. Further studies are needed to elucidate what these components are and their role in the pathogenesis of sporotrichosis. However, a 100 kDa antigenic molecule (an endoplasmic signal peptidase) the 44 kDa, and the GP60-70 protein just had been described as potential vaccine candidates (Della Terra et al. 2017; De Almeida et al. 2018; Lopes-Bezerra 2011). Given the importance of Sporothrix EVs in the immune response and their possible virulence factors, these particles’ purification is essential for understanding the infection caused by these fungi. Next, we will describe the methods of isolation and characterization of Sporothrix vesicles.

2 Sporothrix EVs Isolation and Characterization EVs are released from some fungi, including Sporothrix yeast cells. Isolating these EVS and understanding their biology and their role during fungal disease is essential to control the disease. However, to separate and analyze these EVs from Sporothrix

40

M. A. K. Ikeda and K. S. Ferreira

Fig. 3 Sporothrix brasiliensis and Sporothrix schenckii yeasts EVs isolation. Yeasts were cultivated in Brain Heart Infusion broth for 5 days at 37  C under shaking at 150 rpm. Cell-free supernatant was obtained by centrifugation at 4000  g for 15 min at 4  C and centrifuged at 15,000  g for 15 min at 4  C to remove the debris. The supernatant was ultracentrifuged at 100,000  g at 4  C for 1 h to sediment the EVs. The pellet was resuspended in sterile Phosphate Buffered Saline and ultracentrifuged again at 100,000  g at 4  C for 1 h. The final pellet containing the EVs was resuspended in sterile Phosphate Buffered Saline

can be limited because of the lack of standardization of isolation techniques. Here, we described the most used method for Sporothrix EVs isolation, the ultracentrifugation protocol. This technique is considered the gold standard for isolation EVs, specifically for the smaller subpopulations or exosomes (Mathivanan et al. 2012). However, microvesicles, exosomes, or apoptotic cells can be purified for different methods, such as membrane filtration, chromatography, or chemical precipitation (The’ry et al. 2006; Rekker et al. 2014; Vader et al. 2016). To isolate EVs from S. brasiliensis and schenckii strains, Ikeda and coworkers (Ikeda et al. 2018) followed the protocol described previously for other fungi species (Vallejo et al. 2011; Oliveira et al. 2010). Yeast cells were cultivated in BHI broth at 37  C for 48 h. Then, yeasts were transferred to an Erlenmeyer and grown at the same temperature under shaking at 150 rpm for six days. After centrifugation at 4000  g for 15 min at 4  C and centrifuged at 15,000  g for 15 min at 4  C to remove the debris, the cell-free supernatant was obtained. Getting the sediment of EVs, the supernatant was ultracentrifuged at 100,000  g at 4  C for 1 h. Then, the pellet was resuspended in sterile 1X PBS and ultracentrifuged again at 100,000  g at 4  C for 1 h. The final pellet containing fungal EVs was resuspended in sterile 1X PBS (Fig. 3). Many researchers use the Nanoparticle Tracking Analysis (NTA) to analyze EVs and determine particles’ size. Then, after isolation of the EVs, the size and concentration of extracellular vesicles can be measured using NTA protocols. As we can see in Fig. 4, the images are analyzed with NTA software and show us the size and the number of particles/mL. Also, the software processes the film, where it can individually identify each particle and measure the Brownian motion performed by it. The speed of movement is applied to the Stokes-Einstein equation, which considers the sampling time, temperature, and solution viscosity, resulting in the particle size (Dragovic et al. 2011). Also, Transmission Electron Microscopy (TEM) has been valued for detect and characterized EVs. For this, pellets containing EVs from S. brasiliensis or S. schenckii yeast cells were fixed with 4% of paraformaldehyde and 0.1% glutaraldehyde solution for 1 h at room temperature and then embedded in

Extracellular Vesicles from Sporothrix Yeast Cells

41

Fig. 4 EVs quantification and measure by Nanoparticle Tracking Analysis. On the left side, we have a frame from a video captured by the instrument NanoSight NS300 (Malvern Instruments Ltd.) containing the particles from the sample; they are quantified and measured by the Brownian motion. On the right side, we have a graph generated by the instrument identifying the particles by size in nanometers and the concentration of these particles on the sample by milliliters

Fig. 5 Transmission Electron Microscopy (TEM) of Sporothrix brasiliensis (left) and Sporothrix schenckii (right) yeasts. Pellets containing yeast cells from culture for EVs isolation were examined by TEM. At a magnification of 80.000x, we observed secreted EVs (black arrows) and EVs forming (red arrow)

spurr resin. Ultrafine sections can be stained with uranyl acetate and lead citrate and examined by TEM, as Ikeda described in 2018. This technique allows observed the EVs in the fungal wall, as shown in Fig. 5.

42

M. A. K. Ikeda and K. S. Ferreira

3 Conclusion In summary, EVs are secreted from Sporothrix yeast cells and play a crucial role in the biological process. Also, EVs have been considered as a potential therapeutic tool in modulating the immune response.

References Albuquerque PC et al (2008) Vesicular transport in Histoplasma capsulatum: an effective mechanism for trans-cell wall transfer of proteins and lipids in ascomycetes. Cell Microbiol 10 (8):1695–1710 Almeida SR (2012) Therapeutic monoclonal antibody for sporotrichosis. Front Microbiol 3:2010–2013 Almeida-Paes R et al (2012) Biosynthesis and functions of a melanoid pigment produced by species of the sporothrix complex in the presence of L-Tyrosine. Appl Environ Microbiol 78 (24):8623–8630 Almeida-Paes R, de Oliveira MME, Freitas DFS, do Valle ACF, Zancopé-Oliveira RM, GutierrezGalhardo MC (2014) Sporotrichosis in Rio de Janeiro, Brazil: Sporothrix brasiliensis is associated with atypical clinical presentations. PLoS Negl Trop Dis 8:e3094 Almeida-Paes R et al (2015) Phenotypic characteristics associated with virulence of clinical isolates from the sporothrix complex. Biomed Res Int 2015:1–10 Almeida-Paes R et al (2016) Melanins protect Sporothrix brasiliensis and Sporothrix schenckii from the antifungal effects of terbinafine. PLoS One 11(3):e0152796 Arrillaga-Moncrieff I, Capilla J, Mayayo E, Marimon R, Marine M, Genis J, Cano J, Guarro J (2009) Different virulence levels of the species of Sporothrix in a murine model. Clin Microbiol Infect 15:651–655 Barros MBDL, De Almeida Paes R, Schubach AO (2011) Sporothrix schenckii and Sporotrichosis. Clin Microbiol Rev 24(4):633–654 Batista-Duharte A, Téllez-Martínez D, de Roberto Andrade C, Portuondo DL, Jellmayer JA, Polesi MC, Carlos IZ (2018) Sporothrix brasiliensis induces a more severe disease associated with sustained Th17 and regulatory T cells responses than Sporothrix schenckii sensu stricto in mice. Fungal Biol 122:1163–1170 Chakrabarti A, Bonifaz A, Gutierrez-Galhardo MC, Mochizuki T, Li S (2015) Global epidemiology of sporotrichosis. Med Mycol 53:3–14 Chaves AR et al (2013) Treatment abandonment in feline sporotrichosis - study of 147 cases. Zoonoses Public Health 60(2):149–153 Cleare LG, Zamith Miranda D, Nosanchuk JD (2017) Heat shock proteins in histoplasma and paracoccidioides: a minireview. Clin Vaccine Immunol 24(11):e00221-17 Day AM, Quinn J (2019) Stress-activated protein kinases in human fungal pathogens. Front Cell Infect Microbiol 9:261 De Almeida JRF, Jannuzzi GP, Kaihami GH, Breda LCD, Ferreira KS, de Almeida SR (2018) An immunoproteomic approach revealing peptides from Sporothrix brasiliensis that induce a cellular immune response in subcutaneous sporotrichosis. Sci Rep 8:4192. https://doi.org/10. 1038/s41598-018-22709-8 De Beer ZW, Duong TA, Wingfield MJ (2016) The divorce of sporothrix and ophiostoma: solution to a problematic relationship. Stud Mycol 83(1907):165–191 Della Terra PP et al (2017) Exploring virulence and immunogenicity in the emerging pathogen Sporothrix brasiliensis. PLoS Negl Trop Dis 11(8):e0005903

Extracellular Vesicles from Sporothrix Yeast Cells

43

Dib I et al (2017) Zoonotic epidemic of sporotrichosis: cat to human transmission. PLoS Pathog 13 (1):2–8 Dragovic RA et al (2011) Sizing and phenotyping of cellular vesicles using nanoparticle tracking analysis. Nanomedicine 7(6):780–788 El Andaloussi S et al (2013) Extracellular vesicles: biology and emerging therapeutic opportunities. Nature reviews. Drug Disc 12(5):347–357 Gong Y, Li T, Yu C, Sun S (2017) Candida albicans heat shock proteins and hsps-associated signaling pathways as potential antifungal targets. Front Cell Infect Microbiol 7:520 Gremião IDF, Oliveira MME, Monteiro de Miranda LH, Saraiva Freitas DF, Pereira SA (2020) Geographic expansion of sporotrichosis, Brazil. Emerg Infect Dis 26(3):621–624 Gremião IDF, Martins da Silva da Rocha E, Montenegro H et al (2021) Guideline for the management of feline sporotrichosis caused by Sporothrix brasiliensis and literature revision. Braz J Microbiol 52(1):107–124 Hektoen L (1900) A new pathogenic fungus - the sporothrix of schenck. J Boston Soc Med Sci 4 (7):179 Ikeda MAK, De Almeida JRF, Jannuzzi GP, Cronemberger-Andrade A, Torecilhas ACT, Moretti NS, Da Cunha JPC, Almeida SR, Ferreira KS (2018) Extracellular vesicles from Sporothrix brasiliensis are an important virulence factor that induce an increase in fungal burden in experimental sporotrichosis. Front Microbiol 9:2286 Joffe LS et al (2016) Potential roles of fungal extracellular vesicles during infection. mSphere 1(4): e00099-16 Lopes-Bezerra LM (2011) Sporothrix schenckii cell wall peptidorhamnomannans. Front Microbiol 2:243. https://doi.org/10.3389/fmicb.2011.00243 Macêdo-Sales PA, Souto SRLS, Destefani CA et al (2018) Domestic feline contribution in the transmission of Sporothrix in Rio de Janeiro State, Brazil: a comparison between infected and non-infected populations. BMC Vet Res 14:19 Marimon R et al (2007) Sporothrix brasiliensis, S. globosa, and S. mexicana, three new sporothrix species of clinical interest. J Clin Microbiol 45(10):3198–3206 Marimon R et al (2008) Sporothrix luriei: a rare fungus from clinical origin. Med Mycol 46 (6):621–625 Mathivanan S, Fahner CJ, Reid GE, Simpson RJ (2012) ExoCarta 2012: database of exosomal proteins, RNA and lipids. Nucleic Acids Res 40(Database issue):D1241–D1244 Miranda LHM, Meli M, Conceição-Silva F, Novacco M, Menezes RC, Pereira SA et al (2018) Co-infection with feline retrovirus is related to changes in immunological parameters of cats with sporotrichosis. PLoS One 13(11):e0207644 Montenegro H et al (2014) Feline sporotrichosis due to Sporothrix brasiliensis: an emerging animal infection in São Paulo, Brazil. BMC Vet Res 10(1):269 Moreira JAS, Freitas DFS, Lamas CC (2015) The impact of sporotrichosis in HIV-infected patients: a systematic review. Infection 43(3):267–276 Nimrichter IL et al (2016) Extracellular vesicle-associated transitory cell wall components and their impact on the interaction of fungi with host cells. Front Microbiol 7:1–11 Oliveira DL et al (2010) Extracellular vesicles from Cryptococcus neoformans modulate macrophage functions. Infect Immun 78(4):1601–1609 Pereira SA, Gremião ID, Kitada AA, Boechat JS, Viana PG, Schubach TM (2014) The epidemiological scenario of feline sporotrichosis in Rio de Janeiro, state of Rio de Janeiro. Brazil Rev Soc Bras Med Trop 47:392–393 Ramos-e-Silva M, Vasconcelos C, Carneiro S, Cestari T (2007) Sporotrichosis. Clin Dermatol 25:181–187 Rekker K, Saare M, Roost AM et al (2014) Comparison of serum exosome isolation methods for microRNA profiling. Clin Biochem 47:135–138 Rizzo J, Nimrichter L, Rodrigues ML (2017) What is new? Recent knowledge on fungal extracellular vesicles. Curr Fungal Infect Rep 11:141–147

