Neisseriae 1990: Proceedings of the Seventh International Pathogenic Neisseriae Conference, Berlin, Federal Republic of Germany, September 9–14, 1990 9783110867787, 9783110127126

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Neisseriae 1990

Neisseriae 1990 Proceedings of the Seventh International Pathogenic Neisseria Conference Berlin, Federal Republic of Germany September 9 -14,1990

Editors Mark Achtman • Peter Kohl • Christian Marchal Giovanna Morelli • Andrea Seiler • Burghard Thiesen

W DE

G Walter de Gruyter • Berlin • New York 1991

Editors Mark Achtman, Ph. D. Giovanna Morelli, Dr. rer. nat. Burghard Thiesen, Dr. vet. med. Max-Planck-Institut für molekulare Genetik Ihnestraße 73 D-1000 Berlin 33 Fed. Rep. Germany

Peter Kohl, Dr. med. Ruprecht-Karls-Universität Voßstraße 2 D-6900 Heidelberg Fed. Rep. of Germany Christian Marchai, M. D., Ph. D. Unité des Antigènes Bactériens Institut Pasteur 28 rue du Docteur-Roux F-75724 Paris Cedex 15 France

CIP-Kurztitelaufnahme der Deutschen Bibliothek Neisseriae 1990 : proceedings of the Seventh International Pathogenic Neisseria Conference, Berlin, Federal Republic of Germany, September 9-14,1990 / ed. Mark Achtman Berlin ; New York : de Gruyter, 1991 ISBN 3-11-012712-1 NE: Achtman, Mark [Hrsg.]; International Pathogenic Neisseria Conference

Library of Congress Cataloging-in-Publication Data International Pathogenic Neisseriae Conference (7th : 1990 : Berlin, Germany) Neisseriae 1990 : proceedings of the Seventh International Pathogenic Neisseria Conference, Berlin, Federal Republic of Germany. September 9-14,1990 / editors Mark Achtman... [et al.]. p. cm. Includes bibliographical references and index. ISBN 0-89925-866-2 (U. S.) 1. Neisseria infections-Congresses. I. Achtman, Mark. II. Title. QR201.N45I58 1990 91-3426 616.9'2-dc20 CIP

© Printed on acid free paper which falls within the guidelines of the AN SI to ensure permanence and durability. Copyright © 1991 by Walter de Gruyter& Co., D-1000 Berlin 30. All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced in any form - by photoprint, microfilm or any other means - nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Gerike GmbH, Berlin. Binding: Luderitz & Bauer GmbH, Berlin. - Printed in Germany.

Preface The 7 th International Pathogenic Neisseria Conference was held in Berlin from the 9 th - 14th of September, 1990. It followed former conferences in this series held in (year; organizer): San Francisco (1978; G. Brooks)(l), limed (1980; S. Normark), Montreal (1982; I. W. deVoe), Asilomar (1984; G. Schoolnik)(2), Noordwijkerhout, the Netherlands (1986, J. T. Poolman)(3) and Atlanta (1988, S. Morse)(4). The 8 th will be held in Mexico in 19921. 230 scientists from numerous countries presented 38 talks of 30 minutes and 140 posters at the Berlin meeting. The general topic was the current state of research on pathogenic Neisseriae with a few additions from microbiological analyses of other organisms. The individual sessions revolved around the topics of phase and antigenic variation, the bacterial cell surface, molecular genetic aspects, epidemiology and vaccines, immunogenicity

and

pathogen-host interactions. In addition, there was the traditional Thursday evening party which apparently was a great success, as usual. I must confess that I was so tired from organizing the meeting that I did not have the energy to read all the posters nor was I as attentive as I might have been at all the talks. Thus, for me (and probably for other participants), this book represents a possibility to study the science presented at the meeting in greater detail. In addition, I hope that it can serve as a compact introduction to the topics of current interest in this field for junior scientists just beginning their career and senior scientists coming from a different field 2 . Scientists who did not attend the meeting will not readily be able to reconstruct which articles were presented as talks and which as posters. In my opinion, some posters were more exciting than certain talks and the general level of science was very high. In addition, this book includes several summary chapters by chairmen of individual sessions containing an overview of and introduction to other articles. A few of the presentations given at the meeting are not included. Therefore, there has been no attempt to preserve the organization of the meeting and this volume is presented as an independent effort arising from the meeting but which must stand on its own merits.

'contact Dr. Carlos J. Conde-Glez, Centro de Investigaciones Sobre Enfermedades Infecciosas, Inst. Nacional de Salud Publica, Postal No 222, Officina de Correos No 1, Cuernavaca, Morelos, C.P.62000, Mexico for further information 2 I remember well how much the books arising from the former meetings helped me when I joined this field in 1982

VI

This book is based on photo offset reproduction of camera-ready manuscripts. I decided to use this form of publication rather than type-setting in order to ensure quick publication and reduce costs. That decision then necessitated that the editorial work be performed by the (non-professional) editors, to whom I express my deepest thanks. For some manuscripts from countries where the postal communication is not always unproblematical, I rewrote the manuscripts in order to improve the style and English grammar. I hope that I did not introduce any grave mistakes in the process; the alternative would have been a very long delay in publication to ensure obtaining clean copies. Part of the financial costs of publishing this book were defrayed by a generous gift from Hoefer Scientific Instruments, San Francisco. The meeting itself was only possible due to the generous financial support of the Bundesministerium fur Forschung und Technologie, the Deutsche Forschungsgemeinschaft, the MaxPlanck Gesellschaft and the National Institutes of Health (U.S.A.) as well as gifts from various non-governmental sponsors. I am very grateful for all this support. I also look forward to being a participant at future meetings in this series.

Berlin, February 1991

Mark Achtman

References 1. Brooks, G.F., E. C. Gotschlich, K. K. Holmes, W. D. Sawyer and F. E. Young. 1978. Immunobiology of Neisseria gonorrhoeae. American Society for Microbiology, Washington, D.C. pp. 1-400. 2. Schoolnik, G.K., G. F. Brooks, S. Falkow, C. E. Frasch, J. S. Knapp, J. A. McCutchan and S. A. Morse. 1985. The Pathogenic Neisseriae. ASM Press, Washington, D.C. pp. 1-647. 3. Poolman, J.T., H. C. Zanen, T. F. Meyer, J. E. Heckels, P. R. H. Makela, H. Smith and E. C. Beuvery. 1988. Gonococci and Meningococci. Kluwer Academic Publishers, Dordrecht, pp. 1-842. 4. Morse, S.A., J. S. Knapp, C. V. Broome, W. M. Shafer, J. Cannon, P. F. Sparling, M. Cohen and D. Stephens. 1989. Clinical Microbiology Reviews, Supplement: Perspectives on Pathogenic Neisseriae. ASM Press, Washington, DC. pp. SlS149.

Table of Contents

Epidemiology and Vaccines Epidemiology and vaccines Jones, D. M

3

Properties and epidemiology of 2 epidemic clones of serogroup A Neisseria meningitidis associated with African epidemics since 1980 Achtman, M., Morelli, G., Kusecek, B., Bopp, M., Wang, J. and Caugant, D. A

5

Serotyping of Neisseria meningitidis group B strains in China with monoclonal antibodies by whole cell ELISA Bai, X. Y., Wang, L. Y., Huang, W. X. and Liu, D. Z

11

Group A meningococcus: Epidemiology and development of a protein polysaccharide conjugate vaccine Broome, C. V

17

The resolution of clonal types of Neisseria meningitidis by pulsed field gel electrophoresis Bygraves, J. A. and Maiden, M. C. J

25

A three-year survey of Neisseria gonorrhoeae in Mexico Conde-Glez, C. J., Calderón, E., Náder, E. and Mondragón, V. A

31

Genetic structure and epidemiology of serogroup B Neisseria meningitidis Caugant D. A., Frholm, L. O., Sacchi, C. T. and Selander, R. K

37

Two epidemiological types of meningococcal infection incidence caused by serogroup B meningococci Demina, A. A., Devjatkina, N. P., Martynov, Yu. V., Koroleva, I. S., Molinert, H. T., Novo, M. V., Martines, T. M., and Patton, A. C

43

Prevalence of group A (Clone II-2) Neisseria meningitidis in Scotland Fallon, R. J., Shearer, J. and Thorn, L

49

VIII The use of restriction fragment length polymorphisms for typing Neisseria

meningitidis

Fox, A. J., Jones, D. M., Gray, S. J. and Saunders, N. A

55

Recent meningococcal epidemiology in Norway. Eight years of serotyping for strain characterization. Fr2 fold), but none of the six developed a 2 f o l d or g r e a t e r rise to PIA. T h r e e of the six developed

>2 fold rises against LOSs f r o m PIB strains and f o u r

developed >2 f o l d rises against LOS f r o m the PIA strain.

PIII protein s t i m u l a t e d a

>10 f o l d rise in three of six volunteers i n c l u d i n g 1 who developed a >40 f o l d rise (Fig 2).

Correlation

of PIII antibody

level and bactericidal

activity.

To e v a l u a t e the

possibility that a rise in blocking a n t i b o d y to the PIA resulted f r o m v a c c i n a t i o n , we

A PIII (blocking) Antibody A PI+LOS (bactericidal) Antibody

Fig 3. PIII blocking a n t i b o d y response directed against the PIA strain ( N R L #7122) f o l l o w i n g vaccination. C h a n g e in survival of N. gonorrhoeae is expressed as a f u n c t i o n of the ratio of change in PIII (blocking) a n t i b o d y (IgG) to c h a n g e in PI + LOS (combined [bactericidal] antibodies [IgG]). ^ P I I I / A [PI + LOS]).

233 e x a m i n e d t h e r a t i o of rises in t h e p u t a t i v e b l o c k i n g a n t i b o d y ( P I I I a n t i b o d y ) to r i s e s in t h e p u t a t i v e k i l l i n g a n t i b o d i e s (PI a n d L O S [ c o m b i n e d ] a n t i b o d i e s ) .

We f o u n d t h a t

t h i s r a t i o w a s i n v e r s e l y c o r r e l a t e d w i t h t h e d e g r e e of k i l l i n g b y post v a c c i n a t i o n s e r u m (r=0.84).

T h i s s u g g e s t e d a b l o c k i n g e f f e c t d u e to P I I I a n t i b o d y d e v e l o p m e n t

( F i g 3). T h i s c o r r e l a t i o n w a s not seen w i t h k i l l i n g of P I B s t r a i n s . P r e v i o u s i n v e s t i g a t i o n s (9) h a v e s h o w n t h a t a n t i b o d y d i r e c t e d a g a i n s t P I I I b l o c k s bactericidal activity.

S e l e c t i v e d e p l e t i o n of P I I I a n t i b o d y f r o m n o n k i l l i n g D G I

convalescent serum restores killing activity.

In o r d e r to c o r r o b o r a t e t h e c o r r e l a t i o n s

d e m o n s t r a t e d a b o v e , w e r e m o v e d PIII s p e c i f i c a n t i b o d i e s ( b y a f f i n i t y c h r o m a t o g r a p h y ) f r o m a volunteer's sera whose b a c t e r i c i d a l activity against the PIA strain has decreased 3 fold a f t e r immunization.

In t h e P I I I d e p l e t e d s e r u m (42 d a y s ) , k i l l i n g

w a s r e s t o r e d by 2.5 f o l d . T h e PIII s p e c i f i c a n t i b o d y ( e l u e n t ) was t h e n a d d e d b a c k to t h e PIII d e p l e t e d s e r u m , a n d o n c e a g a i n k i l l i n g w a s r e d u c e d , in t h i s case by 2 f o l d .

Discussion

T h i s v a c c i n e t r i a l h a s s h o w n t h a t PIB a n t i b o d y i n c r e a s e s w e r e s t i m u l a t e d g r e a t e r t h a n 2 - f o l d , in 3 of 6 v o l u n t e e r s . f o r all t h e v o l u n t e e r s .

K i l l i n g a c t i v i t y a g a i n s t t w o PIB s t r a i n s w a s 2 - f o l d or less

H o w e v e r , since PIII a n t i b o d y c o n c e n t r a t i o n s , w h i c h may have

b l o c k e d k i l l i n g , a l s o i n c r e a s e d (> 1 2 - f o l d ) , it is d i f f i c u l t to assess t h e k i l l i n g p o t e n t i a l of P I B a n t i b o d y in t h i s t r i a l .

F u r t h e r m o r e , t h e m o d e s t i n c r e a s e in a n t i b o d i e s to L O S s

f r o m PIB s t r a i n s m a y h a v e also c o n t r i b u t e d to t h e small k i l l i n g e f f e c t seen in t h e s e s e r a , f u r t h e r o b s c u r i n g t h e k i l l i n g e f f e c t of PIB a n t i b o d y . P I A a n t i g e n d i d not s t i m u l a t e a n t i b o d y p r o d u c t i o n .

T h e e f f e c t s of PIII b l o c k i n g

a n t i b o d y w e r e most r e a d i l y a p p a r e n t in a s s a y s t h a t e m p l o y e d t h e P I A s t r a i n .

PIII

blocking antibody may have o f f s e t either baseline bactericidal antibody, bactericidal a n t i b o d y s t i m u l a t e d by L O S , or b o t h .

F u t u r e t e s t i n g w i t h PI p r e p a r a t i o n s , f r e e of

L O S a n d P I I I , w o u l d be n e c e s s a r y in o r d e r to assess t h e b a c t e r i c i d a l p o t e n t i a l of t h i s a n t i g e n as a v a c c i n e c a n d i d a t e .

234 Acknowledgements

This research was supported, in part, by grants AI 19469 (MB), AI 18367 (MB), AI 15633 (PAR) and AI 24760 (PAR) a w a r d e d by t h e N a t i o n a l Institutes of H e a l t h , USA.

References

1.

T e e r l i n k , T., R. Breas, Ron van Eijk, R. Teiesjema, E.C. Beuvery. 1985. In: T h e p a t h o g e n i c neisseriae (G.K. Schoolnik, G.F. Brooks, S. F a l k o w , C.E. F r a s c h , J.S. K n a p p , J.A. M c C u t c h a n , and S.A. Morse eds)., A m e r i c a n Society f o r Microbiology p. 259.

2.