44

M. A. K. Ikeda and K. S. Ferreira

Rodrigues ML et al (2007) Vesicular polysaccharide export in Cryptococcus neoformans is a eukaryotic solution to the problem of fungal trans-cell wall transport. Eukaryot Cell 6(1):48–59 Rodrigues ML et al (2008) Extracellular vesicles produced by Cryptococcus neoformans contain protein components associated with virulence. Eukaryot Cell 7(1):58–67 Rodrigues AM et al (2013) Phylogenetic analysis reveals a high prevalence of sporothrix brasiliensis in feline sporotrichosis outbreaks. PLoS Negl Trop Dis 6:7 Rodrigues ML, Godinho RM, Zamith-Miranda D, Nimrichter L (2015) Traveling into outer space: unanswered questions about fungal extracellular vesicles. PLoS Pathog 11(12):e1005240 Romero-Martinez R et al (2000) Biosynthesis and functions of melanin in Sporothrix schenckii. Infect Immun 68(6):3696–3703 Rossow JA, Queiroz-Telles F, Caceres DH, Beer KD, Jackson BR, Pereira JG, Gremião IDF, Pereira SA (2020) A one health approach to combatting Sporothrix brasiliensis: narrative review of an emerging zoonotic fungal pathogen in South America. J Fungal 6(4):247 Schenck BR (1898) On refractory subcutaneous abscesses caused by a fungus possibly related to the sporotricha. John Hopkins Hosp Bull 9(93):286–290 Schubach A, Barros MB, Wanke B (2008) Epidemic sporotrichosis. Curr Opin Infect Dis 21:129–133 She X, Khamooshi K, Gao Y, Shen Y, Lv Y, Calderone R, Fonzi W, Liu W, Li D (2015) Fungalspecific subunits of the Candida albicans mitochondrial complex I drive diverse cell functions including cell wall synthesis. Cell Microbiol 17(9):1350–1364 Silva MBT, Costa MMM, Torres CCS (2012) Esporotricose urbana: epidemia negligenciada No Rio de Janeiro. Brasil Cadernos Saúde Pública 28:1867–1880 Silveira CP et al (2013) The heat shock protein (Hsp) 70 of Cryptococcus neoformans is associated with the fungal cell surface and influences the interaction between yeast and host cells. Fungal Genet Biol 60:53–63 The’ry C, Amigorena S, Raposo G (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. Chapter 3, Unit 3.22 Tiwari S, Shankar J (2018) Hsp70 in fungi: evolution, function and vaccine candidate. In: Asea A, Kaur P (eds) HSP70 in human diseases and disorders. Heat shock proteins, vol 14. Springer Vader P, Mol EA, Pasterkamp G et al (2016) Extracellular vesicles for drug delivery. Adv Drug Deliv Rev 106:1–9 Vallejo MC et al (2011) The pathogenic fungus paracoccidioides brasiliensis exports extracellular vesicles containing highly immunogenic -galactosyl epitopes. Eukaryot Cell 10(3):343–351 Vickers KC, Remaley AT (2012) Lipid-based carriers of microRNAs and intercellular communication. Curr Opin Lipidol 23(2):91–97

Filamentous Fungi Extracellular Vesicles Teresa Gonçalves, Joana Oliveira, and Chantal Fernandes

Contents 1 2 3 4

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Historical Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biogenesis and Release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EVs Morphology and Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Aspergillus fumigatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Alternaria infectoria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Fusarium oxysporum f. sp. vasinfectum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Trichoderma reesei . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5 Zymoseptoria tritici . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Role of EVs in the Interaction of Filamentous Fungi with Their Hosts and with the Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

46 46 47 48 48 49 49 49 50 50 52 52

Abstract Since the first description of extracellular vesicles in a filamentous fungus, Alternaria infectoria, data has been gathered showing the importance of EVs in the interaction of filamentous fungi with the environment and with the animal and plant hosts. In Aspergillus spp. it was described paracrine effects over host cells, namely regulating the immune response; in phytopathogens, it was described the importance of EVs in infection structures. The study of the filamentous fungi EVs associated with cargos indicates important roles in the breakdown of substrates and the remodeling of the cell wall. Nevertheless, the information about filamentous fungi EVs is still scarce and the biogenesis and release deserve further study.

T. Gonçalves (*) · J. Oliveira · C. Fernandes CNC—Center for Neurosciences and Cell Biology, Coimbra, Portugal FMUC—Faculty of Medicine-University of Coimbra, Coimbra, Portugal e-mail: [email protected] © Springer Nature Switzerland AG 2021 M. Rodrigues, G. Janbon (eds.), Fungal Extracellular Vesicles, Current Topics in Microbiology and Immunology 432, https://doi.org/10.1007/978-3-030-83391-6_5

45

46

T. Gonçalves et al.

1 Introduction Filamentous fungi, or molds, are organisms forming multicellular, septated or not, filamentous structures known as hyphae. These are branched filamentous structures that can differentiate in structures to produce spores, to sexual or nonsexual reproduction. Although usually environmental, living in decaying matter, they are well known as plant and animal pathogens. Like other eukaryotes and prokaryotes (Kuipers et al. 2018), filamentous fungi are able to produce and release extracellular vesicles (EVs). In general, EVs are described as cell-derived double-layer phospholipid membrane vesicles (Margolis and Sadovsky 2019), and are crucial in cell-tocell communication, having an important role in nutrition and physiopathogenesis (Kuipers et al. 2018). EVs are carriers of proteins, lipids, nucleic acids like mRNAs, and non-coding RNAs, polysaccharides, toxins, pigments, prions, and allergens. EVs are commonly classified according to their size and biogenesis as exosomes, microvesicles, and apoptotic bodies (Margolis and Sadovsky 2019). Exosomes have a size between 30–100 nm by endocytic pathway, microvesicles have 100–1000 nm released from cells through membrane shedding, and apoptotic bodies have the larger size, 800–1000 nm, by programmed cell death. Most of the knowledge gathered about fungal EVs has been generated in yeast studies (Rizzo et al. 2020a) but both dimorphic fungi and filamentous fungi release EVs. Regardless of the challenges associated with the study of filamentous fungi EVs biogenesis and cargo, there is a growing perception that these play a critical role in the physiology of fungi and inter-kingdom interactions, in host-pathogen or microbiotas.

2 Historical Aspects Briefly, following the first description of EVs in fungi in 1973 (Takeo et al. 1973), there was evidence of the existence of EVs in diverse species of fungi, where researchers discovered some structures which were released to extracellular space. The first complete characterization of fungal EVs was described in 2007 in Cryptococcus neoformans (Rodrigues et al. 2007). Since then, the interest in this field is continuously growing (Joffe et al. 2016), and many other studies showed the production of EVs in other yeasts and filamentous fungi, as recently reviewed (Rizzo et al. 2020a). Nevertheless, the information about their structural properties, biogenesis and functionality is still low. In what concerns filamentous fungi, to date, the studies are scarce. Alternaria infectoria was the first mold to be described as producing EVs (Silva et al. 2014), followed by studies in Aspergillus fumigatus (Souza et al. 2019; Rizzo et al. 2020b), Aspergillus flavus (Brauer et al. 2020), Trichophyton interdigitale (Bitencourt et al. 2018), Trichoderma reesei (de Paula et al. 2019), Fusarium oxysporum f. sp. vasinfectum (Bleackley et al. 2020), Rhizopus delemar (Liu et al. 2018), and

Filamentous Fungi Extracellular Vesicles

47

Zymoseptoria tritici (Hill and Solomon 2020). Recently, it was described that the phytopathogens Ustilago maydis, Gigaspora rosea, and Rhizophagus irregularis, are able to produce EVs, although these are not characterized (Roth et al. 2019). These fungi are environmental organisms, phytopathogens, and/or agents of opportunistic animal infections. Recently, also emerged the role of some of these fungi in natural mycobiotas, the fungal components of microbiotas, of plants and mammalians, namely in the human lung (Underhill and Iliev 2014), where extracellular vesicles are known to have an important role (Macia et al. 2019).

3 Biogenesis and Release The biogenesis of EVs can proceed through different mechanisms; the endosomal sorting complex required for transport (ESCRT) machinery is considered essential for the biogenesis of exosomes in mammalian cells (Colombo et al. 2019). Then, the EVs have to reach the extracellular milieu, which is particularly difficult in cells bearing rigid cell walls such as fungi. In fungi, it seems that the biogenesis of EVs can proceed through conventional secretory pathways involving the formation of multivesicular bodies (MVBs) that ultimately merge with the plasma membrane (Wolf and Casadevall 2014; Rodrigues et al. 2007) or by evagination of the plasma membrane (Silva et al. 2019). Deletion of ESCRT components in Saccharomyces cerevisiae decreases the number of EVs produced (Zhao et al. 2019), but definitive information regarding the involvement of ESCRT in fungal EVs trafficking is still lacking. In T. reesei, a phytopathogen, it was described that EVs carry proteins that are involved in the trans-Golgi network (TGN)-endosomal system for vesicular transport (de Paula et al. 2019). The presence of glyceraldehyde-3-phosphate dehydrogenase in A. infectoria EVs was interpreted as a probable association within the transport of vesicles along the hyphae (Silva et al. 2014) since it is also associated with axonal transport of organelles and molecules (Zala et al. 2013). In F. oxysporum, the proteomic analysis revealed proteins associated with vesicle transport (Bleackley et al. 2019). In the powdery mildew, fungus Blumeria graminis and Golovinomyces orontii, MVBs were reported in the appressoria and haustoria but until now, EVs were completely characterized in these phytopathogens (Samuel et al. 2015). The fungal cell wall is a rigid and robust structure, but also a dynamic structure, and so the release of EVs most certainly proceeds through transient openings, still to be clearly elucidated. There are three hypotheses in order to explain how EVs cross the fungal cell wall: first, vesicles could move through a guide channel; second, enzymes associated with the degradation and with the synthesis of the fungal cell wall can generate openings, facilitating EVs release; third, the presence of pores in the cell wall by turgor pressure (Wolf and Casadevall 2014; Brown et al. 2015). Proteomics of the EVs produced by filamentous fungi revealed the presence of enzymes (or protein families) suggesting the association with cell wall degradation and synthesis (Silva et al. 2014; Souza et al. 2019; Rizzo et al. 2020b).

48

T. Gonçalves et al.

Nevertheless, the extrusion of EVs out of filamentous fungi hyphal cell walls occurs through mechanisms not clearly elucidated and deserves further studies.

4 EVs Morphology and Content In EVs, biomolecules are packaged inside double-membrane vesicles that are released to the extracellular space. As in yeasts and animal cells, the EVs produced by filamentous fungi are round-shaped structures surrounded by a double bilayer. The EVs’ size and cargo most probably reflect the strain biological properties, the biogenesis pathways, and the environmental growth conditions. The EVs produced by filamentous fungi are double-membrane vesicles with sizes ranging from 20 to 200 nm; the few studies about the content of filamentous fungi extracellular vesicles aimed to study the proteomics of EVs, while lipids, carbohydrates, nucleic acid, and other small molecules are poorly studied:

4.1

Aspergillus fumigatus

There are two main studies characterizing the EVs produced A. fumigatus. The first (Souza et al. 2019) described EVs produced by fungal mycelia as round-shaped vesicles with 100–200 nm, while the second (Rizzo et al. 2020b) studied the EVs produced by fungal protoplasts and found vesicles with an average diameter of 200 nm (by TEM). Fungal mycelia EVs-associated proteome (Souza et al. 2019) revealed 60 proteins, including hydrolases, oxydoreductase, peptidases, transferases, RNA/carbohydrate/protein binding, and structural proteins. About one-fourth of these proteins were extracellular (24%) and 14% were associated with the cell wall. According to their biological function, most of the proteins were associated with carbohydrate metabolism, response to stress, and pathogenesis. In A. fumigatus protoplasts, the authors not only characterized the vesicular proteome but also the carbohydrates constituents of the vesicles; the proteomic analysis of the EVs highlighted how different physiological conditions can modify dramatically the protein content of fungal EVs. In fact, while in fresh protoplasts there were 142 proteins, in cell wall regenerating protoplasts, the authors found 2056 proteins, among which were the 142 present in fresh protoplasts (Rizzo et al. 2020b). These proteins included several protein families related to cell wall synthesis and remodeling, such as glycan synthases or O-mannosyltransferases, amino acids synthesis, or carbohydrate metabolism, among others. In respect to the EVs-associated glycans, besides the detection of galactosaminogalactan (GAG), a component of the fungal extracellular matrix, it was found markers of glucan and GAG (Rizzo et al. 2020b).

Filamentous Fungi Extracellular Vesicles

4.2

49

Alternaria infectoria

This was the first mold where EVs’ production was described (Silva et al. 2014). This a melanized ubiquitous mold, phytopathogenic but also an opportunistic agent of human infection. The small size EVs (20–40 nm as measured by TEM; 50–100 nm measured by Dynamic Light Scattering) carry two prevalent categories among the 20 identified proteins, including the from polysaccharide metabolism group, related to plant host invasion or biosynthesis/degradation of cell wall components, and the nuclear proteins, especially DNA repair enzymes. Besides those, there were also enzymes related to pigment synthesis, adhesion to the host cell, and trafficking of vesicles/organelles/molecules. Among the vesicular proteins, it was described for the first time. The presence of heat shock protein 60 (Hsp60).

4.3

Fusarium oxysporum f. sp. vasinfectum

In this cotton pathogen, it was described the most extensive EV proteome found in filamentous fungi with 482 different proteins; the most prevalent were associated with several functions such as proteases, two frequency/period clock, an HSP70 like protein, and polyketide synthase. The proteome reflects basic cellular processes, such as the metabolism of proteins and nucleotides, lipid biosynthesis, and cell structure. The EVs include a pigment not yet characterized and a mean diameter of 155 nm (Bleackley et al. 2020).

4.4

Trichoderma reesei

The isolation and characterization of EVs produced by this fungus that degrades lignocellulose was studied using either cellulose, glycerol, or glucose as carbon sources (de Paula et al. 2019). The mean size of the EVs was 144 nm, and the number of EVs produced decreased when the fungus was grown in glycerol or glucose. The proteomic analysis revealed that when the fungus was grown in cellulose, the EVs contained 188 proteins produced under a dynamic sequence: 33 proteins exclusively identified after 24 h of growth, 14 after 48 h, 49 after 72 h, 37 after 96 h, and 29 after 120 h. Only one protein, encoding an RNA-binding protein involved with rRNA biogenesis was identified in all time points. The T. reesei EVs cargo included cellulases, related to the main biological trait of this fungus, but also small GTPases, which are involved in polar growth, exocytosis, endocytosis, and secretory-vesicle fusion to the plasma membrane; a vesicleassociated membrane protein, VAMP/Synaptobrevin, involved protein trafficking that is an essential component of trans-Golgi network (TGN)-endosomal system for vesicular transport. Two vesicular heat shock proteins, Hsp70 and Hsp60, were also

50

T. Gonçalves et al.

found, which lead the authors to suggest these proteins might constitute a mechanism to environmental sensing by the fungus (de Paula et al. 2019).

4.5

Zymoseptoria tritici

The EVs produced by this wheat phytopathogen have diameters ranging from 50 to 300 nm with a peak of 100–250 nm in size (Hill and Solomon 2020). In Z. tritici, EVs 210 proteins were found. Among these, the proteins associated with exo/endocytosis proteins, cytoskeleton, proteolytic enzymes, and general metabolism, among others, such as Hsp70 and Hsp90, were identified. The authors proceeded with an analysis of the similarity between the proteome of Z. tritici EVs compared with other fungal EVs concluding that there is a common pattern between the protein content of fungal EVs (Hill and Solomon 2020). Interestingly, it emerged that Hsp70 might be considered a fungal EV marker similarly to its use in mammalian cells EVs (Kowal et al. 2016; Bleackley et al. 2019; Hill and Solomon 2020).

5 Role of EVs in the Interaction of Filamentous Fungi with Their Hosts and with the Environment Extracellular vesicles historically were described as not having biological or physiological importance and the interpretation; instead, the interpretation was that they corresponded to a way of cells to get rid of waste (Margolis and Sadovsky 2019). The perception that microbial extracellular vesicles were merely vehicles of enzymes and other small molecules essential to obtain nutrients from the environment is easily understood. In filamentous fungi, this is even more intelligible because they are saprophytes with the need of secreting enzymes to break down substrates and obtain the needed nutrients. EVs constitute a form of secreting concentrated “catalysts”. This is clearly illustrated by the cargo of EVs produced by filamentous fungi in which the proteomes were described, in particular fungi such as T. reesei, fungi that degrade lignocellulose (de Paula et al. 2019) and have enriched EVs-associated hydrolytic enzymes needed for this hydrolysis in the presence of cellulose. In fact, filamentous fungi that are phytopathogens or ubiquitous concentrate in their EVs-associated proteomes enzymes that are involved in the degradation of complex substrates into smaller molecules that can be taken by the fungus as a nutrient (Silva et al. 2014; Hill and Solomon 2020; de Paula et al. 2019; Bleackley et al. 2019). Another important EV-associated function revealed by the constitution of the filamentous fungi EVs is related to the fungal cell wall remodulation. In fact, some of the proteomes reveal enzymes involved in the metabolism and biosynthesis of important components of the cell wall such as chitin, glucans, or mannan residues. The importance of EVs cargo in the biosynthesis of the fungal cell wall is clearly

Filamentous Fungi Extracellular Vesicles

51

demonstrated by the results obtained by Rizzo and coworkers (Rizzo et al. 2020b) showing that A. fumigatus protoplasts kept in cell wall regenerating conditions have an enriched EV-proteome in enzymes related to cell wall such as glucan synthase or chitin synthases. Moreover, the EVs contained not only mannosyl transferases, involved in the synthesis of galactomannan (GAG), a key component of A. fumigatus extracellular matrix, but also GAG and the monosaccharides needed for its synthesis. Paracrine signs are determinants for the communication between pathogens and the host. The canonical secretion of biomolecules can be considered less efficient than extracellular vesicles, concentrating biomolecules that can trigger a response in the host cells. The pathogen’s EVs with their cargos can circulate to locations different from the infection place. A prove of the dissemination of fungal EVs throughout the human host is the presence of A. fumigatus EVs in the urine of patients with invasive aspergillosis, detected using the antibody MAb476 that recognizes GAG, a component of the A. fumigatus EVs (Dufresne et al. 2012). Furthermore, it is expectable that these circulating EVs will have paracrine effects in cells or tissues located outside the localized infection (e.g. in the lung). In fact, GAG has been associated with the virulence of A. fumigatus, both in animal models and in the human host (Fontaine et al. 2011; Gresnigt et al. 2014). This important aspect of the importance of the interaction of fungi with the plant and animal hosts has been extensively revised (Samuel et al. 2015; Joffe et al. 2016; Kuipers et al. 2018; Zamith-Miranda et al. 2018; de Toledo Martins et al. 2019). Although most of the studies regarding the role of fungal EVs in the hostpathogen interaction come from yeasts, the information gathered so far about how filamentous fungi EVs influence the host is enough to demonstrate its importance, both in the plant and in the animal host. A. fumigatus, considered one of the most important human pathogens causing serious diseases such as bronchopulmonary allergic aspergillosis or invasive aspergillosis (Lamoth 2016), actively produce EVs during infection of the animal/human host. These EVs stimulate phagocytosis, trigger the production of proinflammatory mediators, such as TNF-α and CCL2. Phagocytes sensitized with A. fumigatus EVs, have an increased antifungal phenotype, resulting in higher fungal clearance, compared with phagocytes that were not primed with EVs (Souza et al. 2019). Another Aspergillus species, Aspergillus flavus, an important agent of human aspergillosis, a phytopathogen, and a strong producer of aflatoxin B1, one of the most potent carcinogens, produces EVs that are pro-inflammatory, leading to the production of TNF-α, IL-6, and IL-1β and increasing the fungicidal activity of macrophages (Brauer et al. 2020). T. interdigitale is a filamentous fungus that belongs to the group of dermatophytes, filamentous fungi with a tropism for keratinized structures, infecting the skin, nails, and hair, causing dermatophytosis or tineas. This human fungal pathogen produces EVs and, although their cargo is still not known, that induce a response of macrophages and keratinocytes, mediated by the Toll-like receptor 2, with the production of NO (nitric oxide), TNF-α, IL-6 and IL-1β, and increase the fungicidal capacity of macrophages (Bitencourt et al. 2018).

52

T. Gonçalves et al.

In the last few years, there has been an increasing awareness of the importance of RNAs in infectious mechanisms and there is growing knowledge on the importance of these molecules in fungal infections, namely delivered by yeasts extracellular EVs (Peres da Silva et al. 2015). In the zygomycete Rhizopus delemar, agent of mucormycosis, a severe opportunistic human fungal infection was published an extensive study characterizing the RNAs associated with EVs produced by this fungus (Liu et al. 2018). In this study, it is described using RNASeq of ex-sRNAs, 3.3 and 3.2 million clean reads were found, with further annotation allowing the identification of 560 and 526 miRNAs. By target prediction and analysis, the authors describe that these miRNAs are involved in pathways of metabolism regulation, secondary metabolite biosynthesis, and two-component system signaling. Although there is no direct evidence of these findings in the pathogenesis of R. delemar, the in silico analysis is indicative of the importance of these RNAs in the virulence mechanisms (Liu et al. 2018; Cai et al. 2019). The role of filamentous fungi small RNAs enclosed in vesicles in the interaction of fungi with the plant host seems to be critical for the outcome of infections (Wang and Dean 2020). A recent study shows that the export of small interfering RNA from the fungal insect pathogen Beauveria bassiana inside vesicles attenuates mosquito immunity and facilitates infection (Cui et al. 2019), indicating a possible biocontrol of vector-borne diseases.

6 Concluding Remarks Although the knowledge regarding filamentous fungi EVs has increased, there are limitations and challenges, including (a) the identification of specific fungal EV biomarkers; (b) optimization of EV isolation procedures (fungal growth conditions, isolation, and characterization methodologies); (c) ability to distinguish between fungal and host EVs; and (d) the identification of “hybrid” extracellular vesicles originated in infected cells host cells and bearing both host and fungal antigens (Fleming et al. 2014). Future and further research in filamentous fungi extracellular vesicles importance will certainly elucidate its role in its virulence, either in the place of infection and as paracrine effectors, and will highlight the usage of these vehicles of fungal antigens in the control of diseases and the development of vaccines (Freitas et al. 2019; Silva et al. 2019).

References Bitencourt TA, Rezende CP, Quaresemin NR, Moreno P, Hatanaka O, Rossi A, Martinez-Rossi NM, Almeida F (2018) Extracellular vesicles from the dermatophyte Trichophyton interdigitale modulate macrophage and keratinocyte functions. Front Immunol 9:2343. https://doi.org/10. 3389/fimmu.2018.02343.