Blake, M.S. 1985. /«: T h e pathogenic neisseriae (G.K. Schoolnik, G.F. Brooks, S. F a l k o w , C.E. Frasch, J.S. K n a p p , J.A. M c C u t c h a n , and S.A. Morse eds)., A m e r i c a n Society f o r Microbiology p. 252.

3.

Hook, E.W. III, D.A. Olsen, T.M. Buchanan.

4.

B u c h a n a n , T.M., J.F. H i l d e r b r a n d t .

5.

K n a p p , J.S., M.R. Tarn, R.C. Nowinski, K.K. Holmes, E.G. Sandstrom. I n f e c t . Dis. 150: 44.

6.

Blake, M.S., E.C. Gotschlich.

7.

Lytton, E.J., M.S. Blake.

8.

Westphal, O., O. L u d e r i t z , F. Bister.

9.

Rice, P.A, H.E. Vayo, M.R. Tam, M.S. Blake.

1984. I n f e c t . I m m u n . 43: 706.

1981. I n f e c t . Immun. 32: 985. 1984. J.

1982. I n f e c t . Immun. 36: 277.

1986. J. Exper. Med. 164: 1749. 1952. N a t u r f o r s c h . B7: 148. 1986. J.Exp.Med. 164: 1735

Immunobiology of Neisseria porin proteins and mapping of epitopes with vaccine potential.

J.E. Heckeis, C.R. Tinsley, N. Butt, B. McGuinness, M. Virji, P.R. Lambden, I.N. Clarke. Department of Microbiology, University of Southampton Medical School, Southampton S09 4XY, UK.

Introduction The porin proteins are the most abundant proteins present on the surface of the pathogenic Neisseria and unlike several other major surface antigens do not undergo antigenic shift during infection. In addition to allowing the uptake of essential nutrients, the porins are responsible for serological specificity and appear to play an important role in interaction with host cells (1). Gonococcal strains express a single porin protein PI. Immunological studies of PI have revealed two major classes (PIA and PIB) and these can be further subdivided into a number of different serovars (2). Meningococci express two rather than one porin protein, class 1 and class 2/3 proteins, which are responsible for subtype and serotype specificity respectively (3). A number of studies have shown that monoclonal antibodies directed against the porin proteins protect against challenge in model systems (4,5). These properties have focussed attention on the potential of porin proteins as vaccine components for prevention of both gonococcal and meningococcal disease. This study concerns the immunochemical analysis of such protective epitopes on gonococcal PIB and meningococcal class 1 protein.

Results and Discussion Location of protective epitopes on gonococcal PIB Comparison of the available amino acid sequences of PIB from three gonococcal strains reveals considerable homology with significant variations being confined to two

Neisseriae 1990 © 1991 by Walter de Gruyter & Co., Berlin - New York-Printed in Germany

236

regions centred on residues 196 and 237 (6). A series of overlapping decapeptides have been synthesised corresponding to the variable regions from strain P9 and reacted with both type-specific and cross-reacting monoclonal antibodies. Each of the type-specific antibodies tested reacted with peptides corresponding to sequences from the first variable region, encompassing residues 190-197, while the cross-reacting antibody SM24 recognised the adjacent sequence ^YSIPS 2 0 1 . The similarity of the epitopes recognised by the type-specific monoclonal antibodies was surprising given their differing patterns of reactivity with a panel of strains. Indeed two mAbs (SM21 and SM203) with differing strain specificities both recognised the same hexapeptide 192 YEHQVY 197 . In order to determine the effect of amino acid sequence variation on antigenic specificity, the sequence of the segment of the PIB gene encoding expression of the variable region containing the epitopes was determined for each of the gonococcal strains. A series of peptides based on the decapeptide i^EYEHQVYSIP 200 found in strain P9 were synthesised, in which individual amino acid residues were substituted, singly and in combination, by each of the different residues found in the other strains (Fig 1). 2.0

2.0

I SM203

¡ZZJ SM21 1.0

1.0

0.0^ E. Y. E. . D . HY Y.S Q. . . Y. AA Y. . . S . . . I . . . P . . .

0.0

D D . D .D D .O S . SSD D . DO Y Y Y TTT T

E. Y. E . . D. HY Y.S Q. . . Y. AA Y. . . 3 . . . I . . . P . . .

D D. D.D D .D 8 . 8SDD . D D Y Y Y TTT T

Figure 1. Effect of amino acid substitutions in variable region 1 of gonococcal PIB on recognition by two mAbs. Comparison of the effect of individual amino acid changes revealed the basis of the difference in specificity of mAbs SM21 and SM203. Substitution of Y for 194 H or A for 196 V in peptide ^EYEHQVYSIP 2 0 0 had no effect on binding of mAbs SM21 or SM203 consistent with their reactivity with strains containing that sequence. The two antibodies did show differences in reactivity when other substitutions were made.

237

Antibody SM21 also reacted with peptides containing the substitutions 194S, 196 Y or 196 T but not 194 D while SM203 reacted with all substitutions at either position. The reactivity of SM21 was completely abolished by the substitution of D for consistent with its failure to react with all three strains containing this substitution. Thus the available monoclonal antibodies all recognise peptides within a highly localised region of PIB (Fig 2). Specificity results from the effect of single amino acid changes, so that antigenic differences between strains are revealed by different patterns of reactivity within a panel of antibodies. 190 200 1 | TKKIEYEHQVYSIPSLFVE SM22

...IEYEHQVY

SM21

H V YE/Q/Y Y A

SM203

YEXQX

SM24

YSIPS

• TYPE-SPECIFIC

} CROSS-REACTING

Figure 2. Location and identity of type specific and cross reacting epitopes on gonococcal protein IB. Subtype specific epitopes on meningococcal class 1 protein. Comparison of the predicted amino acid sequence of three meningococcal class 1 proteins also reveals considerable homology with most of the structural diversity confined to two variable regions (VR1 and VR2) centred on residues 32 and 184 (7). Epitope mapping with synthetic peptides has demonstrated that the two variable regions independently generate separate antigenic domains recognised by protective subtype specific monoclonal antibodies. Thus with a subtype Pl.7,16 isolate antibodies with PI.7 subtype specificity react with the VR1 domain while antibodies with P1.16 subtype specificity react with the VR2 domain. The P1.16 subtype could be located in the peptide 181KDTNNN186. It is interesting to note that although the class 1 proteins and gonococcal PIB show significant homology, the variable domains responsible for their respective antigenic specificities are located in quite different regions of the molecules. DNA sequence analysis of the porA gene from a Pl.7,16 isolate (MC58) associated with an outbreak of meningitis in the Gloucester area of England shows

238

a single nucleotide substitution which generates an amino acid change, from D to N, at position 182 in the VR2 domain responsible for the PI, 16 epitope (8). A series of overlapping decapeptides, spanning the VR2 regions of the two strains, were synthesised and reacted with the Pl,16specific monoclonal antibody. In each case peptides containing the D to N substitution failed to react with the antibody (Fig 3).

8 A Y T P A Y Y T

A Y T P A Y Y T K

Y T P A Y Y T K D

T P A Y Y T K D T N

P A Y Y T K D T N N N N N N L N N

Fig 3. Effect of D to N substitution on the binding of a P1.16-specific monoclonal antibody to synthetic peptides corresponding to variable region VR2 of isolates expressing P1.16 and P1.16b. In order to determine the molecular basis and extent of possible variations within the P1.16 epitope, selected B:15:P1.7,16 isolates were subjected to direct sequencing of the segment of the porA gene encoding VR2. Using this information the strains could be divided into two distinct groups one of which had identical sequences to the PI. 16 reference strain whereas the other strains each contained the substitution causing the 182D t o 182N c h a n g e (designated P1.16b). Although the P1.16 antibody (MN5C11G) recognised the native P1.16b protein isolates in ELISA, reactivity was significantly decreased and the antibody was ineffective in bactericidal killing. The occurrence of such antigenic differences between isolates which were previously thought to be identical has important implications for our understanding of the pathogenesis and epidemiology of meningococcal disease. We would predict that strains possessing the PI. 16b epitope may well show differences in their recognition by antibodies resulting from an immune response to the typical P1.16 epitope which would have significant effects on the susceptibility of individuals to meningococcal disease.

239

Immunisation with synthetic peptides The identification of continuous epitopes recognised by protective monoclonal antibodies permits the use of synthetic peptides for vaccination purposes (9). In an attempt to induce an immune response to the conserved, protective SM24 epitope (197YSIPS201), peptides were synthesised corresponding to residues 193-204 and 195206 of PIB from strains R10(Pep 1) and P9 (Pep 2) respectively. The peptides were coupled to KLH as a carrier protein and used for immunisation. In each case an immune response to the immunising peptide was accompanied by production of antibodies reacting with the homologous strain (Fig 4a). Antisera raised against the RIO peptide Pep 1 reacted in a typespecific manner and was bactericidal for the homologous strain, while the antisera raised against the P9 peptide Pep 2 reacted equally with both strains but was not bactericidal for either (Fig 4b). Log (Titre) (a) Pre ¡52 Post •

4 • 3 :

2 -I 21321*2

1 R10P8 Peptide 1

.Ii 2 R10P8

Peptide 2

R10 PO Antl-Peptlde 1

R10 PO Antl-Peptlde 2

Fig 4. Immunisation with PIB synthetic peptides Pep 1 and Pep 2 conjugated to KLH. (a) Immune response (b) Bactericidal effect of sera. Epitope mapping of the immune response to each peptide revealed the reasons for the specificity and bactericidal activity of the antisera. Anti-Pep 1 sera contained antibodies which were directed primarily against the N-terminus of the peptide containing protective but type-specific epitopes. Anti-Pep 2 sera reacted with conserved sequences towards the carboxy terminus of the peptide and did not recognise the centrally located SM24 epitope.

240

Comparative analysis of the sequence of the Neisseria porin sequences has enabled the construction of a model of their organisation on the surface of the bacteria (10). The model predicts eight surface exposed loops and each of the protective epitopes recognised by the monoclonal antibodies discussed above lies at the apex of a loop. It would appear that these regions represent particularly effective targets for the immune response. The anti-Pep 1 sera recognise one such region The ability to identify protective epitopes combined with a working model of the topology of the porin proteins enables the identification of potential targets for immunisation. The studies described above illustrate the potential of synthetic peptides for immunisation but reveal that careful targeting will be required to produce an immune response of the desired specificity. Acknowledgements This work was supported by Medical Research Council Project Grants and by The National Meningitis Trust. References

1. Lynch, E.C., M.S. Blake, E.C. Gotschlich, A. Mauro. 1984. Biophys. J. 45, 104. 2. Knapp, J.S., M.R.T. Tamm, R.C. Nowinski, K.K. Holmes, E.G. Sandstrom, 1984. J. Infect. Dis. 50, 44. 3. Frasch, C.E., W.D. Zollinger, J.T. Poolman. 1985. Rev. Infect. Dis. 7,504. 4. Viiji, M., K., Zak, J.E. Heckels. 1986. J. Gen. Microbiol. 132,1621. 5. Saukkonen, K., H. Abdillahi, J.T. Poolman, M. Leinonen. 1987. Micro. Path. 3,261. 6. Butt, NJ., M. Viiji, F. Vayreda, P.R. Lambden, J.E. Heckels 1990. J. Gen. Microbiol. 136,1871. 7. McGuinness B., A.K. Barlow, I.N. Clarke, J. E. Farley, A. Anilionis, J.T. Poolman, J.E. Heckels. 1990. J. Exp. Med. 171,1871. 8. McGuinness B.T., A.K. Barlow, I.N. Clarke, P.R. Lambden, J.T. Poolman, D.M. Jones, J.E. Heckels. 1990. (submitted for publication). 9. Heckels, J.E., M. Viiji, C.R. Tinsley, 1990. Vaccine 8,225. 10. Van der Ley, P., J.E. Heckels, M. Viiji, P. Hoogerhout, J.T. Poolman (submitted).

Serological responses in Norwegian adult volunteers to a meningococcal 15:P1.16 outer membrane vesicle vaccine (Phase II studies)

E.A. H0iby, E. Rosenqvist, G. Bjune, O. Closs, L.O. Fr0holm National Institute of Public Health, N-0462 Oslo 4, Norway

Introduction Norway has suffered a meningococcal outbreak since the mid 1970s. About 80% of the cases have been caused by serogroup B strains (1,2). The majority of these organisms have carried the 15:P1.16 epitopes and belonged to the ET-5 multilocus enzyme genotype (3). On this basis, and as B polysaccharide does not induce protective antibodies in humans, work to develop and evaluate an outer membrane protein serogroup B vaccine including i.a. immune response studies and protection trials was begun. An outer membrane vesicle preparation (OMV) from a B:15:P1.16 Neisseria meningitidis (strain 44/76) was prepared and three vaccines of different composition were produced. Two of the preparations contained equal amounts of protein (P) and serogroup C capsular polysaccharide (C), and two Al(OH)3 as adjuvant (A). The LPS content is about 7% compared to protein dry weight. The three vaccines are thus designated PA, PCA and PC. The design of the three steps of the phase II trial (II-l to II-3) is shown in Table 1. In phase II-2 and II-3, placebo controls (PLAC.) were included and meningococcal carriage examined. Preliminary data on dose responses for the three vaccines have been given earlier (4). No significant difference in bactericidal response between 25 and 50 fig doses was observed in phase II-2. In this poster report we want briefly to outline some of the main results concerning the two preparations and doses chosen for further studies in phase II-3 (PA and PCA: the dose for both was 25 fig of P; the latter also contained 25 fig of serogroup C polysaccharide). In phase II-3 25 fig doses of PA were given to 120, 25 fig PCA to 50 and adjuvant

Neisseriae 1990 © 1991 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

242 Table 1 Experimental Design in the Three Steps of Phase II Studies (II-l to II-3) with the Norwegian Meningococcal Serogroup B Outer Membrane Vesicle Vaccine STEP

No. of Volunteers

Preparations applied

Doses applied (fig)

II-l

118 Health personnel

PA, PCA, PC

12.5, 25, 50 and 100

II-2*

96 Military recruits

PA

25 and

II-3*

220 Health personnel

PA, PCA

25

50

* placebo controls and examination for meningococcal throat carriage included in study alone (PLAC.) to 50 volunteers in a double blind, randomized manner. Two doses of the same preparation were administered to each adult volunteer at 6 weeks' interval. Sera were drawn on day 0 (before vaccination) and at weeks 2, 4, 6 (revaccination), 8, 12, 26, and after one and two years from some of the volunteers. For some comparisons we also included sera from the 1984 ACYW2bl5-2 vaccine trial (5) where the outer membrane protein preparation was given as one dose containing 52 /¿g outer membrane protein of each of the two serotypes and 40 /¿g of each polysaccharide. This vaccine contained about 3 % LPS and no adjuvant. The vaccine group consisted of 23 military recruits and 10 additional volunteers, while 24 recruits given one dose of 50 ng of each polysaccharide (ACYW) served as the control group in this study. Antibody responses were studied by bactericidal assay (BA) against the vaccine strain using 2 5 % human complement. Sera were titrated twofold starting at a final serum concentration of 50% and survival was tested after an incubation time of 30 minutes. > 5 0 % killing of the inoculum measured as colony forming units at this dilution was recorded as a titer 1/2 = reciprocal log2 titer of 1. A titer of 0 is assigned to a serum with less than 5 0 % killing in 50% serum. ELISA tests with vaccine strain outer membrane protein (OMP) antigen (5), C polysaccharide or vaccine strain meningococcal LPS as antigen were also performed.

243 Results and conclusions PA 2 5 u g

7

dose

t

Figure 1 Log2

—

4- •

Bactericidal

titer 3- •

Hk.O

Kk.4

Wk.6

Time a f t e r

Nk.B 1.

Wk.lS

Phase

II-l

-•r- Phase

II-2

-o-Phase

II-3

Hk.26

vaccination

Fig 1 shows the overall kinetics of bactericidal antibody development in the three different phase II trials for 25 fig PA. The BA titers before and after vaccination differed in the three parts of the phase II study in that the II-2 volunteers (military recruits with more than 50% meningococcal carriage) showed a higher activity at all points of time compared to the older adults involved in trials II-l and II-3 (carriage rates 0.6°C or the sum of temperature rises in three rabbits exceeded 1.40C. All doses of liposomal LOS from 0.1 to 50 pg after intraperitoneal inoculation induced intensive accumulation of PFC in CBA mice on day 6 (up to 62,040 PFC per spleen at the maximum dose). This immune response was tenfold higher than that to free LOS under the same conditions. The study of the dynamics of PFC accumulation revealed two important facts: (i) in comparison with free LOS, liposomal LOS inoculatedat intraperetonealy in a dose of 10 pg induced more prolonged accumulation of PFC (till day 21 i.e. for the period of observation); (ii) the peak of the response was observed later, on day 6 after immunization. Subcutaneous administration of the preparation was found to be less effective than its intraperitoneal and intravenous injection. The adjuvant action of liposomal LOS was retained and caused a sixfold increase in spleen PFC on day 5 at the dose of LOS equal to 10 pg (data not shown). It should be pointed out that free LOS in a dose of 10 pg introduced intravenously produced a toxic effect on mice. The level of serum antibodies was determined by ELISA as described by Ito et al. (9). The results of each serum were compared with standard mouse anti-LOS affinitypurified antibodies. One unit (1U) corresponded to the activity of 1 pg of the reference preparation with peroxidase-conjugated rabbit anti-mouse polyvalent immunoglobulins. The preparations were injected intraperitoneally in a single injection. The results of the experiments on the dynamic response (Table 2) indicate a prominent liposome adjuvant effect. It should be emphasized that on day 7 IgM antibodies prevailed while on day 21 IgG3 antibodies were detected (data not shown).

262 Table 2. The level of antibodies to N. meningitidis in CBA mice after immunization with free or liposome-incorporated LOS LOS preparation

Liposomal LOS

Free LOS

Saline

Dose (jig)

1.0 5.0 25 1.0 5.0 25 -

Level of antibodies (U/mI±m) in serum on different days of immunization day 7 day 14 0.29±0.08 0.36±0.09 2.45±0.42 3.73±0.18 2.42±0.32 2.41±0.19 0.13±0.04 0.18±0.08 0.81±0.16 0.42±0.11 0.89±0.21 0.41 ±0.32 0.09 ±0.04 -

The results obtained in our study indicate that N. meningitidis LOS incorporated into liposomes is able to considerably reduce endotoxin activity and to raise humoral response in mice. The pathophysiological action of LOS is manifested through the cells of monocyto-macrophage system. The following chain of interactions is supposed: LOS hydrophobic part (lipid A) is recognized by special protein receptors on the membrane of macrophages, then the mechanism of intensive synthesis and release of monokines and particularly one of the main mediators of a septic shock (tumour necrosis factor) is started (10). Total LOS detoxification could be achieved through lipid A removal and partial detoxification occured due to its deacylation or dephosphorylation or lipid A shading by hydrophobic compounds, such as polymyxin B or phospholipids (5,11). In the latter case LOS is captured by the phagocyte without recognition and the pathophysiological mechanism of endotoxic shock is not started. Nowadays special attention is drawn to the development of vaccines based on LOS from N. meningitidis and other gram-negative bacteria. The main approach to the solution of this task consists in the removal of lipid A and the conjugation of carbohydrate component with the protein carrier (12). But lipid A possesses adjuvant effect besides its toxic action. Therefore it is most important to preserve lipid A during the construction of new vaccines in case of its detoxification. The main drawback of polysaccharide vaccines used is their inefficiency when administered in young children and the lack of immunological memory. Our results demonstrate that liposomal LOS preserves its capacity of inducing immune response in CBA/N mice lacking LyB5 + B-lymphocytes, i.e. LOS induces response as type 1 thymus independent antigen (data not shown). Liposomes considerably prolong

263 accumulation of PFC and the circulation of antibodies. Therefore it is possible that one injection of liposomal LOS may be sufficient for the development of prolonged immunity.

Acknowledgements. The research was supported by the World Health Organization.

References 1. Saukkonen,K.,M. Leinonen,H. Kayhty,H. AbdillahiJ.T. Poolman. 1988. J.Infect.Dis.l5S:209. 2. Maland,J.A.,S. Smoland. 1986. Acta Pathol.Micribiol.Scand., Sec.B,Microbiol.94:223. 3. Tsai,C.-M.,L.F. Mocca,C.E. Frasch. 1987. Infect.Immun.55:1652. 4. Gregoriadis,G. 1990. Immunol.Today.ll:89. 5. DijkstraJ.J.W. MellorsJ.L. Ryan. 1989. Infect.Immun.57:3357. 6. Trubetskoy,V.S.,N.V. Koshkina,V.G. Omelyanenko,V.L. Lvov, B.A. Dmitriev,A.B. Petrov,V.P. Torchilin. FEBS Lett, (in press). 7. WHO Tech.Rep. 1981. Ser.658.p.l74. 8. Cunningham,A.J. 1965. Nature.207:1106.12. 9.Ito,J.I.,Jr.,A.C. WinderlichJ. Lions,C.E. Davis,D.G. Guiney, A.I. Braude.1980. J.Infect.Dis.l42:532. 10. Beutler,B.,A. Cerani. 1987. N.Engl.J.Med.716:379. 11. Chia,J.K.S. 1989. J.Infect.Dis.l59:872. 12. Jennings,H.J.,C. Lugowski,F.E. Ashton. 1984. Infect.Immun.43:407.

Relative bactericidal activity of IgG antibodies against outer membrane complexes from meningococci, as a function of vaccine type, dose and time after vaccination

E. Rosenqvist, E.A. H0iby National Institute of Public Health, N-0462, Oslo 4, Norway.

Introduction. The bactericidal activity of antibodies against Gram-negative bacteria is a function of several, partly unknown parameters. Some antigens in the outer membrane of Neisseriaceae are known to be more important targets for antibodies than others (1-4). Among the important factors for the bactericidal activity are the isotype and probably also the affinity of the antibody molecules. These parameters may be influenced by mode of presentation of the antigens (e.g. adjuvant) and by the time lapsed after immunization. In this preliminary investigation we have studied the bactericidal activity and the IgG levels in sera collected after vaccination with a new vaccine against group B meningococci and tried to analyse the relation between them.

Results. Sera collected at various times from 127 adult volunteers vaccinated with three different 15:P1.16 meningococcal outer membrane protein vaccines, in phase II1 of the clinical trial in Norway (5), were studied. Twp of the preparations contained equal amounts of protein (P) and serogroup C capsular polysaccharide (C) and two aluminum hydroxide (A) as adjuvant. The vaccines are designated PCA, PA and PC. Doses from 12.5 to 100 microgram protein were administered twice at six weeks interval, and sera collected at week 0, 4, 6 (revaccination), 8, 12 and 26 were analysed.

Neisseriae 1990 © 1991 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

266 The bactericidal titers were determined as the serum dilution giving 60 % kill of the vaccine strain 44/76. We used human serum without antibody activity as complement source. The total amount of IgG binding to outer membrane complexes (OMC) from the same strain was determined by ELISA. The mean level of IgG against OMC and the bactericidal titers, as a function of time after vaccination, showed apparently parallel slopes with a primary and secondary response and a maximum two weeks after revaccination (Fig 1).

Mc.O

Hk.4

Nk.6 Hk.B Hk.12 Time a f t e r vaccination-1

We.26

Fig. 1. Kinetics of antibody responses in adult humans analysed by a bactericdal assay and ELISA. Only the results from vaccinees who received 25 ug doses of vaccine are shown. When the mean log2 IgG values from OMC ELISA were plotted as a function of bactericidal titers, approximately parallel lines were observed (Fig 2). However, the curves for sera collected at different point of times after vaccination were positioned differently along the IgG axis, indicating that the same amount of IgG detected in ELISA could result in different bactericidal activity, depending on the time after vaccination.

267 10-

Wk.6 Hk.8 — Hie. 12 •0- Wk.26 —

Fig. 2. Mean Log2 values of IgG against OMC from strain 44/76 as a function of the bactericidal titer with the same strain. The different curves are from sera collected at various times after vaccination. Results from all the vaccines are used. The efficiency of the IgG antibodies to induce complement-mediated cell lysis was calculated for each serum as the bactericidal titer (log2 of serum dilution) divided by the level of IgG binding to outer membrane complex antigen (OMC) from the same strain. The resulting number we defined as the relative bactericidal activity (RBA). The variation in RBA as a function of time and vaccine type is illustrated in Fi

8 3-

0.03 0.025 • PCA. PC. • PA.

0.015 0.01

0.005

Wk.6 Kk.8 Nk.12 Time after vaccination-!

Wk.26

Fig. 3. Relative bactericidal activities (RBA) of sera from vaccinees as a function of time after vaccination and vaccine type. Only the results from vaccinees without bactericidal activities before vaccination are shown. We found that RBA increased up to week 6 after vaccination, when the second dose of vaccine was administered. Minimum values were observed at week 8 and

268 then RBA again increased up to week 26. Standard error of the means in RBA are about 0.002 The two formulations of vaccines containing adjuvant (PA and PCA) gave significantly higher RBA values than the vaccine without adjuvant (PC). We also found RBA to be a function of the vaccine dose. The lowest RBA values were observed with the highest dose. The dose effect was most pronounced for the vaccine without group C polysaccharides (PA). This phenomenon was seen at all points of time after vaccination as shown in Fig 4.

0.02--

—

Hk.4 Hk.B

RBA

— Hie. 12

a- Wk.26

0.01

12.5 ug

25 ug

50 ug

100 ug

Fig 4. Relative bactericidal activity (RBA) as a function of vaccine dose and time after vaccination. Results from all vaccinees are included. Also the correlation coefficients (r) between IgG from OMP ELISA and the bactericidal assay varied depending on vaccine type, dose and time after vaccination. The correlation was at its maximum at week six and at a miniumum at week eight, and highest for the vaccinees who had received the two vaccines containing C-polysaccharides. The highest correlation coefficient (r= 0.9],) was observed six weeks after the first vaccination for those who received PCA vaccine, while the lowest (r= 0.45) was observed two weeks after the second injection, for those who received PA. Discussion. Both the vaccine and the outer membrane vesicle preparation used as antigen in ELISA, contain many different antigens. The major components in the vaccine are

269 the class 1, 3 and 5 outer membrane proteins. In addition several minor proteins and about 7% lipopolysaccharide (LPS) are present. We do not know the antibody responses to the individual antigens or how this changes with time. The decrease in RBA and in the correlation coefficients, observed after revaccination, may be explained by assuming that the second immunization stimulates the production of IgG molecules binding to the ELISA antigen, but which are functionally less effective in the bactericidal test. We have found that antibodies against LPS are induced by vaccination and shown that anti-LPS antibody levels do not correlate well with the corresponding bactericidal activity. If the amount of anti-LPS antibodies, relative to the other antibodies, varies with time and vaccine type, this could influence the RBA value. Minor antigens in the vaccine which may induce blocking antibodies, e.g. class 4 protein molecules, may possibly become more important after immunization with the highest doses of vaccine. The changes in RBA with time may be an effect of changes in affinity and/or subtype of the IgG molecules as well. In progressing from a primary to secondary response, there is a well documented increase in antibody affinity (6). Such changes may also affect the ability of antibody molecules to activate complement and possibly the bactericidal activity. Further analyses of specific antigen binding, complement activation and antibody subclass distribution and affinity studies are in progress.

References 1. Saukkonen, K., H. Abdillahi, J.T. Poolman and M. Leinonen.1987. Microb. Pathogen. 3:261. 2. Brodeur, B.R., Y. LaRose, P. Tsang, J.Hamel, F.A. Ashton and A. Ryan. 1985. Infect. Immun. 50:510. 3. Virji, M. and J. Heckels. 1989. J. Gen. Microbiol. 135:1895. 4. Rice, P.A., H.E. Vayo, M.R. Tam and M.S. Blake. 1986. J. Exp. Med. 164:17351748. 5. H0iby, E.A., E. Rosenqvist, G. Bjune, O. Closs, A. Halstensen, H. Sjursen, J. Fuglesang and L.O. Fr0holm. 1988. 6th Int. Path. Neiss. Conf., Atlanta. Abst. VC29. 6. Eisen H.N. and G. W. Siskind. 1964. Biochemistry 3:996.