Filamentous Fungi Extracellular Vesicles

53

Bleackley MR, Dawson CS, Anderson MA (2019) Fungal extracellular vesicles with a focus on proteomic analysis. Proteomics 19(8):e1800232. https://doi.org/10.1002/pmic.201800232 Bleackley MR, Samuel M, Garcia-Ceron D, McKenna JA, Lowe RGT, Pathan M, Zhao K, Ang C-S, Mathivanan S, Anderson MA (2020) Extracellular vesicles from the cotton pathogen Fusarium oxysporum f. sp. vasinfectum induce a phytotoxic response in plants. Front Plant Sci 10:1610. https://doi.org/10.3389/fpls.2019.01610 Brauer VS, Pessoni AM, Bitencourt TA, de Paula RG, de Oliveira Rocha L, Goldman GH, Almeida F (2020) Extracellular vesicles from Aspergillus flavus induce M1 polarization in vitro. mSphere 5:e00190-20. https://doi.org/10.1128/mSphere.00190-20. Brown L, Wolf JM, Prados-Rosales R, Casadevall A (2015) Through the wall: extracellular vesicles in gram-positive bacteria, mycobacteria and fungi. Nat Rev Microbiol 13(10):620–630. https:// doi.org/10.1038/nrmicro3480. Cai Q, He B, Weiberg A, Buck AH, Jin H (2019) Small RNAs and extracellular vesicles: new mechanisms of cross-species communication and innovative tools for disease control. PLoS Pathog 15(12):e1008090. https://doi.org/10.1371/journal.ppat.1008090. Colombo C, Moita C, van Niel G, Kowal J, Vigneron J, Benaroch P, Manel N, Moita LF, Théry C, Raposo G (2019) Analysis of ESCRT functions in exosome biogenesis, composition and secretion highlights the heterogeneity of extracellular vesicles. J Cell Sci 126:5553–5565. https://doi.org/10.1242/jcs.128868 Cui C, Wang Y, Liu J et al (2019) A fungal pathogen deploys a small silencing RNA that attenuates mosquito immunity and facilitates infection. Nat Commun 10:4298. https://doi.org/10.1038/ s41467-019-12323-1 de Paula R, Antoniêto ACC, Nogueira KMV et al (2019) Extracellular vesicles carry cellulases in the industrial fungus Trichoderma reesei. Biotechnol Biofuels 12:146. https://doi.org/10.1186/ s13068-019-1487-7 de Toledo Martins S, Szwarc P, Goldenberg S, Alves LR (2019) Extracellular vesicles in fungi: composition and functions. Curr Top Microbiol Immunol 422:45–59. https://doi.org/10.1007/ 82_2018_141. Dufresne SF, Datta K, Li X, Dadachova E, Staab JF et al (2012) Detection of urinary excreted fungal galactomannan-like antigens for diagnosis of invasive aspergillosis. PLoS One 7(8): e42736. https://doi.org/10.1371/journal.pone.0042736. Fleming A, Sampey G, Chung M-C, Bailey C, van Hoek ML, Kashanchi F, Hakami RM (2014) The carrying pigeons of the cell: exosomes and their role in infectious diseases caused by human pathogens. Pathog Dis 71:109–120. https://doi.org/10.1111/2049-632X.12135 Fontaine T, Delangle A, Simenel C, Coddeville B, van Vliet SJ, van Kooyk Y, Bozza S, Moretti S, Schwarz F, Trichot C, Aebi M, Delepierre M, Elbim C, Romani L, Latgé JP (2011) Galactosaminogalactan, a new immunosuppressive polysaccharide of Aspergillus fumigatus. PLoS Pathog 7:e1002372. https://doi.org/10.1371/journal.ppat.1002372. Freitas MS, Bonato VLD, Pessoni AM, Rodrigues ML, Casadevall A, Almeida F (2019) Fungal extracellular vesicles as potential targets for immune interventions. mSphere 4:e00747-19. https://doi.org/10.1128/mSphere.00747-19. Gresnigt MS, Bozza S, Becker KL, Joosten LAB, Abdollahi-Roodsaz S, van der Berg WB, Dinarello CA, Netea MG, Fontaine T, De Luca A, Moretti S, Romani L, Latge JP, van de Veerdonk FL (2014) A polysaccharide virulence factor from Aspergillus fumigatus elicits antiinflammatory effects through induction of interleukin-1 receptor antagonist. PLoS Pathog 10: e1003936. https://doi.org/10.1371/journal.ppat.1003936. Hill EH, Solomon PS (2020) Extracellular vesicles from the apoplastic fungal wheat pathogen Zymoseptoria tritici. Fungal Biol Biotechnol 7:13. https://doi.org/10.1186/s40694-020-00103-2 Joffe LS, Nimrichter L, Rodrigues ML, Del Poeta M (2016) Potential roles of fungal extracellular vesicles during infection. mSphere 1(4):e00099-16. https://doi.org/10.1128/mSphere.00099-16. Kowal J, Arras G, Colombo M, Jouve M, Morath JP, Primdal-Bengtson B, Dingli F, Loew D, Tkach M, Thery C (2016) Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes. PNAS 113:E968–E977

54

T. Gonçalves et al.

Kuipers ME, Hokke CH, Smits HH, Nolte-’t Hoen ENM (2018) Pathogen-derived extracellular vesicle-associated molecules that affect the host immune system: an overview. Front Microbiol 9:2182. https://doi.org/10.3389/fmicb.2018.02182 Lamoth F (2016) Aspergillus fumigatus-related species in clinical practice. Front Microbiol 7:683. https://doi.org/10.3389/fmicb.2016.00683. Liu M, Bruni GO, Taylor CM et al (2018) Comparative genome-wide analysis of extracellular small RNAs from the mucormycosis pathogen Rhizopus delemar. Sci Rep 8:5243. https://doi.org/10. 1038/s41598-018-23611-z Macia L, Nanan R, Hosseini-Beheshti E, Grau GE (2019) Host- and microbiota-derived extracellular vesicles, immune function, and disease development. Int J Mol Sci 21(1):107. https://doi. org/10.3390/ijms21010107. Margolis L, Sadovsky Y (2019) The biology of extracellular vesicles: the known unknowns. PLoS Biol 17(7):e3000363. https://doi.org/10.1371/journal.pbio.3000363 Peres da Silva R, Puccia R, Rodrigues ML, Oliveira DL, Joffe LS, César GV, Nimrichter L, Goldenberg S, Alves LR (2015) Extracellular vesicle-mediated export of fungal RNA. Sci Rep 5:7763. https://doi.org/10.1038/srep07763. Rizzo J, Chaze T, Miranda K, Roberson RW, Gorgette O, Nimrichter L, Matondo M, Latgé J-P, Beauvais A, Rodrigues ML (2020a) Characterization of extracellular vesicles produced by Aspergillus fumigatus protoplasts. mSphere 5:e00476-20. https://doi.org/10.1128/mSphere. 00476-20 Rizzo J, Rodrigues ML, Janbon G (2020b) Extracellular vesicles in fungi: past, present, and future perspectives. Front Cell Infect Microbiol 10:346. https://doi.org/10.3389/fcimb.2020.00346 Rodrigues ML, Nimrichter L, Oliveira DL, Frases S, Miranda K, Zaragoza O, Alvarez M, Nakouzi A, Feldmesser M, Casadevall A (2007) Vesicular polysaccharide export in Cryptococcus neoformans is a eukaryotic solution to the problem of fungal trans-cell wall transport. Eukaryot Cell 6(1):48–59. https://doi.org/10.1128/EC.00318-06. Roth R, Hillmer S, Funaya C et al (2019) Arbuscular cell invasion coincides with extracellular vesicles and membrane tubules. Nat Plants 5:204–211. https://doi.org/10.1038/s41477-0190365-4 Samuel M, Bleackley M, Anderson M, Mathivanan S (2015) Extracellular vesicles including exosomes in cross kingdom regulation: a viewpoint from plant-fungal interactions. Front Plant Sci 6:766. https://doi.org/10.3389/fpls.2015.00766 Silva BM, Prados-Rosales R, Espadas-Moreno J, Wolf JM, Luque-Garcia JL, Gonçalves T, Casadevall A (2014) Characterization of Alternaria infectoria extracellular vesicles. Med Mycol 52(2):202–210. https://doi.org/10.1093/mmy/myt003. Silva VKA, Rodrigues ML, May RC (2019) Deciphering fungal extracellular vesicles: from cell biology to pathogenesis. Curr Clin Micro Rpt 6:89–97. https://doi.org/10.1007/s40588-01900128-1 Souza JAM, Baltazar LM, Carregal VM, Gouveia-Eufrasio L, de Oliveira AG, Dias WG, Campos Rocha M, Rocha de Miranda K, Malavazi I, Santos DA, Frézard FJG, de Souza DDG, Teixeira MM, Soriani FM (2019) Characterization of Aspergillus fumigatus extracellular vesicles and their effects on macrophages and neutrophils functions. Front Microbiol 10:2008. https://doi. org/10.3389/fmicb.2019.02008. Takeo K, Uesaka I, Uehira K, Nishiura M (1973) Fine structure of Cryptococcus neoformans grown in vitro as observed by freeze-etching. J Bacteriol 113(3):1442–1448. https://doi.org/10.1128/ JB.113.3.1442-1448.1973 Underhill DM, Iliev ID (2014) The mycobiota: interactions between commensal fungi and the host immune system. Nat Rev Immunol 14(6):405–416. https://doi.org/10.1038/nri3684. Wang M, Dean RA (2020) Movement of small RNAs in and between plants and fungi. Mol Plant Pathol 21:589–601. https://doi.org/10.1111/mpp.12911

Filamentous Fungi Extracellular Vesicles

55

Wolf JM, Casadevall A (2014) Challenges posed by extracellular vesicles from eukaryotic microbes. Curr Opin Microbiol 22:73–78. https://doi.org/10.1016/j.mib.2014.09.012. Zala D, Hinckelmann MV, Yu H, Lyra da Cunha MM, Liot G, Cordelières FP, Marco S, Saudou F (2013) Vesicular glycolysis provides on-board energy for fast axonal transport. Cell 152 (3):479–491. https://doi.org/10.1016/j.cell.2012.12.029. Zamith-Miranda D, Nimrichter L, Rodrigues ML, Nosanchuk JD (2018) Fungal extracellular vesicles: modulating host-pathogen interactions by both the fungus and the host. Microbes Infect 20(9–10):501–504. https://doi.org/10.1016/j.micinf.2018.01.011. Zhao K, Bleackley M, Chisanga D, Gangoda L, Fonseka P, Liem M, Kalra H, Al Saffar H, Keerthikumar S, Ang CS, Adda CG, Jiang L, Yap K, Poon IK, Lock P, Bulone V, Anderson M, Mathivanan S (2019) Extracellular vesicles secreted by Saccharomyces cerevisiae are involved in cell wall remodelling. Commun Biol 2:305. https://doi.org/10.1038/s42003-0190538-8

Extracellular Vesicles and the Propagation of Yeast Prions Mehdi Kabani

Contents 1 Packaging of Sup35p Prion Particles within Extracellular (EV) and Periplasmic (PV) Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Limitations to Current Prion Propagation Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 What Can EV Tell Us About the Molecular Nature of Propagons? . . . . . . . . . . . . . . . . . . . . . . . . . 4 A Yeast Propagation Model Where EV Shield Propagons from SPQC and Anti-Prion Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

58 59 60 61 62 63

Abstract Infectious proteins or prions are self-replicating transmissible aggregates responsible for heritable traits in yeasts and amyloid diseases in mammals. Extensive investigations into the many prions discovered in the yeast Saccharomyces cerevisiae, and most importantly the [PSI+] prion, shaped our understanding of the cellular mechanisms involved in amyloidosis. [PSI+] arises from the assembly of the translation terminator Sup35p into insoluble fibrillar aggregates leading to nonsense suppression phenotypes. We recently found that infectious Sup35p particles traffic via extracellular (EV) and periplasmic (PV) vesicles in a growth phase and glucose-

This chapter is an updated and shortened version of the following review: International Journal of Molecular Sciences, 2021, 22(1), 90 Extracellular Vesicles-Encapsulated Yeast Prions and What They Can Tell Us about the Physical Nature of Propagons Mehdi Kabani Copyright: © 2020 Mehdi Kabani This is an open-access article distributed under the terms of the Creative Commons Attribution License 4.0 (https://creativecommons.org/licenses/by/4.0). To view the original article, visit https://doi.org/10.3390/ijms22010090

M. Kabani (*) Université Paris-Saclay, CEA, CNRS, Molecular Imaging Research Center (MIRCen), Laboratoire des Maladies Neurodégénératives (UMR9199), Fontenay-aux-Roses, France e-mail: [email protected] © Springer Nature Switzerland AG 2021 M. Rodrigues, G. Janbon (eds.), Fungal Extracellular Vesicles, Current Topics in Microbiology and Immunology 432, https://doi.org/10.1007/978-3-030-83391-6_6

57

58

M. Kabani

dependent manner. In this chapter, I will summarize these findings and explain how they fit in current models of yeast prions transmission.