Efficacy of Human Monoclonal Antibodies to the Group B Polysaccharide of Neisseria Meningitidis in Experimental Infant Rat Model

K. Saukkonen, M. Leinonen National Public Health Institute, Helsinki, Finland H.V. Raff Oncogen, Seattle, USA

INTRODUCTION Neisseria meningitidis group B (MenB) is the predominant cause of meningococcal disease during non-epidemic periods (1,2). At present no effective vaccine against MenB exists. The capsular polysaccharides (CPSs) of other meningococcal groups A, C, Y and W135, have been successfully used as vaccines in adults and children over two years old (3). The CPSs of MenB and E. coli K1 are immunochemically identical polymers of alpha(2-8)-linked N-acetylneuraminic acid, are exceptionally poor immunogens (4), and provide resistance to phagocytosis (5). The antibody response is mainly of the IgM class (6). Passive immunotherapy for treating serious bacterial infections has been suggested as a potential adjunct treatment with conventional antimicrobial treatment (7). Murine monoclonal antibodies (MAbs) against bacteria most frequently causing neonatal and adult meningitis, and bacteremia are shown to possess prophylactic and therapeutic activity, but also as foreign particles, mouse antibodies administered to humans, have serious shortcomings (8). Thus human MAbs specific to bacterial pathogens would be preferable therapeutic

Neisseriae 1990 © 1991 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

272

agents against bloodborne infections. It has been demonstrated before that human MAb against MenB and E.coli K1 or group B streptococcus (9, 10) increase the survival of infected infant rats. In this report we used the infant rat infection model (11) to further specify if these antibodies could inhibit the meningitis, bacteremia and death in animals infected with MenB or E. coli KI.

MATERIALS AND METHODS The details of the infant rat model for meningococcal infection have been described previously (11). Five days old rat pups (outbred Wistar) were used for the study. Two N. meningitidis group B strains B:15:P1.15:L1,8 (355), and B:2b:P1.2:L2 (3006) were obtained from Dr. J.T. Poolman, RIVM, The Netherlands, one N. meningitidis group C strain (5352) obtained from Dr. Aulikki Sivonen, University of Helsinki, Finland. Bacteria were grown as described earlier (11) and given intraperitoneally; the challenge dose was 10® bacteria / animal with MenB and MenC. The development of bacteremia and meningitis was followed by sampling blood and cerebrospinal fluid (CSF) 6 hours after the challenge and is reported as viable bacteria (cfu)/ml of blood or CSF. Monoclonal antibodies were given intraperitoneally in different dilutions made in phosphate buffered saline, pH 8.4 (PBS) one hour before the bacterial challenge. At least three pups were used for every dilution, time point and sample. Two of the MAbs were specific to the meningococcal group B CPS and one human MAb against group B streptococci serving as the negative control. The IgM MAbs are opsonic for both the E. coli Kl and MenB isolates (9).

273

RESULTS Two MAbs to MenB CPS were tested for their protective activity in infant rats infected with meningococal and E. coli K1 strains. Bacteria were quantitated in the blood and CSF of infant rats infected intraperitoneal^ with 10® MenB bacteria (B:15:P1.15:L1,8 and B:2b:P1.2: L2). When either of the two anti-CPS MAbs (5E1 and 9B10) were administered one hour before the bacterial challenge, at 6 hours post challenge, the bacterial counts in the blood were at least 3 logs lower than in the animals receiving control MAb (Fig. IB). The same effect was also seen in the CSF (Fig. 1A). The protective efficacy of the MAbs was determined against the two MenB strains, and a MenC strain. Both MAbs were highly protective against the MenB strains, with 0.13ug/animal providing 100% protection (Fig.lC ). As expected, neither MAb protected against the MenC strain. The MAbs also protected against an E.coli K1 clinical isolate (data not shown). Therefore, the mortality rates correlated with the bacterial counts in body fluids.

DISCUSSION In the present study we show that the human MAbs to meningococcal group B and E. coli K1 capsular polysaccharides are highly protective against bacterial challenge in infant rats. The human antibodies 9B10 and 5E1 (10) are the first EBV-transformed lymphoblastoid cell lines continously producing antibodies protective for MenB and E. coli K1 bacteria. The antibodies (IgM) were biochemically and serologically shown to react only with purified K1 and group B meningococcal capsule preparations and to bind 100% of tested E. coli K1 clinical isolates (9).

274

A

2e+5

MenB: 15 0

MenB:2b

&

MenC

2e+5

1

1e+5 5e+4 Oe+O

0.013

0.013

0.013

0.13

Antibody dose (ug^ml)

1.3

Fig.l. Protective efficacy of human Mabs to MenB CPSs after bacterial challenge with MenB:15, MenB:2b or MenC. Numbers of viable bacteria in CSF (A) and blood (B) 6 hrs after ip challenge with 10^ bacteria/rat. Survival was determined 12 hrs after the challenge.

275

Murine MAbs that possess protective activity for E. coli K1 and MenB have been used in animal models, and provide a basis for comparing prophylactic use of MAbs with protective activity for infections. Mouse MAbs against MenB LPS serotypes or outer membrane proteins and CPS (11, 12, 13) have been tested in several experiments and found to be protective against MenB and E.coli K1 infections, but it is obvious that the problems are abundant using mouse MAbs in humans. Our results show that human MAbs for MenB and E. coli K1 CPSs are protective against meningitis and bacteremia, as well as death. Even as low amount as 0.13 ug/ animal was able to abolish the bacteria from the circulation and spinalfluidin 6 hours vs. the controls. The problem arises with the fact that the MenB and E. coli K1 CPSs crossreact (14) with polysialic antigens present in human tissues during embryonal development and that has been taken as a warning against attempts to increase their immunogenicity. The reactivity of human MAbs 9B10 and 5E1 has not been determined and extensive screening of fetal, neonatal and adult tissues with these antibodies will be necessary to ensure the absence of potentially harmful crossreactions.

ACKNOWLEDGMENTS We thank Ms. Raili Kalliokoski for excellent technical assistance.

REFERENCES 1. PELTOLA, H. 1983. Rev. Inf. Dis. 5: 71-91. 2. POOLMAN, JT, I. Lind, and K. Jonsdottir. 1986. Lancet, ii: 555-557. 3. PELTOLA, H, A. Safara, and H. Kayhty. Pediatrics. 1985. 76: 91-96. 4. WYLE, FA, M. Artenstein, and B. Brandt. 1972. J. Infect. Dis. 126: 514-522.

276

5. CROSS, AS, P. Gemski, J. Sadoff, F. Orskov, and I. Orskov. 1984. J. Infect. Dis. 149:184-193. 6. LEINONEN, M, and K. Frasch. 1982. Infect. Immun. 38: 1203-1207. 7. STIEHM, ER, and R. Kobayashi. 1980. In: Immunoglobulins: characterictics and uses of intravenous preparations. Eds. BM Alving and JS Finlayson. Washington, DC: US department of Health and Human Services, pp. 89-98. 8. LEVY, R, and R. Miller. 1983. Fed. Proc. 42: 2650-2656. 9. RAFF, HV, P. Siscoe, E. Wolff, G. Maloney, and W. Shuford. 1988. J. Exp. Med. 168: 905-917. 10. RAFF, HV, D. Devereux, W. Shuford, D. Abbott-Brown, And G. Maloney. 1988. J. Inf. Dis. 157: 118-125. 11. SAUKKONEN, K, H. Abdillahi, JT. Poolman, and M. Leinonen. 1987. Microbial Pathogenesis. 3: 261-267. 12. SAUKKONEN, K, M. Leinonen, H. Kayhty, H. Abdillahi, and JT. Poolman. 1988. J. Infect. Dis. 158:209-212. 13. FROSCH, M, I. Gorgen, G. Boulnois, K. Timmis, and D. Bitter-Siiermann. 1985. Proc. Natl. Acad. Sci. USA. 82: 1194-1198. 14. FINNE, J, M. Leinonen, and PH Makela. 1983. Lancet. ii:355-357.

Immunomodulating Complex of Oligopeptide Antigen and Liposomal Form of Neisseria meningitidis Lipooligosaccharide

B.F. Semenov, A.B. Petrov, T.A. Chulok Mechnikov Research Institute for Vaccines and Sera, Moscow, USSR 103064 V.P. Torchilin, V.S. Trubetskoy, N.V. Koshkina USSR Cardiology Research Centre, Moscow, USSR 121552 V.T. Ivanov, T.M. Andronova, B.B. Ivanov Shemyaking Institute of Bioorganic Chemistry,Moscow, USSR 117871

Introduction The development of immunomodulating methods remains an important condition for the preparation of synthetic oligopeptide vaccines. It is known that for inducing response to oligopeptides their covalent binding with protein carrier is necessary (1). This carrier serves as the source of epitopes for T-lymphocytes. One of the drawbacks of thymus-dependent antigens is the realization of the function of IRgenes on the level of T-lymphocytes. In genetically heterogeneous populations, e.g. in humans, a considerable part of individuals will probably give only a faint response or no response at all to a particular protein carrier. Beside thymusdependent antigens, there are immunogens capable of inducing a protective response

without

the

participation

of T-cells

(2).

We

suppose

that

the

immunomodulating complex (IMC) in which oligopeptide (B-epitope) is associated with liposomal lipooligosaccharide (LOS) without covalent linkage may induce response to oligopeptide as thymus-independent antigen type 1 (TI-1).

Results and Discussion. As oligopeptide, we used the B-epitope of Plasmodium falciparum CS-protein (NANP)3 (3). For the effective incorporation of oligopeptide several hydrophobic

Neisseriae 1990 © 1991 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

278 derivatives were obtained. The liposomal preparation consisting ofN. meningitidis LOS, hydrophobically modified (NANP) 3 and phospholipids was prepared by the method of dehydration-rehydration vesicles (4). The optimum proportion of these components capable of ensuring the stability of such complex was found to be 1:1:100 by weight respectively. The level of serum antibodies was determined in ELISA as described by Hoffman et al. (5) with anti-mouse IgG and IgM peroxidase conjugates. The result was considered positive when the optical density was higher than that for intact mouse serum diluted 1:200. The immune response was evaluated in CBA, CBA/N, BALB/c, C57BL/6 mice immunized intraperitoneal^. Our results indicate that following a single immunization of mice with IMC a prominent immune response could be registered in all animals tested. The table 1 shows that the titre of IgM and IgG antibodies in CBA mice was much higher than those obtained to oligopeptide conjugated with keyhole limpet hemocyanin (KLH) on day 7 after intraperitoneal immunization. In addition, a noticeable switching from the synthesis of IgM to IgG antibodies was observed. Table 1. The level of antibodies to P.falciparum CS-protein after the injection of different forms of oligopeptide (NANP) 3 Preparation

(NANP)3-IMC

Antibody titre Day 7 97,284

IrM

Day 14 480

Day7 9,602

If?G

Day 14 560

(NANP)3-KLH conjugate

640

433

466

720

(NANP) 3

100 kD.

411

binding site for Fe 3+ . This protein appears to be periplasmic and may serve in transport of iron across this space. The characterization of some of the iron-inducible proteins at the DNA level has been completed (18,19), or is in progress (see the following chapters). The protein deduced from the DNA sequence of FrpA, found only in the meningococcus, has strong homology to certain toxins containing repeated sequences (rtx toxin family) such as the E. coli hemolysin and the Bordetella pertussis adenyl cyclase. The possible toxic effects of this protein on host cells are just beginning to be explored. Table II

Mutation or Strain Designation

Ability to grow on Transferrin, Lactoferrin or Citrate

Characteristics of Mutant

TF

LF

Cit

-

+

+

Fails to bind human TF to intact organisms; Lacks TBP1 100 kD receptor protein.

tifi

-

+

+

Low expression of TBP1; presumed to be a promoter or regulatory mutation.

M

+

-

+

Fails to bind LF to intact organisms.

trf2, t i f i , trf4, JM&

tip,

Cannot utilize any Fe3+ source, but grows on Fe2+ i.e. hemin; Lacks both FRPB and TBP2 (normal TF receptor function).

FAM11

tlu Judl4

-

-

+

Binds TF and LF normally; lesion not defined.

+

Binds TF normally and removes the iron from TF. However, the iron is not internalized. Maybe similar to tlu.

(1)

Jud6 and FAM11 are mutants of N. meningitidis, while all the others are mutants of N. gonorrhoeae.

412

The third approach, the isolation of mutants with a defect in iron metabolism has been also vigorously pursued. The strategy for the isolation of these mutants was to use the toxicity of streptonigrin in the presence of iron (20) to select for mutant strains unable to use one or another source of iron. The properties of the mutants which have been characterized (21) to some extent are summarized in Table II. Among these mutants, some (i.e. trfl - ttf5) have defined defects that can explain the observed phenotypes. For others (i.e. FAM11, tlu and fudl4), the relationship of the defects observed with the phenotype is not clear at this time, but their availability represents an important step for future studies. There is considerable interest in exploring the potential of one or the other of these proteins as vaccines. The human immune response to infection indicates that the ironinducible proteins are expressed in vivo and there is agreement that this approach merits serious attention.

Affiliations of the Authors C. Cornelissen and P.F. Sparling, Department of Medicine, University of North Carolina, Chapel Hill, NC; S. A Hill and J. Swanson, Laboratory of Microbial Structure and Function, Rocky Mountain Laboratories, NIAID, Hamilton, MT; J.M. Koomey, Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI; C. Marchai, Unité des Antigènes Bactériens, Institut Pasteur, 75724 Paris Cedex 15, France; T.F. Meyer, Max-Planck-Institut für Biologie, Abteilung Infektionsbiologie, Tübingen, Germany; Atlanta, GA;

S.A. Morse, Centers for Disease Control,

S. Normark, Department of Molecular Microbiology, Washington

University, St. Louis, MO; A.B. Schryvers, Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada;

and H.S. Seifert,

Department of Microbiology and Immunology, Northwestern University Medical School, Chicago, IL.