1 Packaging of Sup35p Prion Particles within Extracellular (EV) and Periplasmic (PV) Vesicles Prions are self-templating conformations of proteins that cause dominant and heritable epigenetic traits in mammals, filamentous fungi, and yeasts (Cox 1965; Aigle and Lacroute 1975; Prusiner 1982; Wickner 1994; Coustou et al. 1997). Many prions were described in the yeast Saccharomyces cerevisiae, constituting as many valuable models to investigate fundamental mechanisms of structural inheritance (reviewed in Sindi and Serio (2009), Kabani and Melki (2011), Liebman and Chernoff (2012), Kabani and Melki (2016), and Wickner et al. (2020)). Most prion proteins contain so-called prion domains that are often but not always Asn/Gln-rich and that allow them to assemble into highly ordered self-replicating β-rich amyloid aggregates (Kabani and Melki 2011, 2016; Wickner et al. 2020; Kabani 2021). Prion assembly into fibrils occurs by nucleated polymerization, a process that can be reproduced in vitro using purified recombinant proteins (Glover et al. 1997; Taylor et al. 1999; Thual et al. 1999; Krzewska and Melki 2006; Patel and Liebman 2007; Kabani and Melki 2011; Liebman and Chernoff 2012). Within cells, yeast prions encompass a heterogeneous continuum of molecular species from small oligomers to larger fibrillar aggregates (Sindi and Serio 2009; Kabani 2021). The equilibrium between the native and prion forms is controlled by molecular chaperones (e.g., Hsp104, Hsp70, Hsp40), proteolytic systems, and other components of the protein quality control machinery (Kabani and Melki 2011; Liebman and Chernoff 2012; Winkler et al. 2012; Wickner et al. 2020; Kabani 2021). The transmission of prions to the bud upon cell division involves cytosolic diffusible particles that were named propagons (Cox et al. 2003). The exact molecular nature of propagons is, however, not clearly defined (Kabani 2021). The assembly of the essential translation terminator Sup35p into self-perpetuating aggregates is at the origin of the [PSI+] prion (Cox 1965; Patino et al. 1996). [PSI+] is the most studied and best-characterized yeast prion at the genetic, cellular, biochemical, and structural levels (Kabani and Melki 2011; Kabani 2020, 2021). We found that infectious Sup35p prion particles are packaged within EVs with a diameter ranging from ~30–100 nm, reminiscent of exosomes, and secreted in the extracellular medium (Kabani and Melki 2015, 2016; Kabani 2020). Under limiting glucose growth conditions, high amounts of infectious Sup35p prion particles are associated with periplasmic vesicles (PVs) (Kabani et al. 2020; Kabani 2020). PVs are up to three orders of magnitude more abundant than EVs, suggesting they may constitute a cytoplasmic/periplasmic pool of macromolecules that can be reused by the cells (Huang and Chiang 1997; Giardina et al. 2014a, b; Kabani et al. 2020; Winters et al.

Extracellular Vesicles and the Propagation of Yeast Prions

59

2020). On a technical note, isolating yeast EVs is a time-consuming process that requires the filtration and ultracentrifugation of substantial volumes of culture supernatants to achieve satisfactory yields (Kabani and Melki 2015). Conversely, high amounts of PVs can be extracted from small-scale yeast cultures by the combined action of high pH and β-mercaptoethanol (Alibhoy et al. 2012; Kabani et al. 2020; Kabani 2021). It should be taken into account that PVs and EVs exhibit significant differences in terms of size and protein cargo and that their secretion is differentially regulated by glucose availability (Kabani et al. 2020).

2 Limitations to Current Prion Propagation Models Most laboratory [PSI+] prion strains have remarkably high mitotic stabilities and form phenotypically homogeneous colonies (Cox 1965; Derkatch et al. 1996; Byrne et al. 2009; Derdowski et al. 2010; Wang et al. 2017). These [PSI+] variants grow at the same rates as isogenic prion-free [psi ] cells and even confer a selective advantage over extended cultivation periods (Wang et al. 2017). Prion loss occurs at very low frequencies, depending on the genetic background and the prion variant, resulting in the appearance of rare sectored colonies or [psi ] cells in liquid cultures (Cox 1965; Derkatch et al. 1996; Byrne et al. 2009; Derdowski et al. 2010; Wang et al. 2017). So how does this exceptional prion stability fit with what we know about the context in which the transmission of propagons occurs during cell division (Kabani 2021)? i-A number of criteria were experimentally and mathematically determined to describe propagons (Sindi and Serio 2009; Byrne et al. 2009; Derdowski et al. 2010). Yeast cells contain ~100–1000 propagons, with a minimal size of ~4–30 Sup35p molecules, that are passively but efficiently transmitted to daughter cells, while mother cells retain most of the larger aggregates (Tanaka et al. 2006; Bagriantsev et al. 2008; Byrne et al. 2009; Derdowski et al. 2010; Villali et al. 2020). ii-Bud formation and cell division are highly regulated processes (Juanes and Piatti 2016; Bhavsar-Jog and Bi 2017). The emerging bud is separated from the mother cell by a septin ring, and the division site is crowded with macromolecules, such as the dividing nucleus, organelles (e.g., mitochondria, endoplasmic reticulum, peroxisomes), actin cables and vesicles (Sentandreu and Northcote 1969; Knoblach and Rachubinski 2015) (Fig. 1). iii-Cell division is asymmetric: mother cells retain most aging determinants including misfolded and aggregated proteins and damaged organelles. The daughter is essentially free of aging determinants that might reduce its lifespan (HiguchiSanabria et al. 2014). Spatial protein quality control (SPQC) mechanisms and antiprion systems ensure that protein aggregates and prions are sequestered within protein inclusions (e.g., IPOD, JUNQ) or cleared by proteolytic machineries (e.g., proteasome, autophagy) (Hill et al. 2017; Sontag et al. 2017). Hence, a model where propagons in equilibrium with both monomers and larger aggregates are passively transmitted to the emerging bud by cytosol repartition does

60

M. Kabani

Fig. 1 Hypothetical model of yeast prion propagation. “Naked” or vesicle-embedded propagons (blue barrels), but not larger assemblies, may escape spatial protein quality control (SPQC; red hexagons) mechanisms (e.g., molecular chaperones, proteasome, autophagy) to reach the bud. Prion-containing vesicles could reach the bud via actin cable-mediated transport or diffusion through the cell-wall/periplasm and subsequent reinternalization. Yeast prions packaged within vesicles are also secreted in the extracellular medium (adapted from Kabani and Melki (2016) and Kabani (2020, 2021))

not quite add up with these antiaging protecting mechanisms (Chernova et al. 2017; Hill et al. 2017; Sontag et al. 2017; Wickner et al. 2020; Kabani 2021). By definition, propagons contain the structural information required to propagate the prion conformation and should be preferential targets of anti-prion systems (Chernova et al. 2017; Hill et al. 2017; Sontag et al. 2017; Wickner et al. 2020). Of course, some prion particles may escape quality control mechanisms and diffuse through the mother-bud junction despite crowding. This may suffice to allow prion maintenance during multiple cell divisions as only one propagon is able to perpetuate the prion conformation (Tanaka et al. 2006). Nevertheless, given the efficiency of protein quality control mechanisms, we would expect in this case higher [PSI+]-loss frequencies than the ones we previously observed (Wang et al. 2017; Kabani 2021).

3 What Can EV Tell Us About the Molecular Nature of Propagons? Recombinant Sup35p fibrils are infectious: they efficiently induce [PSI+] formation when introduced inside [psi ] recipient cells using a spheroplasts transformation procedure (King and Diaz-Avalos 2004; Tanaka et al. 2004). Cell extracts from

Extracellular Vesicles and the Propagation of Yeast Prions

61

[PSI+] cells can also be used as infectious material in such assays allowing us to assess both qualitatively and quantitatively how mutations or growth conditions affect the number and infectious properties of prion particles (Kabani et al. 2011, 2014; Wang et al. 2019). Susan Liebman and colleagues were able to purify SDS-resistant particles from [PSI+] cells expressing hexahistidine-tagged Sup35p (Bagriantsev et al. 2008). These particles have the aspect of ~20 nm barrels composed of ~4–20 Sup35p molecules associated with molecular chaperones, mainly Hsp70-family members (Bagriantsev et al. 2008). These barrel-like structures, which could be small amyloid fragments, were able to assemble into ~100–200 nm detergent-labile bundles (Bagriantsev et al. 2008). The [PSI+]-inducing activity of these purified Sup35p aggregates in protein transformation assays increased after SDS treatment, hinting that the ~20 nm barrel-like Sup35p polymers are the infectious unit (Bagriantsev et al. 2008). In agreement with these results, we recently reported significant changes in the size, amount, and infectious properties of SDS-resistant Sup35p particles in cell growth- and metabolic status-dependent manner (Wang et al. 2019; Kabani et al. 2020). These changes may reflect the growth phase-dependent organization of Sup35p prion particles into larger aggregates as suggested by fluorescence microscopy data (Wang et al. 2019). We showed that the infectivity of EVs and cell lysates prepared from [PSI+] cells and normalized for Sup35p levels is similar (Kabani and Melki 2015). Thus, EVs (and PVs) appear to contain true propagons (Kabani 2020; Kabani 2021). I previously estimated the average volume of ~50–100 nm EVs and compared it to those of the particles described by Liebman and colleagues (Bagriantsev et al. 2008; Kabani and Melki 2015; Kabani 2021). While EVs could contain one or several SDS-resistant Sup35p particles, they cannot accommodate larger aggregates (Bagriantsev et al. 2008). Protease protection assays showed that most Sup35p is inside EVs (Kabani and Melki 2015). We cannot, however, exclude that larger Sup35p prion particles stick to the vesicle surface or bind to peripheral vesicle proteins (Hill et al. 2016). Purifying Sup35p prion particles from cell lysates and isolated EVs (or PVs), and comparing them at the ultrastructural level would be of great interest to characterize Sup35p propagons at the molecular level (Kabani 2021).

4 A Yeast Propagation Model Where EV Shield Propagons from SPQC and Anti-Prion Systems Different prion variants and genetic backgrounds, as well as metabolic and environmental factors, affect the transmission of propagons, resulting in a wide range of prion mitotic stabilities (Derkatch et al. 1996; Schlumpberger et al. 2001; Brachmann et al. 2005; Sharma et al. 2009; McGlinchey et al. 2011; Huang et al. 2013; Wang et al. 2017). As discussed above, given the efficiency and diversity of

62

M. Kabani

protein quality control, anti-prion, and antiaging systems, we expect the appearance of cured cells at each cell division (Chernova et al. 2017; Hill et al. 2017; Sontag et al. 2017; Wickner et al. 2020). However, some commonly used laboratory [PSI+] variants are transmitted through generations with an efficiency only seen for essential macromolecules such as chromosomes. Whereas anti-prion systems may be most efficient in ridding cells from the most harmful aggregates (Wickner et al. 2020), neutral or beneficial prions may be poorer substrates for such systems (Kabani 2021). As discussed above, EVs and PVs are physically able to contain and transport small SDS-resistant infectious prion particles (Bagriantsev et al. 2008; Kabani and Melki 2015; Kabani et al. 2020). These vesicles may accumulate in the cytosol as intracellular vesicle clusters (Winters et al. 2020), forming a pool of “hidden” propagons (Kabani and Melki 2015; Kabani et al. 2020). In light of these findings, I propose a model where vesicles play a significant role in prion propagation, accounting for the very high mitotic stability of some prion variants (Fig. 1) (Kabani and Melki 2016; Kabani 2020, 2021). Secretory vesicles are addressed to the emerging bud to deliver the macromolecules and enzymes required to build the newly forming plasma membrane and cell-wall (Sentandreu and Northcote 1969). Actin cables convey vesicles and organelles from mother to daughter cells (Fig. 1) (Pruyne et al. 2004). Remarkably, yeast prions formation relies on the actin cortical cytoskeleton and the endocytic-vacuolar pathways (Ganusova et al. 2006; Manogaran et al. 2011; Speldewinde et al. 2017; Dorweiler et al. 2020). Propagons-containing vesicles could join this flow of vesicles transported across the bud neck and deliver prion particles to daughter cells (Fig. 1) (Kabani 2020, 2021). Such a mechanism would ensure efficient and stable transmission of propagons at each cell division (Fig. 1). Vesicles would serve as a cloak protecting propagons from the action of molecular chaperones, proteolytic machineries, and other spatial quality control and anti-prion systems (Kabani 2020, 2021). Another possible route of prion dissemination is via the periplasm/cell-wall where PVs could avoid molecular crowding and “checkpoint” controls at the bud neck (Kabani et al. 2020; Kabani 2021). PVs would simply diffuse to daughter cells and deliver their cargo when reinternalized depending on growth conditions and the metabolic state of the cells (Fig. 1) (Kabani 2020, 2021). Because EVs can cross the cell-wall barrier to reach the extracellular space (Kabani and Melki 2015), it is plausible to envision they can be recaptured by neighboring cells (Zhao et al. 2019). Hence, we raised the possibility that prions could be horizontally transmitted from cell-to-cell in the absence of cell division or mating (Kabani and Melki 2015, 2016).

5 Concluding Remarks Understanding when and how yeast prions particles are packaged inside vesicles could be of great significance in the context of human neurodegenerative diseases, such as Creutzfeldt-Jakob, Parkinson’s, and Alzheimer’s diseases (Kabani and Melki 2016; Kabani 2020, 2021). EVs were shown to contribute to the intracerebral

Extracellular Vesicles and the Propagation of Yeast Prions

63

spreading of the causing agents of these diseases, namely neuropathological protein aggregates formed by the prion protein PrP, α-synuclein or Tau, respectively, (Fevrier et al. 2004; Alvarez-Erviti et al. 2011; Rajendran et al. 2014; Dujardin et al. 2014; Minakaki et al. 2018). Fundamental cellular mechanisms controlling protein synthesis, folding, degradation, and trafficking are conserved from yeast to humans (Tuite 2019; Dhakal and Macreadie 2020; Kabani 2021; Ishikawa 2021). This is exemplified by recent findings showing that aggregated Sup35p prion domains (Sup35NM) heterologously expressed in mammalian cells are released and traffic from cell-to-cell via EVs (Liu et al. 2016). Therefore, yeasts constitute cost-effective models to investigate the mechanisms and molecular determinants regulating the export of neuropathological prion-like protein aggregates in EVs (Kabani 2020, 2021). Note This chapter is an updated and shortened version of a recently published review (Kabani 2021). Funding MK is supported by the Centre National de la Recherche Scientifique (CNRS). Conflicts of Interest The author has no conflict of interest.

References Aigle M, Lacroute F (1975) Genetical aspects of [URE3], a non-mitochondrial, cytoplasmically inherited mutation in yeast. MGG. Mol Gen Genet 136:327–335. https://doi.org/10.1007/ BF00341717 Alibhoy AA, Giardina BJ, Dunton DD, Chiang HL (2012) Vps34p is required for the decline of extracellular fructose-1,6- bisphosphatase in the vacuole import and degradation pathway. J Biol Chem 287:33080–33093. https://doi.org/10.1074/jbc.M112.360412 Alvarez-Erviti L, Seow Y, Schapira AH, Gardiner C, Sargent IL, Wood MJA, Cooper JM (2011) Lysosomal dysfunction increases exosome-mediated alpha-synuclein release and transmission. Neurobiol Dis 42:360–367. https://doi.org/10.1016/j.nbd.2011.01.029 Bagriantsev SN, Gracheva EO, Richmond JE, Liebman SW (2008) Variant-specific [PSI+] infection is transmitted by Sup35 polymers within [PSI+] aggregates with heterogeneous protein composition. Mol Biol Cell 19:2433–2443. https://doi.org/10.1091/mbc.E08-01-0078 Bhavsar-Jog YP, Bi E (2017) Mechanics and regulation of cytokinesis in budding yeast. Semin Cell Dev Biol 66:107–118 Brachmann A, Baxa U, Wickner RB (2005) Prion generation in vitro: amyloid of Ure2p is infectious. EMBO J 24:3082–3092. https://doi.org/10.1038/sj.emboj.7600772 Byrne LJ, Cole DJ, Cox BS, Ridout MS, Morgan BJT, Tuite MF (2009) The number and transmission of [PSI+] prion seeds (propagons) in the yeast Saccharomyces cerevisiae. PLoS One 4:e4670. https://doi.org/10.1371/journal.pone.0004670 Chernova TA, Wilkinson KD, Chernoff YO (2017) Prions, chaperones, and proteostasis in yeast. Cold Spring Harb Perspect Biol 9. https://doi.org/10.1101/cshperspect.a023663 Coustou V, Deleu C, Saupe S, Begueret J (1997) The protein product of the het-s heterokaryon incompatibility gene of the fungus Podospora anserina behaves as a prion analog. Proc Natl Acad Sci U S A 94:9773–9778

64

M. Kabani

Cox BS (1965) PSI, a cytoplasmic suppressor of super-supressor in yeast. Heredity (Edinb) 20:505– 521 Cox B, Ness F, Tuite M (2003) Analysis of the generation and segregation of propagons: entities that propagate the [PSI+] prion in yeast. Genetics 165:23–33 Derdowski A, Sindi SS, Klaips CL, DiSalvo S, Serio TR (2010) A size threshold limits prion transmission and establishes phenotypic diversity. Science (80- ) 330:680–683. https://doi.org/ 10.1126/science.1197785 Derkatch IL, Chernoff YO, Kushnirov VV, Inge-Vechtomov SG, Liebman SW (1996) Genesis and variability of [PSI] prion factors in Saccharomyces cerevisiae. Genetics 144:1375–1386 Dhakal S, Macreadie I (2020) Protein homeostasis networks and the use of yeast to guide interventions in alzheimer’s disease. Int J Mol Sci 21:1–45 Dorweiler JE, Oddo MJ, Lyke DR, Reilly JA, Wisniewski BT, Davis EE, Kuborn AM, Merrill SJ, Manogaran AL (2020) The actin cytoskeletal network plays a role in yeast prion transmission and contributes to prion stability. Mol Microbiol. https://doi.org/10.1111/mmi.14528 Dujardin S, Bégard S, Caillierez R, Lachaud C, Delattre L, Carrier S, Loyens A, Galas MC, Bousset L, Melki R, Aurégan G, Hantraye P, Brouillet E, Buée L, Colin M (2014) Ectosomes: a new mechanism for non-exosomal secretion of tau protein. PLoS One 9. https://doi.org/10. 1371/journal.pone.0100760 Fevrier B, Vilette D, Archer F, Loew D, Faigle W, Vidal M, Laude H, Raposo G (2004) Cells release prions in association with exosomes. Proc Natl Acad Sci U S A 101:9683–9688. https:// doi.org/10.1073/pnas.0308413101 Ganusova EE, Ozolins LN, Bhagat S, Newnam GP, Wegrzyn RD, Sherman MY, Chernoff YO (2006) Modulation of prion formation, aggregation, and toxicity by the actin cytoskeleton in yeast. Mol Cell Biol 26:617–629. https://doi.org/10.1128/MCB.26.2.617-629.2006 Giardina BJ, Stanley BA, Chiang HL (2014a) Glucose induces rapid changes in the secretome of Saccharomyces cerevisiae. Proteome Sci 12(9). https://doi.org/10.1186/1477-5956-12-9 Giardina BJ, Stein K, Chiang H-L (2014b) The endocytosis gene END3 is essential for the glucoseinduced rapid decline of small vesicles in the extracellular fraction in Saccharomyces cerevisiae. J Extracell vesicles 3:1–11. https://doi.org/10.3402/jev.v3.23497 Glover JR, Kowal AS, Schirmer EC, Patino MM, Liu JJ, Lindquist S (1997) Self-seeded fibers formed by Sup35, the protein determinant of [PSI+], a heritable prion-like factor of S. cerevisiae. Cell 89:811–819 Higuchi-Sanabria R, Pernice WMA, Vevea JD, Alessi Wolken DM, Boldogh IR, Pon LA (2014) Role of asymmetric cell division in lifespan control in Saccharomyces cerevisiae. FEMS Yeast Res 14:1133–1146. https://doi.org/10.1111/1567-1364.12216 Hill SM, Hao X, Grönvall J, Spikings-Nordby S, Widlund PO, Amen T, Jörhov A, Josefson R, Kaganovich D, Liu B, Nyström T (2016) Asymmetric inheritance of aggregated proteins and age reset in yeast are regulated by Vac17-dependent vacuolar functions. Cell Rep 16:826–838. https://doi.org/10.1016/j.celrep.2016.06.016 Hill SM, Hanzén S, Nyström T (2017) Restricted access: spatial sequestration of damaged proteins during stress and aging. EMBO Rep 18:377–391. https://doi.org/10.15252/embr.201643458 Huang PH, Chiang HL (1997) Identification of novel vesicles in the cytosol to vacuole protein degradation pathway. J Cell Biol 136:803–810. https://doi.org/10.1083/jcb.136.4.803 Huang VJ, Stein KC, True HL (2013) Spontaneous variants of the [RNQ+] prion in yeast demonstrate the extensive conformational diversity possible with prion proteins. PLoS One 8: e79582. https://doi.org/10.1371/journal.pone.0079582 Ishikawa T (2021) Saccharomyces cerevisiae in neuroscience: how unicellular organism helps to better understand prion protein? Neural Regen Res 16:489–495 Juanes MA, Piatti S (2016) The final cut: cell polarity meets cytokinesis at the bud neck in S. cerevisiae. Cell Mol Life Sci 73:3115–3136 Kabani M (2020) Hiding in plain sight: vesicle-mediated export and transmission of prion-like proteins. Microb Cell 7:199–201. https://doi.org/10.1111/mmi.14515