413

References 1. Kellogg, D.S., Jr., I.R. Cohen, L.C. Nonns, A.L. Schroeter, G. Reising. 1968. J. Bacteriol. Pö:596-605. 2. Swanson, J.L., S.J. Kraus, E.C. Gotschlich. 1971. J. Exp. Med. 754:886-906. 3. Swanson, J.L., K. Robbins, O. Barrera, D. Corwin, J. Boslego, J. Ciak, M.S. Blake, J.M. Koomey. 1987. J. Exp. Med. 765:1344-1357. 4. Meyer, T.F., N. Mlawer, M. So. 1982. Cell 50:45-52. 5. Sparling, P.F., J.G. Cannon, M. So. 1986. J. Infect. Dis. 755:196-201. 6. Meyer, T.F., C.P. Gibbs, R. Haas. 1990. Ann. Rev. Microbiol. 44:451-477. 7. Swanson, J., J.M. Koomey. 1989. In: Mobile DNA (D.E. Berg and M.M. Howe eds. American Society for Microbiology, Washington, p. 743-761. 8. Hill, S.A., S.G. Morrison, J. Swanson. 1990. Molec. Microbiol. 4:1341-1352. 9. Koomey, M., E.C. Gotschlich, K. Robbins, S. Bergstrom, J.L. Swanson. 1987. Genetics 777:391-398. 10. Gibbs, C.P., B.-Y. Reimann, E. Schultz, A. Kaufmann, R. Haas, T.F. Meyer. 1989. Nature 558:651-652. 11. Seifert, H.S., R.S. Ajioka, C. Marchai, P.F. Sparling, M. So. 1988. Nature 336:392-395. 12. Swanson, J., S. Morrison, O. Barrera, S. Hill. 1990. J. Exp. Med. 777:2131-2139. 13. Taha, M.K., M. So, H.S. Seifert, E. Billyard, C. Marchal. 1988. EMBO. J. 7:4367-4378. 14. Weinberg, E.D. 1978. Microbiol. Rev. 42:45-66. 15. West, S.E., P.F. Sparling. 1985. Infect. Immun. 47:388-394. 16. Schryvers, A.B., B.C. Lee. 1989. Can. J. Microbiol. 55:409-415. 17. Schryvers, A.B., G.C. Gonzalez. 1990. Can. J. Microbiol. 55:145-147. 18. Berish, S.A., T.A. Mietzner, L.W. Mayer, C.A. Genco, B.P. Holloway, S.A. Morse. 1990. J. Exp. Med. 777:1535-1546.

414

19. Berish, S.A., D.R. Kapczynski, S.A. Morse. 1990. Nucleic Acids Res. 78:4596. 20. Cohen, M.S., Y. Chai, B.E. Britigan, W. McKenna, J. Adams, T. Svendsen, K. Bean, D.J. Hassett, P.F. Sparling. 1987. Antimicrob. Agents. Chemother. 37:15071513. 21. Blanton, K.J., G.D. Biswas, J. Tsai, J. Adams, D.W. Dyer, S.M. Davis, G.G. Koch, P.K. Sen, P.F. Sparling. 1990. J. Bacteriol. 772:5225-5235.

Interaction Between Iron Regulated Outer Membrane Protein Of Neisseria meningitidis and Human Transferrin Binding Activity.

D.A. Ala'Aldeen, Heather A. Davies, R.A. Wall and S.P. Boniello. Microbial Pathogenicity Research Group, MRC Clinical Research Centre, Watford Road, Harrow, Middlesex HAI3UJ.

Introduction Neisseria meningitidis is able to chelate iron from human transferrin (HTF). Previous workers have reported that a ca. 70 kilodalton (kDa) iron regulated outer membrane protein (FeRP-70) is a highly specific receptor for HTF [1], We have examined the interaction between the iron regulated outer membrane proteins (OMP's) and HTF, using rabbit anti-HTF and gold-labelled HTF. Also, we used monospecific rabbit anti-FeRP70 in competitive experiments to determine the role of FeRP-70 in HTF- binding.

Materials and Methods The following different serogroups

of N. meningitidis were used: A (2), B (6), C

(2), W135 (2), 29E (1), H (1), I (1) and

K (1).

Commensal Neisseriae were N.

polysacchareae and N. lactamica. Meningococci were grown on Mueller Hinton agar

(Oxoid), incorporating 25 uM

desferrioxamine for iron limitation, for 24h in 5% C02. OMP's were extracted by the lithium acetate chloride (LAC) extraction method [2] and diluted to yield 1 mg/ml protein. SDS-PAGE was performed in 10% linear acrylamide gels [3]. FeRP-70 purification was achieved by elution of the protein

from

10% SDS-PAGE [4], and lyophilisation.

Polyclonal monospecific anti-FeRP-70 (R-70) was raised by injecting a New Zealand white rabbit with the eluted FeRP-70 preparations and the sera was absorbed overnight with iron-replete homologous (GN) strain before use (yielding AR-70) [4].

Neisseriae 1990 © 1991 by Walter de Gruyter& Co., Berlin • New York - Printed in Germany

416 OMPs were Western blotted with AR-70 (dilution of 1:4000), or with HTF (dilution 250 ug/ml) followed by rabbit anti HTF (dilution of 1:1000) and the reactions visualised by peroxidase-conjugated goat anti-rabbit. Gold-labelled human transferrin (Au-HTF) was prepared according to Frens [5]. The conjugation of HTF (Sigma) was carried out as described in the Janssen handbook [6]. OMPs were Western blotted with Au-HTF (dilution 1 ug/ml) and then subjected to silver enhancement of the gold particles [7].

Results and Discussion SDS-PAGE analysis of the purified (eluted) FeRP-70 demonstrated the migration of this protein at the same molecular weight level as the original protein. Injecting rabbits with this material raised a polyclonal monospecific antisera which recognised FeRP-70, a co-migrating protein and one protein expressed under iron repleted conditions on the homologous strain. The latter two were easily excluded with the absorption which involved incubation of the serum with live intact cells overnight. AR-70 demonstrated the presence of cross-reactive FeRP-70 in different serotypes and serogroups

of meningococci as well as commensal Neisseriae (Fig.l).

This cross-

reaction is complete, unlike the rabbit sera previously raised against intact whole cells [2].

H

I

K L P

GN

Fig. 1. AR-70 Western blotted against OMP's of different strains of N. meningitidis and commensal neisseriae, grown in the presence of 25 uM desferoxamine. Serogroups H, I, K and C (GN, homologous) as well as commensal Neisseriae N. lactamica (L) and N. polysacchareae (P) (*) FeRP-70.

417

Fig.2. Competetive Western blotting between HTF and AR-70 of bacteria grown in the absence (-) or presence (+) of desfeirioxamine. (a) shows silver enhanced reaction of Au-HTF with an iron regulated OMP from strain GN (C NT) at ca. 80 kDa following prior incubation of the membrane with AR-70. The reaction of AR-70 with the FeRP-70 was subsequently visualised with peroxidase conjugated goat antirabbit (b). The HTF-binding proteins of different strains of N. meningitidis and some commensal Neisseriae grown under iron restriction are also shown (c). FeRP-70 was subsequently visualised on this membrane with AR-70 and showed that none of the strains possessed an HTF-binding protein at the same position as the FeRP-70 (not shown).

In our previous reports, we showed complete cross-reaction of human acute and convalescent sera with FeRP-70 of different serogroups and serotypes [2], It appears that such fully cross-reactive antibodies can be raised in rabbits by injection with purified, reduced and denatured proteins, as opposed to incomplete cross-reactivity following the injection of intact whole cells [2], Finally, it is concluded that the 70 kDa iron regulated protein of N. meningitidis a human transferrin receptor and that

is not

HTF-binding protein is present on all examined

strains, but at different molecular weight levels. Although commensal Neisseriae appear to possess HTF-binding protein, it is not known whether they are able to chelate transferrin-bound iron. However, this binding implies that this ability of HTF-binding is not an essential virulence factor in strict terms. characterisation.

The HTF-receptor requires further

418

Acknowledgement

This work was supported by a grant from the Sir Halley Stewart's Trust.

References 1. Schryvers A. and Morris L. (1988). Molecular Microbiol. 2,281-288. 2. Ala'Aldeen DA, Wall RA and Bordello SP. (1990) / . Med. microbiol. 32, 275-282. 3. Laemmli U.K. (1970). Nature (London). 227, 680-685. 4. Ala'Aldeen D.A., Heather A. Davies, Wall R.A. and Borriello S.P. (1990). FEMS Microbiol. Lett. 69, 37-42. 5. Frens G. (1973). Nat. Phys. Sei. 241, 20-22. 6. Janssen manual (1985). Colloidal gold sols for macromolecular

labelling.

7. Moeremans M., Daneeis G., Van Dijck A., Langanger G. and De Mey J. (1984) J. Immunol. Meth., 74, 353-360.

Gonococcal unprocessed prepilin is transported to E.coti inner membrane

B. Dupuy, M.K. Taha and C. Marchai Unité des Antigènes Bactériens, Institut Pasteur, Paris, France.

Introduction Pili of Neisseria gonorrhoeae are important virulence determinants in that they mediate adhesion to human epithelial cells ( 1,2 ). Piliated gonococci have an advantage over non piliated variants in their attachement to various susceptible cell lines ( 3,4 ). Pili are composed of identical pilin subunits, with molecular weight varying from 17.000 to 22.000 D(5). Pilins from enterobacteria follow a classical secA dependent exportation pathway, synthetized as precursors with 20 to 30 amino acid long N-terminal signal sequences that are cleaved during cytoplasmic membrane translocation ( 6 ) . This signal sequence ressembles that of the majority of exported bacterial polypeptides, and contains a hydrophobic core preceded by a polar N-terminal short sequence and followed by the recognition site for signal peptidase. In contrast to this class of pilin, the sequence of gonococcal pilin deduced from that of pilin gene (pilE) predicts the existence of a short 7 amino-acid N-terminal extention which is absent in the mature polypeptide, Met-Asn-Thr-Leu-Gln-Lys-Gly- ( 7 ). Mature pilin contains a N-terminal methyl phenylalanine that is followed by a stretch of predominantly hydrophobic residues, which possibly represents the actual uncleaved signal sequence. These particularities are also found in pilins of various bacterial pathogens, including Pseudomonas aeruginosa, Bacteroides nodosus, Moraxella bovis and non

liquefaciens,

Neisseria meningitidis and Vibrio cholerae ( 8 ). This methyl phenyalanine class of pilin is then likely to follow a similar secretion pathway, and such specific functions have recently been demonstrated in Pseudomonas aeruginosa (9 ). To analyse the first step of gonococcal pilus biogenesis, i.e. pilin export, we took advantage of the fact that gonococcal pilin gene is expressed in E.coli but pilus biogenesis is not achieved and adddressed the following questions : 1) is gonococcal pilin unusual export signal functional in E.coli and 2) is the gonococcal pilin gene product cleaved at its usual site in E.coli ? Therefore we have determined the cellular location of the product of the gono-

Neisseriae 1990 © 1991 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

420 coccal pilE gene cloned in E.coli and purified this polypeptide from E.coli to determine its N-terminal sequence.

Results andDiscussion pNGllOO recombinant plasmid is a pBR322 derivative and contains the gonococcal pilin gene (pi/El) cloned from strain MS11 (10 ). This gene is expressed in E.coli, its own promoter is recognized and pNGl 100 determines pilin synthesis to a level of approximately 1% of total cellular proteins ( 1 0 ) . To determine the cellular location of this gonococcal pilin gene product expressed in E.coli by immunodetection, we have raised specific antibodies against gonococcal pili purified as previously described ( 11 ) by two subcutaneous injections to rabbits. Anti-pili antibodies were purified by preadsorption against E.coli DH5 proteins coupled to activated CH-Sepharose 4B (Pharmacia). E.coli DH5 cells harboring pNGllOO were grown to exponential phase, separated from the culture supernatant and sonicated. Soluble fraction representing cytoplasm plus periplasm, total membrane fraction as well as culture supernatant were analysed by western blot for their pilin content.

45K 34 7 K

^ •

24K

•

18.4K

•

14.3K

• I

1

2

3

4

5

6

Figure 1 : Western blot analysis of gonococcal pilin in E.coli DH5 (pNGl 100) fractions : (I) : purified pilin, (1) : culture supernatant, (2) : crude extract, (3) : soluble fraction = cytoplasm + periplasm, (4) : osmotic shock, (5) : total membranes, (6) : outer membrane fraction n°10 from Fig.2, (7): inner membrane fraction n° 31 from Fig.2. Samples from 2 to 8 correspond to 10^ cells equivalents except for sample 3 which is a crude extract of 10^ cells. No pilin was detected either in the supernatant or in the soluble fraction and 80 to 95% of pilin observed in a crude extract was present in the total membrane fraction (Fig. 1).

421 Identical washed cells were also passed through a French press, inner and outer membrane fractions were separated by isopycnic sucrose gradient centrifugation ( 1 2 ) and all gradient fractions were then analysed for the presence of the inner membrane NADH oxydase activity ( 1 2 ) and the outer membrane lambda receptor activity ( 1 3 ) .

34.7 K

-

24K

•

14.3K

I

1

4

7 10 13 16 19 22 2 5

28

31

34

Figure 2 : Gonococcal pilin immunodetection in inner and outer membranes of E.coli DH5 (pNGllOO). Isopycnic sucrose gradient fractions of membranes submitted to a French press were assayed for there protein content and NADH oxydase or lambda receptor (LamB) activities (upper part). The same fractions were analysed by western blot for their gonococcal pilin content (lower part). The fractions were subsequently analysed for presence of pilin by western blot procedure and Fig.2 indicates that the amount of pilin detected in the fractions is strictly proportional to

422

the NADH activity. As fractions 13 to 19 contain both inner and outer membrane markers, they are likely to contain vesicles of unseparated membranes. These results indicate that gonococcal pilin synthetized by E.coli. is transported to the cytoplasmic membrane and that it contains a signal sequence which is efficient in E.coli. But translocation through this membrane is interrupted as no pilin was detected in periplasmic, outer membrane or extracellular fractions. If gonococcal pilin remains associated with E.coli inner membrane, this may be the result of the existence of a gonococcal specific secretion machinery that participates in pilin secretion and which is absent in E.coli. In particular, we wanted to analyse here if gonococcal prepilin peptidase-like activity was present in E.coli or if prepilin would be cleaved at a non orthodox site, for instance by E.coli signal peptidase. To determine the N-terminal amino-acid sequence of E.coli inner membrane associated pilin, proteins from DH5 (pNGl 100) total membrane fraction were solubilized in 1% cholate buffer and dialysed against a 7M urea buffer before pilin immunoprecipitation using an excess of antibodies and protein A Sepharose CL-4B (Pharmacia). About 10 picomole of pilin was electroblotted onto immubilon P (Millipore) from a semipreparative 12% SDS -polyaerylamide gel and the blotted material was submitted to automated Edman degradation. 18 amino acids were sequenced (NH2"Met-Asn-Thr-Leu-Gln-Lys-Gly-Phe-Thr-Leu-De-Glu-Leu-Met-Ile-Val-IleAla-) and they exactly correspond to the N-terminus of the primary pilin gene product ( 7 ). Consequently, gonococcal pilin associated with E.coli inner membrane appears to be the precursor form of pilin and it seems likely that E.coli either is deficient in an activity which cleaves the seven-amino-acid residue peptide of prepilin, or contains such a peptidase in a more peripheral compartiment. This absence of processing may not be the reason for export interruption of prepilin in the inner membrane as export, maturation and assembly of gonococcal pilin may require a multicomponent system. A mutant in aeruginosa

Pseudomonas

deficient in prepilin processing was actually unaffected in the

compartimentalization of pilin ( 9 ) and this processing would rather be a prerequisite for pilin subunits assembly. Other gonococcal functions could be involved in normal export and we are currently developping a strategy using pilin-PhoA fusions to identify such functions necessary for gonococcal pilus biogenesis.

Acknowledgements This work was supported by I.N.S.E.R.M (CRE 893009), C.N.R.S. (UA 040557) and

423

C.C.A.R. (27790)

References

1.

Swanson, J. 1973. J.Exp.Med. 127: 571-589.

2.

Buchanan, T. M. 1977. In: The gonococcus (R.R. Roberts ed.), John wiley & Sons, Inc., New york p.255-272.

3.

Ward, M. E„ P. J. Watt, J. N. Robertson. 1974. J. Infect. Dis. 129: 650-659.

4.

Mardh, P. A., L. Westrom. 1976. Infect. Immun. 13: 661-666.

5.

Roberston, J. N„ P. Vincent, M. E. Ward. 1977. J. Gen. Microbiol. 102: 169-177.

6.

Oliver, D. 1985. Annu. Rev. Microbiol. 39: 615-648.

7.

Meyer, T. F„ E. Billyard, R. Haas, S. Storzbach, M. So. 1984. Proc. Natl. Acad. Sci. USA. 81: 6110-6114.

8.

Elleman, T. C. 1988. Microbiol. Rev. 52: 233-247.

9.

Nunn, D„ S. Bergman, S. Lory. 1990. J. Bacteriol. 172: 2911-2919.

10. Meyer, T.F., N. Mlawer, M. So. 1982. Cell. 30: 45-52. 11. Brinton, C.C., J. Bryan, J. A. Dillon, N. Guernia, L. J. Jackobson, A. Labik, S.Lee, A. Levine, S. Lim, J. McMichael, S. A. Polen, K. Rogers, A. C.-C. To, S. C.-M. To. 1978. In: Immunology of Neisseria gonorrhoeae . (G.F. Brooks, E.C. Gotschlich, K. K Holmes, W.D. Sawyer, F.E. Young eds.). American Society For Microbiology, Washington DC. p. 155-178 12. Osborn, M. J., J. E. Gonder, E. Parisi, J. Carson. 1972. J. Biol. Chem. 247: 3962-3972. 13. Marchal, C„ M. Hofnung. 1983. EMBO. J. 2: 81-86.

Illegitimate Recombination and Gonococcal Pilin Gene Variation

Stuart A. Hill, Sandra G. Morrison and John Swanson NIAID Laboratory of Microbial Structure and Function, Rocky Mountain Laboratories, Hamilton, MT 59840

Introduction Gonococcal pilin gene variation is believed to occur by a unidirectional, intra-genomic transfer of genetic information from one of several silent pilin genes (piiS) that replaces the resident information at the single pilin expression locus (pilE); the outcome resembles classical gene conversion (1-3).

However, inter-genomic recombination between pilS and

pilE mediated by DNA transformation has recently gained favor (4, 5). The studies of Swanson et al. (6) support an alternative mechanism (s) to DNA transformation for pilin variation when they

demonstrated

equivalent frequencies and types of pilin gene changes in transformationdefective gonococci (dud phenotype [7]) when compared with wild-type. In the present study we present evidence that internal deletions of piZE can influence pilus+ (P+) to pilus- (P-) transitions and that repair of internal deletions produce novel intact piE's. The gonococcal pilE locus is particularly prone to deletions with two types predominating (8, 9).

(i)

The most common piiE

deletion

removes pilE promoter regions, resulting in gonococci that are permanently nonpiliated (P-n phenotype. Fig. 1A). P-n formation is mediated by an illegitimate recombinatorial event between direct repeats (data not shown) (10) and occurs whether gonococci contain a wild-type or mutant recA allele (data not shown),

(ii) An alternative pilE deletion

results in internally deleted pilEs (P-a phenotype), such that 5' and 3' flanking regions remain intact ( Fig. IB) (9). Such deletions can affect P-

Neisseriae 1990 © 1991 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

426

to P+ reversions and P+ to P- phase transitions in gonococci (9). In all cases, the deleted segments were bracketed by direct oligonucleotide repeats (arrows. Fig. 1), with a single repeat element remaining in the internally deleted piIE. A.

m-%

U

pllS

mr-urn

promoter!«»« piiH (P-n) b.

m-Q

U pllx

HIIIIIIK^a-» -*-

"V —-G—U

m - ^

intamally dalatad plix (P-a) Fig. 1. Schematic representation of (A) P-n formation and (B) P-a formation. In both cases deletions occur between direct oligonucleotide repeats (arrows) with a single repeat element remaining in the deleted pi£E.

Results and Discussion Internal pilE deletions (P-a phenotype) can be repaired by novel pilS sequence resulting in intact pilin genes containing unique sequence (9). MS 11 P-a variant (4:8) is internally deleted for approximately the entire hypervariable region and for sequence that encodes the upstream cysteine residue (Fig. 2 and 3). Three P+ revertants were derived from

427

4:8, mRNA's were sequenced from each revertant and each was found to consist of an intact pilE containing novel pilS sequence (Fig. 3). In each case, the 5' end was derived from the parental 4:8 sequence, with the 3' end being derived from the pilS located 3' to pilE

(see Fig. 2); the

stretch of nucleotides that repaired the internal deletion apparently arose from some unknown pilS (see Figs. 2 and 3).

MSll

4.8

parent

—Q-C Xntarnally ^Mnt 1 repairing pilS sequenc*

H Fig. 4. P i E Deletion/Repair Model. A schematic representation of the formation and repair of an internally deleted p i E (P-a), which results in a functionally intact pilin gene containing unique sequence. The various shading patterns indicate sequence changes at p i E in the semivariable and hypervariable regions of the gene after an internal deletion between the direct repeat (arrows).

430

Acknowledgement We would like to thank Susan Smaus for secretarial assistance in the preparation of this manuscript.

References 1. Haas, R. and T. F. Meyer. 1986. Cell 44, 107-115. 2. Meyer, T. F. 1987. Trends Genet. 3, 319-324. 3. Swanson, J. and J. M. Koomey. 1989. In: Mobile DNA, (D. E. Berg, and M. M. Howe, eds.). American Society for Microbiology, Washington, DC. pp. 743-761. 4. Seifert, H. S., R. S. Ajioka, C. Marchal, P. F. Sparling, and M. So. 1988. Nature 336, 392-395. 5. Seifert, H. S. and M. So. 1988. Microbiol. Rev. 52, 327-336. 6. Swanson, J., Morrison, S., Barrera, O. & Hill, S. (1990) J. Exp. Med. 171, 2131-2139. 7. Biswas, G. D., S. A. Lacks, and P. F. Sparling. 1989. J. Bacterid. 171, 657-664. 8. Swanson, J., S. Bergström, O. Barrera, K. Robbins, and D. Corwin. 1985 J. Exp. Med. 162, 729-744. 9. Hill, S. A., S. G. Morrison, and J. Swanson. 1990. Molec. Microbiol. 4, 1341-1352. 10. Segal, E., E. Billyard, M. So, S. Storzbach, and T. F. Meyer. Cell 40, 293-300.

1985.

11. Ehrlich, D. 1989. In: Mobile DNA, (D. E. Berg, and M. M. Howe, eds.). American Society for Microbiology, Washington, DC. pp. 799-832.

Translatìonal Frameshifting in pilC Encoding a Gonococcal Outer Membrane Protein Results in Pili Phase Variation.

A.B. Jonsonn, G. Nyberg Department of Microbiology and Immunology, Umea University, S-901 87 Umea, Sweden. S. Normark Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110.

Introduction The only documented virulence factor in Neisseria gonorrhoeae is the expression of pili (1). Gonococcal pili are structurally related to pili expressed by Bacteroides nodosus, Moraxella bovis, Pseudomonas aeruginosa and Vibrio cholerae (2). It is widely held that these so called type 4 pili promote bacterial attachment to host cells. In no case has a specific receptor been identified for type 4 pili and it is still unclear whether pilus attachment is mediated by the major pilin subunit or by a minor pilus associated adhesin. However, most recent data favor the hypothesis that the major pilin subunit carries receptor binding properties. The biogenesis pathway for type 4 pili is yet unknown but it seems likely that it shows similarities to the pathway worked out for E. coli pili such as type 1 and Pap. Pap pili are heteropolymeric fibers containing one major and several minor subunits one of which is the receptor binding adhesin (3). Both major and minor subunits are secreted across the cytoplasmic membrane by a normal SecA dependant process. Prior to processing of the signal peptide the subunits form dimeric complexes with a periplasmic chaperone protein that probably enhances post secretional release and

Neisseriae 1990 © 1991 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

432

folding (4). The successful assembly of Pap pili requires the expression of an 88kD large outer membrane protein. The exact role of this protein is unknown, but it is thought to act as an assembly center for subunit-chaperone complexes and/or a pore for the assembling pilus (5). We anticipated that the assembly of gonococcal pili also depended on a large molecular weight outer membrane protein and have therefore characterized a llOkD protein that is present in highly purified preparations of pili from N. gonorrhoeae strain MS 11.

Results and Discussion Pili purified from strain MS11(P + ) contains minor amounts of a llOkD protein. Antiserum was generated against the gel purified protein and used in Western blots against different strains of N. gonorrhoeae.

All but one strain contained one or two

proteins around 1 lOkD in size that reacted with the antiserum. The strain that did not react was a nonpiliated P"n variant. Western blots showed that outer membranes prepared from both MS 11 (P + ) and MS 11 (P'n) contained the llOkD protein. A Xgtll library from strain MS 11 (P + ) was screened by the llOkD antiserum. One clone was found reacting with the antiserum. The 800bp insert from this clone was used to search a plasmid library prepared from strain MS 11. Several overlapping clones were found one of which, pABJ04, expressed minor amounts of a llOkD protein in E. coli minicells. This protein was denoted PilC. Its structural gene was subsequently sequenced. The 5' end of pilC including the initiation codon and the major part of the signal sequence was not in frame with the rest of the pilC gene.

Moreover, the aminoterminal

sequence of the gel purified PilC from MS 11 was not absolutely identical to that deduced from the nucleotide sequence. This difference in sequence was explained by

433

the presence of two pilC loci in the MSI 1 genome. To see if the cloned pilC 1 allele was expressed or not in strain MS 11 we generated mini Tn3 insertions in pilC 1 by allelic replacements. MSI 1 mutants in pilCl still expressed PilC suggesting that pilCl is the active locus in the MS 11(P+) variant under study. PilCl on pABJ04 contained a tract of twelve G- residues in the region encoding the signal peptide. An addition of one G or loss of two would place the pilC open reading frame in frame with its AUG initiation codon. PCR amplification and subsequent DNA sequencing was performed on the 5' region of pilC from strain MSI 1. Sequences corresponding to pilCl contained 11 or 12 G's and were therefore always out of frame whereas sequences corresponding to pilC2 contained 12 or 13 G's in the G tract. Since strain MS 11 (P + ) expresses PilC from pilCl we suggest that the cells under study contain predominantly 13G's in pilCl and 12 in pilCl. We obtained similar results with PCR amplified fragments from other strains of N. gonorrhoeae.

One strain did not express the 1 lOkD PilC protein nevertheless it

contained two pilC loci. However, we obtained no amplified fragments where pilC was in frame with its initiation codon. These data clearly indicated that pilC expression was controlled by frequent frameshift mutations in the G- tract. Since, we were able to obtain frameshifting also in recA E. coli, we believe the mechanism to be due to recombination indépendant slipped strand mispairing as has been proposed for the phase- and antigenic variation of opa genes (6). If PilC is essential for pili biogenesis and if pilC undergoes phase variation one would expect some phaseswitching of pili expression to be due to switching in pilC expression. We have isolated a number of nonpiliated (P~) variants from MSI 1 (P+). Five out of eight P" descendants that still expressed pilin did not react with the PilC antiserum in Western blots. Piliated (P + ) revenants were isolated from P", PilC" clones. In all cases did we regain expression of PilC. The loss of piliation in PilC" clones could be due to coupled rearrangements in the pilE gene resulting in an assembly defective pilin. If so P+, PilC + , revenants should be

434 expected to contain alterations in pilE. We have sequenced the pilE gene from one such P", PilC —> P+, PilC + pair and found no differences in the coding sequence. We therefore suggest that successful assembly of gonococal pili requires the expression of PilC, and that pili phase variation can be caused by varied expression of PilC. It is not known why most gonococcal isolates contain two loci of pilC. We know that pilC\ and pilCl in MS 11 are highly homologous in their 5' regions but differ markedly, in their central and 3' regions. It may be that the benefit of two loci is to increase the antigenic variability of PilC. Alternatively, the two loci may encode proteins with slightly different properties. Perhaps the assembly of only certain variants of pili can take place with either of the two loci.

Acknowledgements The molecular cloning of pilCl from a plasmid library was performed by Ann-Beth Jonsson during a stay in the laboratory of Dr. Thomas Meyer, Max-Planck-Institut fur Biologie, Tubingen, West Germany. The help of Drs. Thomas Meyer and Carol Gibbs are gratefully acknowledged.

References 1.

Kellogg, D.S., J.R. Cohen, L.C. Norins, A.L. Schroeter, G. Reising. 1968. J. Bacteriol. 96: 596.