Extracellular Vesicles and the Propagation of Yeast Prions

65

Kabani M (2021) Extracellular vesicles-encapsulated yeast prions and what they can tell us about the physical nature of propagons. Int J Mol Sci 22:1–13 Kabani M, Melki R (2011) Yeast prions assembly and propagation: contributions of the prion and non-prion moieties and the nature of assemblies. Prion 5:277–284. https://doi.org/10.4161/pri.5. 4.18070 Kabani M, Melki R (2015) Sup35p in its soluble and prion states is packaged inside extracellular vesicles. MBio 6. https://doi.org/10.1128/mBio.01017-15 Kabani M, Melki R (2016) More than just trash bins? Potential roles for extracellular vesicles in the vertical and horizontal transmission of yeast prions. Curr Genet 62. https://doi.org/10.1007/ s00294-015-0534-6 Kabani M, Cosnier B, Bousset L, Rousset JP, Melki R, Fabret C (2011) A mutation within the C-terminal domain of Sup35p that affects [PSI+] prion propagation. Mol Microbiol 81:640– 658. https://doi.org/10.1111/j.1365-2958.2011.07719.x Kabani M, Redeker V, Melki R (2014) A role for the proteasome in the turnover of sup35p and in [PSI+] prion propagation. Mol Microbiol 92:507–528. https://doi.org/10.1111/mmi.12572 Kabani M, Pilard M, Melki R (2020) Glucose availability dictates the export of the soluble and prion forms of Sup35p via periplasmic or extracellular vesicles. Mol Microbiol. https://doi.org/ 10.1111/mmi.14515 King CY, Diaz-Avalos R (2004) Protein-only transmission of three yeast prion strains. Nature 428: 319–323. https://doi.org/10.1038/nature02391 Knoblach B, Rachubinski RA (2015) Sharing the cell’s bounty - organelle inheritance in yeast. J Cell Sci 128:621–630 Krzewska J, Melki R (2006) Molecular chaperones and the assembly of the prion Sup35p, an in vitro study. EMBO J 25:822–833. https://doi.org/10.1038/sj.emboj.7600985 Liebman SW, Chernoff YO (2012) Prions in yeast. Genetics 191:1041–1072. https://doi.org/10. 1534/genetics.111.137760 Liu S, Hossinger A, Hofmann JP, Denner P, Vorberg IM (2016) Horizontal transmission of cytosolic sup35 prions by extracellular vesicles. MBio 7. https://doi.org/10.1128/mBio. 00915-16 Manogaran AL, Hong JY, Hufana J, Tyedmers J, Lindquist S, Liebman SW (2011) Prion formation and polyglutamine aggregation are controlled by two classes of genes. PLoS Genet 7:e1001386. https://doi.org/10.1371/journal.pgen.1001386 McGlinchey RP, Kryndushkin D, Wickner RB (2011) Suicidal [PSI+] is a lethal yeast prion. Proc Natl Acad Sci U S A 108:5337–5341. https://doi.org/10.1073/pnas.1102762108 Minakaki G, Menges S, Kittel A, Emmanouilidou E, Schaeffner I, Barkovits K, Bergmann A, Rockenstein E, Adame A, Marxreiter F, Mollenhauer B, Galasko D, Buzás EI, SchlötzerSchrehardt U, Marcus K, Xiang W, Lie DC, Vekrellis K, Masliah E, Winkler J, Klucken J (2018) Autophagy inhibition promotes SNCA/alpha-synuclein release and transfer via extracellular vesicles with a hybrid autophagosome-exosome-like phenotype. Autophagy 14:98–119. https://doi.org/10.1080/15548627.2017.1395992 Patel BK, Liebman SW (2007) “Prion-proof” for [PIN+]: infection with in vitro-made amyloid aggregates of Rnq1p-(132-405) induces [PIN+]. J Mol Biol 365:773–782. https://doi.org/10. 1016/j.jmb.2006.10.069 Patino MM, Liu JJ, Glover JR, Lindquist S (1996) Support for the prion hypothesis for inheritance of a phenotypic trait in yeast. Science (80- ) 273:622–626 Prusiner SB (1982) Novel proteinaceous infectious particles cause scrapie. Science (80- ) 216:136– 144 Pruyne D, Legesse-Miller A, Gao L, Dong Y, Bretscher A (2004) Mechanisms of polarized growth and organelle segregation in yeast. Annu Rev Cell Dev Biol 20:559–591 Rajendran L, Bali J, Barr MM, Court FA, Kramer-Albers E-M, Picou F, Raposo G, van der Vos KE, van Niel G, Wang J, Breakefield XO (2014) Emerging roles of extracellular vesicles in the nervous system. J Neurosci 34:15482–15489. https://doi.org/10.1523/JNEUROSCI.3258-14. 2014

66

M. Kabani

Schlumpberger M, Prusiner SB, Herskowitz I (2001) Induction of distinct [URE3] yeast prion strains. Mol Cell Biol 21:7035–7046. https://doi.org/10.1128/MCB.21.20.7035-7046.2001 Sentandreu R, Northcote DH (1969) The formation of buds in yeast. J Gen Microbiol 55:393–398. https://doi.org/10.1099/00221287-55-3-393 Sharma D, Martineau CN, Le Dall MT, Reidy M, Masison DC, Kabani M (2009) Function of SSA subfamily of Hsp70 within and across species varies widely in complementing Saccharomyces cerevisiae cell growth and prion propagation. PLoS One 4. https://doi.org/10.1371/journal.pone. 0006644 Sindi SS, Serio TR (2009) Prion dynamics and the quest for the genetic determinant in protein-only inheritance. Curr Opin Microbiol 12:623–630. https://doi.org/10.1016/j.mib.2009.09.003 Sontag EM, Samant RS, Frydman J (2017) Mechanisms and functions of spatial protein quality control. Annu Rev Biochem 86:97–122. https://doi.org/10.1146/annurev-biochem060815-014616 Speldewinde SH, Doronina VA, Tuite MF, Grant CM (2017) Disrupting the cortical actin cytoskeleton points to two distinct mechanisms of yeast [PSI+] prion formation. PLoS Genet 13. https://doi.org/10.1371/journal.pgen.1006708 Tanaka M, Chien P, Naber N, Cooke R, Weissman JS (2004) Conformational variations in an infectious protein determine prion strain differences. Nature 428:323–328. https://doi.org/10. 1038/nature02392 Tanaka M, Collins SR, Toyama BH, Weissman JS (2006) The physical basis of how prion conformations determine strain phenotypes. Nature 442:585–589. https://doi.org/10.1038/ nature04922 Taylor KL, Cheng N, Williams RW, Steven AC, Wickner RB (1999) Prion domain initiation of amyloid formation in vitro from native Ure2p. Science (80- ) 283:1339–1343 Thual C, Komar AA, Bousset L, Fernandez-Bellot E, Cullin C, Melki R (1999) Structural characterization of Saccharomyces cerevisiae prion-like protein Ure2. J Biol Chem 274: 13666–13674 Tuite MF (2019) Yeast models of neurodegenerative diseases. In: Progress in molecular biology and translational science, vol 168. Elsevier, Amsterdam, pp 351–379 Villali J, Dark J, Brechtel TM, Pei F, Sindi SS, Serio TR (2020) Nucleation seed size determines amyloid clearance and establishes a barrier to prion appearance in yeast. Nat Struct Mol Biol 27: 540–549. https://doi.org/10.1038/s41594-020-0416-6 Wang K, Melki R, Kabani M (2017) A prolonged chronological lifespan is an unexpected benefit of the [PSI+] prion in yeast. PLoS One 12:e0184905. https://doi.org/10.1371/journal.pone. 0184905 Wang K, Melki R, Kabani M (2019) Growth phase-dependent changes in the size and infectivity of SDS-resistant Sup35p assemblies associated with the [PSI+] prion in yeast. Mol Microbiol 112. https://doi.org/10.1111/mmi.14329 Wickner RB (1994) [URE3] as an altered URE2 protein: evidence for a prion analog in Saccharomyces cerevisiae. Science (80- ) 264:566–569 Wickner RB, Edskes HK, Son M, Wu S, Niznikiewicz M (2020) How do yeast cells contend with prions? Int J Mol Sci 21:4742. https://doi.org/10.3390/ijms21134742 Winkler J, Tyedmers J, Bukau B, Mogk A (2012) Chaperone networks in protein disaggregation and prion propagation. J Struct Biol 179:152–160. https://doi.org/10.1016/j.jsb.2012.05.002 Winters CM, Hong-Brown LQ, Chiang HL (2020) Intracellular vesicle clusters are organelles that synthesize extracellular vesicle-associated cargo proteins in yeast. J Biol Chem 295:2650–2663. https://doi.org/10.1074/jbc.RA119.008612 Zhao K, Bleackley M, Chisanga D, Gangoda L, Fonseka P, Liem M, Kalra H, Al Saffar H, Keerthikumar S, Ang CS, Adda CG, Jiang L, Yap K, Poon IK, Lock P, Bulone V, Anderson M, Mathivanan S (2019) Extracellular vesicles secreted by Saccharomyces cerevisiae are involved in cell wall remodelling. Commun Biol. https://doi.org/10.1038/s42003-0190538-8

Contributions of Extracellular Vesicles to Fungal Biofilm Pathogenesis Marienela Heredia and David Andes

Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 EV Function in Bacterial Biofilms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Fungal Extracellular Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Role of C. albicans EVs in Matrix Biogenesis and Drug Resistance . . . . . . . . . . . . . . . . . . . . . . . 5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

68 68 70 72 74 75

Abstract Extracellular vesicles (EVs) are produced by all kingdoms of life and have been increasingly recognized as a key aspect of microbial pathogenicity. These membrane-bound compartments serve as secretory vehicles for the delivery of macromolecules to the extracellular environment. Studies over the past several decades have revealed that microbial EVs are highly suited to the biology and environmental context of the organism secreting them. Fungal EVs have been described in at least 12 species and have diverse functions. These functions include, but are not limited to, molecular transport across the cell wall, immunomodulation, cell-cell communication, export of virulence factors and nucleic acids, extracellular matrix (ECM) production, and induction of drug resistance. This chapter will explore the contributions of EVs to fungal pathogenesis and virulence, with a detailed focus on the role of C. albicans biofilm EVs in matrix biogenesis and antifungal resistance. Brief commentary on EV function in bacterial biofilms will also be provided for comparison, and suggestions for areas of future investigation in this field will be discussed.

M. Heredia · D. Andes (*) Departments of Medicine and Medical Microbiology and Immunology, University of Wisconsin, Madison, WI, USA e-mail: [email protected] © Springer Nature Switzerland AG 2021 M. Rodrigues, G. Janbon (eds.), Fungal Extracellular Vesicles, Current Topics in Microbiology and Immunology 432, https://doi.org/10.1007/978-3-030-83391-6_7

67

68

M. Heredia and D. Andes

1 Introduction The biofilm lifestyle has been widely recognized as a major aspect of microbial life and pathogenicity. First observed in bacterial populations, biofilms consist of matrixenclosed microbes that aggregate and adhere to biotic or abiotic surfaces (HallStoodley et al. 2004; Sharma et al. 2019). In the case of pathogens, this mode of community growth enhances tolerance to antimicrobial drugs and thus, poses a major challenge for the development of targeted therapeutics (Koo et al. 2017; Sharma et al. 2019). While biofilm drug resistance is multifaceted, the extracellular matrix (ECM) of biofilm-forming pathogens plays a key role by shielding biofilm cells from a myriad of environmental insults (Sharma et al. 2019). The impact of the ECM on biofilm formation has been widely investigated in both bacterial and fungal pathogens, and their macromolecular components have been extensively characterized. The observation of a lipid component within the ECM led to the discovery of extracellular vesicles (EVs), whose biochemical properties and functions are as diverse as the microbes producing them. EVs are nanosized, membrane-bound compartments responsible for carrying and delivering macromolecular cargo into the extracellular space (Zaborowski et al. 2015; Abels and Breakefield 2016). They are produced across all kingdoms of life and have been characterized in numerous model systems by their size, cargo, and mechanisms of biogenesis (Juan and Fürthauer 2018). EVs have been implicated in cell-cell communication, signal transduction, quorum sensing, and host-pathogen interactions. EVs are specifically suited to the biology and environmental context of the organism secreting them, resulting in diverse, species-dependent functions (Zarnowski et al. 2018). This chapter will review general EV contribution to fungal pathogenesis, with a major focus on the role of EVs in biofilm matrix biogenesis and drug resistance in the most common fungal pathogen, C. albicans. Brief coverage on EV function in bacterial biofilms will be provided for comparison.

2 EV Function in Bacterial Biofilms First identified in Escherichia coli in the 1960s, EVs have been well characterized in gram-negative bacteria (Bishop and Work 1965, Knox et al. 1966; Work et al. 1966; Knox et al. 1967). These EVs, called outer membrane vesicles (OMVs), originate in the outer membrane and serve primarily as secretory vehicles (Brown et al. 2015; Gill et al. 2019). OMVs carry a variety of cargo crucial to pathogenicity, including virulence factors, immunomodulatory factors, toxins, nutrient-scavenging factors, and nucleic acids (Brown et al. 2015; Gill et al. 2019). OMVs were first suggested to form part of biofilm matrices by Schooling and Beveridge and have since been implicated in both matrix accumulation and biofilm production in various pathogenic and nonpathogenic species (Schooling and Beveridge 2006). For example, OMV secretion has been shown to play a crucial role in the ECM formation of Helicobacter pylori strain TK1402 biofilms (Yonezawa et al. 2009). Additionally,

Contributions of Extracellular Vesicles to Fungal Biofilm Pathogenesis

69

OMVs secreted by the soil bacterium Pseudomonas putida under varying environmental stress conditions cause an increase in cell surface hydrophobicity, leading to enhanced biofilm formation (Baumgarten et al. 2012). In its pathogenic relative, Pseudomonas aeruginosa, OMVs were found to be a major constituent of the biofilm ECM (Toyofuku et al. 2012). In this same study, the authors observed that approximately 30% of biofilm matrix proteins were outer membrane proteins also found in OMVs, suggesting that they may be responsible for the delivery of matrix components to the ECM. It was first proposed that the mechanism behind P. aeruginosa biofilm OMV production and matrix component release was via explosive cell lysis (Turnbull et al. 2016). However, Cooke and colleagues recently reported that biogenesis via explosive cell lysis only accounts for a small, unique population of P. aeruginosa biofilm OMVs; rather, biogenesis of these vesicles is largely dependent on the Pseudomonas Quinolone Signal (PQS), which was previously believed to only contribute to the production of P. aeruginosa planktonic OMVs (Cooke et al. 2019). In contrast to OMVs secreted by gram-negative bacteria, EVs were not studied in gram-positive bacteria or mycobacteria until more recently due to the interference of thicker, more complex cell walls surrounding these microbes (Brown et al. 2015). As a result, EV function within these bacterial biofilms has not been well characterized. The first study to characterize EVs in mycobacterial biofilms was conducted by Marsollier and colleagues, who first observed small vesicles embedded within the biofilm ECM of Mycobacterium ulcerans, the causative agent of Buruli ulcer in humans. They found that the toxin mycolactone, a key virulence factor in the pathogenesis of M. ulcerans, is exported out into the biofilm ECM by EVs (Marsollier et al. 2007). In Streptococcus mutans, a major cause of human dental caries, extracellular DNA (eDNA) was found to be exported via lysis-independent membrane vesicles, among other modes of release (Liao et al. 2014). Notably, eDNA produced by S. mutans biofilms was significantly higher than that produced by planktonic cells, and depletion of eDNA with DNAse I abrogated biofilm formation (Liao et al. 2014). Recently, EVs produced by the opportunistic pathogen, Staphylococcus aureus, were reported to influence mixed bacterial communities, specifically by inhibiting the adhesion and growth of other bacterial species on polystyrene surfaces (Im et al. 2017). The involvement of EVs in bacterial pathogenesis has made them attractive vaccine platforms for disease prevention, and several studies have been conducted using planktonic bacterial EVs to further explore this possibility (Tan et al. 2018; Fingermann et al. 2018). For example, mycobacterial EVs administered systemically in mice were found to induce a protective immune response against M. tuberculosis infection (Prados-Rosales et al. 2014). Additionally, Wang and colleagues discovered that EVs secreted by a community-associated methicillin-resistant S. aureus (MRSA) mutant strain expressing attenuated cytolysins are immunogenic in murine models and protective against MRSA infection (Wang et al. 2018). Future work in this area may take a similar approach by establishing the immunogenic potential of biofilm EVs and exploring their capacity for preventing biofilm-associated bacterial infections.

70

M. Heredia and D. Andes

3 Fungal Extracellular Vesicles The existence of fungal EVs was first proposed in 1973 in the fungal pathogen, Cryptococcus neoformans (Takeo et al. 1973). Several key investigations over the years have been conducted to characterize fungal EVs with the goal of better understanding how they contribute to pathogenesis and virulence. Over time, basic aspects of fungal EVs, such as composition and immunobiological effects, have been examined in numerous fungal pathogens, including Histoplasma capsulatum, C. albicans, Paracocciodiodes brasiliensis, Aspergillus fumigatus, and Malassezia sympodialis (Albuquerque et al. 2008; Vargas et al. 2015; Vallejo et al. 2011, 2012a, b; Souza et al. 2019; Gehrmann et al. 2011). However, the contributions of these EVs in the context of fungal biofilms had yet to be considered. The first major investigation of fungal EVs was conducted by the Casadevall group over a decade ago in C. neoformans. In this study, C. neoformans EVs were found to carry glucuronoxylomannan (GXM), a key component of the cryptococcal polysaccharide capsule shed into the ECM, as well as other key lipids (Rodrigues et al. 2007). GXM is required for C. neoformans biofilm formation and virulence (Martinez and Casadevall 2005); therefore, this finding highlights the importance of EVs in C. neoformans pathogenesis and lends to the idea that EV cargo may, in many cases, include matrix components. Further studies in C. neoformans identified more cargo proteins, including some with functions in virulence and protection against oxidative stress, and revealed that protein composition does not significantly differ with size or mode of release (Rodrigues et al. 2008; Wolf et al. 2014). C. neoformans EVs were further linked to virulence in a genetic study by Panepinto and colleagues; upon RNAi knockdown of the exocyst complex component, Sec6, defects in EV-mediated secretion of virulence factors led to attenuated virulence (Panepinto et al. 2009). These factors include those required for laccase, urease, and polysaccharide secretion. EV contribution to C. neoformans pathogenesis was further supported by the discovery that microvesicles, a class of EVs, facilitate cryptococcal traversal of the blood-brain barrier (BBB) and accumulate at sites of cystic lesions in a murine model of cryptococcal meningoencephalitis (Huang et al. 2012). EVs from the Cryptococcus spp., C. gattii, have also been isolated and studied in the context of infection. A study by Bielska and colleagues found that the proliferation of individual cells within host phagocytes is mediated by the release and exchange of EVs by C. gattii. These EVs are taken up by infected host phagocytes and are trafficked to the phagosome of ingested cryptococci, where they trigger rapid proliferation of the ingested cell. Additionally, EV protein and RNA cargo are necessary for the induction of rapid proliferation within host macrophages (Bielska et al. 2018). These findings implicate C. gattii EVs in longdistance message relay and delivery of virulence factors required for pathogenesis. Taken together, these key studies in C. neoformans highlight the importance of studying EV secretion and cargo on a molecular level to better understand how EVs contribute to fungal infection.

Contributions of Extracellular Vesicles to Fungal Biofilm Pathogenesis

71

In addition to C. neoformans vesicles, EVs from other fungal pathogens have also been studied. Albuquerque and colleagues confirmed the production of EVs in H. capsulatum, C. albicans, Candida parapsilosis, Sporothrix schenckii, and Saccharomyces cerevisiae (Albuquerque et al. 2008). Using lipidomic and proteomic approaches to further examine H. capsulatum EVs, the authors discovered diverse macromolecular cargo involved in key processes, such as metabolism, cell recycling, virulence, and stress signaling. In addition to their EV composition analyses, the authors demonstrated that H. capsulatum EVs are immunogenic and associated with pathogenesis, as they react with sera from patients with histoplasmosis (Albuquerque et al. 2008). Recent investigations revealed that both the innate immune system and the extracellular environment affect H. capsulatum EV content and characteristics. Treatment of H. capsulatum cells with heat shock protein Hsp60binding monoclonal antibodies alters vesicle size and protein cargo in an antibody concentration-dependent manner; further, these differences in EV loading modulated by opsonization can potentially impact susceptibility to innate immune defenses (Matos Baltazar et al. 2016; Baltazar et al. 2018). Differences in environmental conditions, such as pH, oxygen concentrations, and nutrient availability also modulate EV cargo loading and secretion; EVs isolated from H. capsulatum cultured in three different types of media demonstrated marked differences in their protein, lipid, and carbohydrate metabolite profiles (Cleare et al. 2020). The first C. albicans EV proteome analyses were conducted by Gil-Bona and colleagues, who observed enrichment in EV-associated proteins with the cell wall and pathogenesis functions (Gil-Bona et al. 2015). The following year, a more comprehensive EV composition study was reported by the Nimrichter group using planktonic cells (Vargas et al. 2015). Proteomic analyses revealed EV proteins with functions in pathogenesis, cell organization, lipid and carbohydrate metabolism, and stress response, among other categories. Lipid analyses detected the presence of ergosterol, lanosterol, and glucosylceramide as major EV components. This same study also established the immunogenicity of C. albicans EVs and showcased their potential as a useful therapeutic platform (Vargas et al. 2015). Recently, stressinduced C albicans EVs produced by both model strain ATCC 90028 and clinical vulvovaginal candidiasis (VVC) isolate were reported to carry 34 proteins linked to pathogenesis and virulence (Konečná et al. 2019). This finding further showcases the importance of EVs in the context of fungal infection, particularly via the delivery of virulence-associated protein cargo. In addition to C. albicans, proteomic analyses have recently been conducted in the clinically relevant non-albicans species, C. parapsilosis, Candida glabrata, and Candida tropicalis (Karkowska-Kuleta et al. 2020). In addition to pathogenic fungi, EVs have also been studied in the nonpathogenic yeast, S. cerevisiae. The initial discovery of EVs produced by S. cerevisiae demonstrated that fungal EVs do not solely contribute to pathogenesis; rather, they are also required for other key aspects of fungal biology (Albuquerque et al. 2008). Investigations into the secretory mechanisms governing S. cerevisiae vesiculogenesis revealed that EV secretion is mediated by both conventional Golgi-derived exocytosis and multivesicular body (MVB) formation (Oliveira et al. 2010). While further

72

M. Heredia and D. Andes

investigation is required to understand how EVs function in S. cerevisiae biofilms, vesicle secretion has recently been linked to cell wall remodeling and drug resistance (Zhao et al. 2019). Deletion of key cell wall biosynthesis genes led to increased production of EVs containing the cell wall remodeling proteins, Fks1 and Chs3. Interestingly, these same EVs exhibited a protective effect over yeast cells against the antifungal drug caspofungin. These findings lend to the idea that EVs may contribute to the maintenance of cell wall integrity, especially in the presence of cell wall damaging agents. Besides carrying proteins, carbohydrates, and lipids, fungal EVs have also been shown to carry nucleic acids to the extracellular space. Specifically, H. capsulatum, C. neoformans, S. cerevisiae, and C. albicans EVs have been reported to export fungal RNA (Alves et al. 2019; Peres da Silva et al. 2015). The functions of these RNA cargoes are still unknown, as they have not been well studied in any model fungus. It has been suggested that these RNA molecules may function in cell-cell signaling as they do in mammalian systems (Peres da Silva et al. 2015; O’Brien et al. 2020); therefore, the study of extracellular RNA in the context of fungal infection may constitute a novel and exciting area for future investigations.

4 Role of C. albicans EVs in Matrix Biogenesis and Drug Resistance The first major investigation aimed at characterizing fungal biofilm EVs was conducted by Zarnowski and colleagues, who previously observed that the C. albicans ECM contained a significant amount of phospholipid content (Zarnowski et al. 2014). To further explain this observation, they observed