2.

Elleman, T.C. 1988. Microbiol. Review 52: 233.

3.

Lindberg, F.P., B. Lund, S. Normark. 1987. Nature 328: 84.

4.

Hultgren, S.J., F. Lindberg, G. Magnusson, J. Kihlberg, J.M. Tennent, S. Normark. 1989. Proc. Natl. Acad. Sci. USA 86: 4357.

5.

Norgren, M., M. Baga, J.M. Tennent, S. Normark. 1987. Molec. Microbiol. 1: 169.

6.

Murphy, G.L., T.D. Connell, D.S. Barrit, M. Koomey, J. G. Cannon. 1989. Cell 56: 539.

Iron and meningococcal

disease. S e r u m l a c t o f e r r i n in m e n i n g o c o c c a l

disease

patients a n d controls.

Anne-Brit Kolst0 The Biotechnology Centre of Oslo and Institute of Pharmacy, Department of Microbiology, University of Oslo, N 0371 Oslo 3 Teije O. R0d Department of Infectious Disease Control, National Institute of Public Health, N 0462 Oslo 4, and Kaptein W . W i l h e l m s e n og Frues Bakteriologiske Institutt, University of Oslo, Rikshospitalet, N 0027 Oslo 1, Norway

Introduction The relation between iron and meningococcal disease may be studied by looking at the levels of iron and iron-binding proteins in serum during infection. In a previous report, S-(serum) ferritin, S- iron, and S- TIBC (total iron binding capacity) values in patient samples drawn on admission to hospital were studied (1). Values of S- lactoferrin and possible connections to other iron-binding proteins are discussed in the present report.

Materials and M e t h o d s Data and serum samples were collected from December 1981 to April 1982, as described previously (1, 2, 3). Serum samples were collected from 115 meningococcal disease patients, 61 patient controls, and 293 population controls. Samples f r o m patients were drawn on hospital admission and after one and six weeks. S - lactoferrin was measured by enzyme-linked immunosorbence assay (ELISA) as described by Otnaess et al. (4). S- ferritin, S- iron, and S- TIBC was determined as described previously (1). Levels of white blood cells were determined as described by Otnaess et al. (4). Statistical analyses were performed by using the p r o g r a m m e Data Desk, Odesta Corporation, Northbrook, IL 60062, U.S.A. Means were compared by using Student t-test and analysis of variance. Correlations were studied using Spearman rank correlation test.

Results Patient s a m p l e s c o m p a r e d to population controls. The mean S- lactoferrin value in patient samples drawn on admission to hospital was significantly higher (P=0.013) than the mean value in samples from population controls. A significant difference between mean Slactoferrin values was not found (P>0.05) when patient samples drawn after 1 week were

Neisseriae 1990 © 1991 by Walter de Gruyter & Co., Berlin - New York - Printed in Germany

436

Admittance to hospital

S rasasi

ssusa^—,

,

.rei.

Q HZL

44 - |

—

—

CM

CM

IO

—

—

cm

cm

m

JEZL a
in in m

\r co o co

cn co in cd

r--

CO

CO

CT>

(T>

m

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m

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cr>

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S- lactoferrin, mg/1

F i g u r e 1. S - lactoferrin in s a m p l e s f r o m m e n i n g o c o c c a l d i s e a s e p a t i e n t s and p o p u l a t i o n controls. Patient s a m p l e s w e r e d r a w n on admission to hospital, and after 1 and 6 w e e k s .

437 compared to samples from population controls. S i x weeks after admission to hospital the mean S - lactoferrin value was significantly lower ( P = 0 . 0 1 3 ) than the mean value in samples from population controls (Table 1). T h e distribution o f S - lactoferrin levels in patients and population controls is shown in Fig. 1. T a b l e 1. S - lactoferrin (mg/1) in meningococcal disease patients and population controls. Patients Admission to hospital After one week After six weeks Population controls

N 49 27 23 220

range 5-99 5-70 2-66 1-79

median 21 17 12 19

mean 29.8 20.6 15.3 23.3

SD 22.6 13.9 14.2 14.6

Number o f persons (N), range, mean scrum level, median, and standard deviation ( S D ) are given. Samples from patients were drawn on admission to hospital, after one week, and after six weeks.

D e v e l o p m e n t o f s e q u e l a e c o m p a r e d to f a v o u r a b l e o u t c o m e . In a previous study (1), patients developing sequelae were found to have particularly high values o f S - ferritin, and particularly low values o f S - T I B C . In the present study, no significant difference in Slactoferrin values was found ( P > 0 . 0 5 ) when patients developing sequelae were compared to patients with a favourable outcome (Table 2). T a b l e 2. S - lactoferrin in meningococcal disease patients developing sequelae and patients with a favourable outcome. Stage of sampling Admission to hospital After one week After six weeks

Outcome Favourable outcome Sequelae Favourable outcome Sequelae Favourable outcome Sequelae

N 22 8 14 7 12 7

range 7-99 5-99 5-38 8-42 3-66 6-16

mean 28.6 31.5 17.3 16.4 19.3 10.9

median 26.5 16 15.5 12 11.5 12

SD 21.3 31.0 9.80 11.8 18.4 3.63

Number of persons (N), range, mean scrum level, median, and standard deviation ( S D ) are given.

Correlations

between

S-

ferritin,

S-

iron,

S-

lactoferrin,

S-

TIBC

values.

Correlations between S- lactoferrin values in patient samples drawn on admission to hospital (Table 3), and between S - ferritin, S - iron, S- lactoferrin, and S - T I B C values in population controls (Table 4) were studied using Spearman rank correlation test. Correlations o f S lactoferrin values in patient samples drawn on admission and after 1 week and six weeks were all above 0 . 9 5 (Table 3). S - ferritin, S - iron, S- lactoferrin, and S- T I B C values in patient samples drawn on admission to hospital were studied. A considerable negative correlation, - 0 . 5 0 6 , was observed between S - ferritin and S- T I B C . T h e other correlations were between 0 . 2 6 1 and - 0 . 1 2 3 (Table 3). Correlations between S- ferritin, S- iron, S- lactoferrin, and S - T I B C values in samples from healthy control persons were between 0 . 1 8 0 and - 0 . 2 5 4 (Table 4). W h i t e blood cell counts. The correlation between S - lactoferrin and the number o f white blood cells in patient samples on admission was negligible ( - 0 . 0 4 5 ) . A g e , p o p u l a t i o n c o n t r o l s . Analysis o f variance showed significant differences between mean S - lactoferrin levels in population control samples from different age groups ( P = 0 . 0 1 7 ) . L e v e l s were found to increase with age (Table 5 and Fig. 2). S - ferritin levels

438 Table 3. Correlations between S- ferritin, S- iron, S- lactoferrin, S- TIBC values in patient samples. S- ferritin S- lactoferrin S- iron S- lactoferrin 0.230 S- iron 0.097 -0.123 S- TIBC -0.506 0.261 0.028 The correlations were performed on the values in samples drawn on admission to hospital, and were studied using Spearman rank correlation test.

Table 4. Correlations between S- ferritin, S- iron, S- lactoferrin, S- TIBC values in population controls. S- lactoferrin S- iron S- TIBC

S- ferritin 0.057 0.180 -0.254

S- lactoferrin

S- iron

0.039 -0.042

-0.139

Correlations were studied using Spearman rank correlation test.

were also found to increase with age (P=0.0001), whereas S- TIBC decreased with age. The mean S- iron levels did not show significant age differences (P=0.1902). Sex, population controls. Significant differences in S- lactoferrin values between females and males were not found in any age group (P>0.05). The mean S- ferritin value in males in the age group 24-49 years was significantly higher (P=0.0001) than the corresponding value in females. Significant differences in mean Siron, S- lactoferrin, or S- TIBC values were not found in any age group. 800 + c"

80_E

'E* 25 m+f 59 31 11 - 7 6 3 87.5 135.3 m 11 22 - 175 111.1 39 54.5 f 20 11 - 7 6 3 74.6 32.5 163.9 62.2 25-49 m+f 20 11 - 155 49.5 49.7 m 7 40 - 155 112.0 139 46.2 f 35.4 13 11 - 8 2 25 24.8 ¿50 m+f 11 12 - 7 6 3 133.7 68 215.9 4 22 - 175 109.5 m 120.5 75.0 f 7 12 - 7 6 3 147.4 58 272.5

N mean median Sex range m+f 12 1 -44 23.3 21 m 15.7 3 12 - 18 17 f 9 1 -44 25.8 28 1-11 m+f 64 1 -41 21.9 21 m 1 -40 33 21.2 20 f 31 51 - 9 6 22.6 24 12-24 m+f 105 24 1 - 124 25.8 m 1 - 24 61 26.0 25 f 44 1 - 55 25.5 23.5 m+f 31 13 - 4 7 >25 25.8 23 m 11 13 - 47 27.7 24 f 20 13 - 4 3 24.7 22.5 26.4 25-49 m+f 20 13 - 4 7 23.5 m 7 21 - 47 32.4 27 23.2 f 13 13 - 4 3 21 >50 m+f 11 13 - 4 3 24.6 23 4 13 - 2 3 m 19.5 21 f 7 18 - 4 3 27.6 25

S-

S- TIBC,

Age 0

lactoferrin,

Age 0

Sex m+f m f 1-11 m+f m f 12-24 m+f m f >25 m+f m f 25-49 m+f m f >50 m+f m f

N 17 5 12 67 34 33 106 61 45 30 9 21 20 6 14 10 3 7

mg/1

range 3 -44 3 - 32 5 -44 3 - 51 1 -40 3 - 51 1 -79 1 -79 7 -74 8 -53 12 - 53 8 -53 8 - 53 14 - 53 8 - 53 12 - 4 7 12 - 2 9 16 - 4 7

mean 17.1 13.6 18.6 20.1 20.2 19.9 25.6 24.3 27.4 25.9 28.0 25.0 24.9 30.5 22.5 27.8 23.0 29.9

median 14 14 13 18 18 15 20 20 21 23 28 21 22 31 19.5 28.5 28 35

SD 12.9 11.5 13.7 12.4 11.3 13.7 15.9 15.4 6.48 3.15 13.4 13.3 13.8 15.1 13.1 12.1 9.54 13.2

Age 0

Age 0

Sex m+f m f 1-11 m+f m f 12-24 m+f m f >25 m+f m f 25-49 m+f m f >50 m+f m f

SD 11.9 3.21 12.7 8.53 8.39 8.75 14.0 15.8 11.3 9.75 10.7 9.27 10.3 10.5 8.97 8.98 4.51 9.83

(imol/1 N 12 9 3 64 33 31 105 61 44 31 11 20 20 7 13 11 4 7

range m e a n 51 - 87 67.2 51 - 87 65.4 5 9 - 80 72.3 51 - 96 67.3 5 2 - 92 65.9 51 - 96 68.6 44 - 90 65.0 44 - 86 64.5 4 8 - 90 65.7 47 - 76 62.0 51 - 73 63.2 4 7 - 76 61.3 47 - 76 62.3 5 6 - 73 63.0 4 7 - 76 61.9 4 8 - 70 61.4 51 - 68 63.5 4 8 - 70 60.1

median 66.5 61 78 67 67 69 65 65 65 62 64 61.5 61.5 61 62 66 67.5 57

SD 11.5 11.7 11.6 9.19 8.27 10.0 8.69 8.56 8.92 7.61 6.82 8.10 7.64 6.53 8.40 7.89 8.35 8.01

Levels in both sexes combined (m+f), and separate levels in males (m) and females (f), are reported. Number of persons (N), range, mean, median, and suindard deviation (SD) arc given.

440 Discussion S- lactoferrin values in patient samples drawn on admission to hospital were significantly higher than values in samples f r o m population controls, whereas patient samples d r a w n after six w e e k s w e r e significantly lower. In a study of S- lactoferrin in neutropenia, lactoferrin displayed a clear correlation with neutrophil counts (5). S- lactoferrin is considered to derive f r o m leakage of more mature granulopoietic cells of blood and marrow (5). S- lactoferrin has been f o u n d to be extremely low in profound neutropenia, and neutrophilic granulocytes and their precursors are probably major sources of S-lactoferrin (6). In a study of the neutrophil lactoferrin content (7), the level in m a l e s and postmenopausal females was found to be significantly higher than in pre-menopausal females. In the present study, the mean level of serum values reported in post-menopausal f e m a l e s (35.0 mg/1, age>50) was higher than in pre-menopausal females (27.4 mg/1, age 12-24 ; 22.5 mg/1, age 25-49), but differences were not significant (P4 (lOOmM), pH 6, temperature below 37°C, high osmotic pressure (NaCl or KC1 at 300mM, sucrose at 600mM, urea at 25 mg/ml ). The addition of choline, proline or glutathion in a medium with high osmolality did not restore normal growth in contrast with the situation found in E. coli (13,14). Some of these conditions correspond to those found in urine (proline, isoleucine, cysteine auxotrophy or high concentration of glycine ) which appears unfavourable for growth of N. gonorrhoeae. CAT assays after 24 h of growth on minimal or complete medium with the different fusions pilE-CAT are indicated in Table 1. TABLE 1: CAT Activity (Units / mg protein) on Minimal and Complete Medium G Strains

Minimal medium

Complete medium G

MS11.CAT-1

300

235

C6-1. CAT-1

250

200

MS 11.CAT-2

200

150

MS11.CAT-2X

460

346

1 CAT unit = 1 nmole of chloramphenicol acetylated per minute at room temperature.

The addition of some amino acids (3mM) in minimal medium (alanine, asparagine, aspartic acid, glutamic acid, glutamine, methionine) lowered CAT activity to less than 50% of the control in minimal medium. These results differ from those described by Kamal et al. (10) who have used a complex medium and observed the phenotype of colonies, except for alanine, aspartic acid and methionine. The effects of pH, temperature and limiting factors are reported in Table 2. Low pH which corresponds to the mean value of urine and the limitation of dextrose decreases the

443

expression of the piTE-CAT fusions.

TABLE 2 : Percentage CAT Activity in Various Conditions Strains MSI 1.CAT-1

pH 6

pH 8

T30°

30

94

60

FeN(

Dextrose

Cystine

limiting

limiting

59

98

86

C6.1.CAT-1

37

85

45

ND

ND

76

MS11.CAT-2

41

107

80

64

64

62

MS11.CAT-2X

17

108

60

ND

ND

80.

Cat activity is expressed as % of the activity observed on standard minimal medium.

The reduction of CAT activity is more pronounced in condition of high osmolality especially in minimal medium (Table 3). Effects of compounds that are osmoprotectants for Enterobacteriaceae and Pseudomonadaceae were tested and choline, proline or glutathion did not restore normal growth and we are testing other products like betaine. TABLE 3 : Percentage CAT Activity in High Osmolality in minimal (M) or G medium Strains

+KC1 (300 mM) G

+NaCl

+Sucrose

Urea

(300 mM)

(600 mM)

(400 mM)

M

G

M

G

M

M

17

42.5

15

58

22

2

21

31

MS11. CAT-1

51

16

45

C6. 1. CAT-1

66

23

33

MS 11. CAT-2

41

14

25

16

5

2

42

MS11; CAT-2X

88

27

68

21

26

30

43

To analyse whether some of the conditions that affect pilin gene expression involve the pilA-pilB

regulatory system, we have tested the effect of changes in osmolarity on

gonococcal protein expression by comparing a pilB' mutant to wild type. We have proposed that PilB plays the role of a sensor and affects PilA activity in response to environmental variations. We have compared crude extracts from pilB+and pilB' strains grown at low or high osmolarity, on a Coomassie blue stained SDS-PAGE gel (Figure 1).

444 Three situations appear : some proteins are induced by high osmolality in a pilB dependent manner, others are induced by high osmolality in a pilB independent manner and a last group are induced by low osmolality in a pilB independent manner.

~«66K -«45 K -«34 K

M

K

S

C4-1

K

M

S

C4-1(pi|B")

M

K

M S 11

S

M

K

MS11(pilB~)

Fig 1. Crude extracts of pil B + and pil B~ strains grown at low or high osmolality were compared on Coomassie blue stained SDS-PAGE gel

These results indicate that pilB participates in the adaptative response to osmotic stimuli. Its direct effect on piliation is currently being analysed by western blot and electron microscopy. In all conditions tested, the expression of the CAT-piYE fusions is reduced or invariant. Culture conditions that would increase the expression of pilin have not yet been found. These preliminary results indicate that Neisseria gonorrhoeae shares some common features with Vibrio cholerae (15) in which expression of virulence factors including pili is regulated in response to a wide spectrum of compounds : nicotinic, phenolic and amino acids, temperature , pH and osmoactive products.

445 Acknowledgements This work was supported by I.N.S.E.R.M (CRE 893009), C.N.R.S. (UA 040557) and C.C.A.R. (27790)

References 1. Swanson, J. 1973. J. Exp. Med. 127 : 571-589. 2. Pearce, W. A., T.M. Buchanan. 1980. In : Bacterial adherence ( Beachey, E. ed.), Chapman and Hall, New York p. 291-344. 3.

Bergström, S., K. Robbins, Koomey, J.M., Swanson, J. 1986. Proc. Natl. Acad. Sei. USA, «5:3890-3894.

4. Haas, R„ H. Schwarz, T.F. Meyer. 1987. Proc. Natl. Acad. Sei. USA, 54:9079-9083. 5.

Segal, E„ E. Billyard, M. So, S. Storzbach.T.F. Meyer. 1985. Cell, 40:293-300.

6. Taha, M.K., M. So, H.S. Seifert, E. Billyard, C. Marchal. 1988. Embo. J.7 :4367-4378. 7. Taha, M.K., B. Dupuy, W. Saurin, M. So, C. Marchal. 1990. Mol.Microbiol. (in press). 8.

Keevil, W. C„ N. C. Major, D. B. Davies, A. Robinson. 1986. J. Gen. Microbiol. 132: 3289-3302.

9.

Keevil, W. C„ D. B. Davies, B. J. Spillane, E. Mahenthiralingam. 1989. J. Gen. Microbiol. 135 :851-863.

10. Kamal, U„ S. Saikh, S. Mitra, F. K. Bhattacharyya. 1989. J . Clinic. Microbiol. 27 : 1090-1094. 11. Hendry, A. T . , I. O. Stewart. 1979. Can. J. Microbiol. 25 :512-521. 12. Bhatnagar, R. K., A. Berry, A. T. Hendry, R. A. Jensen. 1989. Molec. Microbiol. 3 :429-435. 13. Csonka, L. 1989. Microbiol. Rev. 53 : 121-147. 14. Laggan, D„ T. M. Loggan, D. G. Lynn, W. Epstein. 1990. J. Bacterid. 172 :3631-3636. 15. Miller, V. L . , J. J. Mekalanos. 1988. J. Bacteriol. 170: 2575-2583.

Antigen Variation and Transformation-Mediated Horizontal Exchange in the Pathogenic Neisseria

T.F. Meyer Max-Planck-Institut für Biologie, Abteilung Infektionsbiologie, Spemannstrasse 34, D-7400 Tübingen, F.R.G.

Introduction Two basic mechanisms have been exploited by microorganisms to modulate their response to environmental changes, gene regulation via the interaction of proteins with DNA and gene rearrangements involving sequence alterations at the DNA level. In Neisseria gonorrhoeae both principles can be observed. In particular the analysis of gene rearrangements underlying the variation of surface proteins make this species an interesting model (for review see Ref. 1). The molecular processes involved in antigen variation are again two fold and can be discriminated on the basis of whether RecA protein is required or not. This short review stresses some aspects of the RecA dependent processes in Neisseria gonorrhoeae that, for example, concern the variation of pilin. Recent data from this laboratory suggest that transformation-mediated exchanges play a central role in these recombination processes and consequently in the modulation of virulence determinants.

Results and Discussion Generation of Variant Pilin Forms in N. gonorrhoeae The phenomenon of pili phase and antigenic variation of N. gonorrhoeae pili has become evident from the work of several groups during the past two decades. Gonococci are able to switch off and on the production of pili and can further vary the functional and antigenic properties of these organelles, which are important in the binding of gonococci to human tissues. The high capacity of gonococcal pili to undergo

Neisseriae 1990 © 1991 by Walter de Gruyter& Co., Berlin • New York - Printed in Germany

448

variation is based on the existence of a family of genes (pi/) which code for the major (possibly sole) subunit of pili, the pilin. A gonococcus possesses one (occasionally two) structural genes (pilE) on its chromosome which are responsible for pilin production and several silent loci (pilS), which code for variant pilin sequences but which are not expressed. In the majority of cases phase and antigenic variation of gonococcal pili is due to changes in the pilin subunit and hence associated with changes in the pilin expression gene (pilE). These changes are caused by intragenic recombination events between pilE and one or several of the variant silent pilS genes. Such recombination events seemed to occur in a non-reciprocal manner, suggesting gene conversion as the principle. As discussed below, gene conversion may however, not be the real mechanism of pilin variation. Based on the fact that a gonococcal cell usually contains more than 10 different silent pilus genes, each consisting of 6 minicassette units which can be reassembled in the pilE locus, it is conceivable that a defined strain can generate more than one million variant pilin types. The outcomes of such reorganizations are not only differences in the immunological and functional properties of pili (i.e. antigenic variation) but occasionally the loss of pili formation (i.e. phase variation). The latter usually occurs in either of two ways (i) if the resulting pilin is processed in such a way that a small secreted pilin (S-pilin) is produced which is unable to polymerize into pili or (ii) if the pilin produced is extra-long (L-pilin) such that it is neither assembled into pili nor extracellularly secreted (2). Thus, both phase and antigenic variation of pili can be explained by recombinatory changes in the pilin expression gene.

The Transformation-Mediated Pathway of Pilin Variation An interesting feature associated with gonococcal L-variants is their increased resistence to certain antibiotics, including kanamycin and penicillin (2, 3). This observation led us to monitor the rate of phase transitions by determining the spontaneous generation of kanamycin resistant variants.

449

Several years ago Norlander and collegues observed that the rate of pilus-specific colony variation in a gonococcal culture was reduced on the addition of pancreatic DNase I (4). This finding could be interpreted as indicating that free DNA played a role in pilin variation, a notion that initially received little attention. With our simplified screening method for the identification of phase variants it was possible to determine precisely the frequency of pilin phase variation and also to study the potential effect of DNase I. Preliminary experiments supported the original observations that under certain growth conditions, the addition of DNase I resulted in a reduction in the number of L-variants obtained, although the change to the L-phase was never completely repressed. Similar results were also obtained by Seifert and coworkers who showed, using an indicator gene, that the transfer of pilin genes between two gene loci is reduced in a transformation deficient gonococcal mutant (5). These observations independently led to the conclusion that in a gonococal culture, free (DNase I-accessible) DNA is present that can contribute to the pilin variation. This appears plausible in the light of two commonly observed phenomena: firstly that gonococci are prone to autolysis in culture (6), thus liberating the whole chromosome, and secondly that Neisseria are naturally competent for the uptake of sequence specific DNA (7). Assuming that transformation dependent recombination is the only mechanism, the question is raised of why pilin variation occurs, when this pathway has been blocked. We therefore studied L-variants obtained either in the presence or the absence of DNase I, with respect to their hybridisation patterns for silent and expressed pilin genes. Suprisingly, those L-variants obtained in the presence of DNase I had not undergone the normal non-reciprocal recombination but instead had undergone reciprocal intrachromosomal recombination between two pilin genes. On the basis of these observations we could conclude that two distinct pathways for the recombination of pilin genes exist; reciprocal, intrachromosomal recombination and transformation mediated recombination. The latter process, which was previously interpreted as gene conversion, appears to be more frequent and only when it is blocked by the action of DNase I or by mutations preventing transformation does the intrachromosomal process become apparent (3). Therefore, when pilin variation via transformation is unfavourable in the infected host, the pathogen can choose an alternative intrachromosomal route. This conclusion is supported by recent observations with

450

transformation defective (dudI) gOnococci which suggest that transformation competence is not a prerequisite for pilin variation (8).

Horizontal transformation-mediated exchange in vivo The elucidation of the above described transformation process involved in gonococcal pilin variation is entirely based on in vitro experiments. It is thus difficult to assess whether under natural conditions DNA can be transferred from one cell to another without being destroyed by DNase. Recent work from this and other laboratories, however, suggests that horizontal exchange of genomic DNA segments does indeed occur between Neisseria strains in vivo. During our investigations on the genetics of IgA protease, another neisserial virulence factor, evidence was obtained for the occurence of horizontal exchange in vivo. The gene for IgA protease (iga) is, in contrast to the pil or opa genes, only found as a single copy on the chromosome. The iga genes, however, exist in different alleles among independent isolates (9). Comparative sequence analysis of four such alleles revealed a partial but perfect conservation of polymorphic sequences in some, but not all, of the investigated genes. It is inconceivable that the observed mosaic distribution of polymorphic regions in the iga gene arose by spontaneous mutation, e.g., driven by convergent selective forces. Therefore an extensive genetic exchange between gonococc, during the recent evolution of the iga genes must be assumed. Due to the extreme host specificity of the gonococcus these genetic exchanges likely occurred in individuals experiencing simultaneous infections with different strains. The discovery of specific DNA sequences that function as signals for the specific uptake of DNA by Neisseria suggests that the exchange between iga genes occurred by transformation. This is supported by the fact that transformation signals are located at the end of all iga genes so far studied, in association with the transcriptional terminators (9, 10). Similar observations were made for horizontal exchange between different Neisseria species. In this case transfers involving the chromosomal penA determinant have occurred from the commensal bacterium Neisseria flavescens to Neisseria meningitidis and independently to N. gonorrhoeae (11). These species also prefer humans as their sole host.

451

Transfer of Chromosomal Markers by Co-Cultivation of Neisseria Strains In order to test whether transformation-mediated exchanges can be demonstrated in vitro, simple co-cultivation experiments were performed. Two genetically defined N.gonorrhoeae strains, a donor that carried an erm r marker linked to the chromosomal iga gene and - in order to avoid its own transformation - a disrupted recA gene, and a transformation-competent recipient that carried two other chromosomal markers, were grown separately in GC medium containing low concentrations of MgC^. At an optical density of about 0.2 O.D. the cultures were combined and grown further. To determine the transfer frequency of the erm r marker from the donor to the recipient, aliquots were taken at several time points. As early as 15 min after combining the cultures, efficient marker exchange was observed, reaching the upper level of 10-5 erm r recipients per total recipient cells after 1 h. This dramatic marker exchange was completely inhibited in the presence of 40/.g/ml DNase I in the medium, suggesting that the observed marker exchange has occurred via transformation. Similar results have been obtained by co-cultivating N. gonorrhoeae or commensal Neisseria donors with N. meningitidis strains as recipients (T. F. Meyer and E. Schultz, to be published).

Conclusion Transformation-mediated processes probably play a central role in not only the variation of N. gonorrhoeae pilin expression but also in the horizontal exchange and modulation of other virulence determinants of this species and its closest relatives. This notion is based on three lines of evidence (i) the demonstration for a transformationmediated pathway of pilin variation in gonococci, (ii) the generation of mosaic genes in vivo and, (iii) the high efficiency of transformation-dependent horizontal exchange of virulence markers in vitro.

Acknowledgements I would like to thank Elke Schultz for excellent technical assistence and Dr. Brian Robertson for critical comments on the manuscript.

452

References 1.

Meyer, T. F., C. P. Gibbs, R. Haas. 1990. Ann. Rev. Microbiol. 44:451.

2.

Haas, R., H. Schwarz, T.F. Meyer. 1987. Proc. Natl. Acad. Sei. USA «4:9079.

3.

Gibbs, C.P., B.-Y. Reimann, E. Schultz, A. Kaufmann, R., Haas, T.F. Meyer. 1989. Nature 535:651.

4.

Norlander, L., J. Davies, A. Norquist, S. Normark. 1979. J. Bact. 138:162

5.

Seifert, H.S., R.S. Ajioka, C. Marchal, P.F. Sparling, M. So. 1988. Nature 336:392.

6.

Heebeler, B.H., F.E. Young. 1975. J. Bact. 722:385.

7.

Sparling, P.F. 1966. J. Bact. 92:1364.

8.

Swanson, J., S. Morrison, O. Barrera, S. Hill. 1990. J. Exp. Med. 777:2131.

9.

Halter, R., J. Pohlner, T.F. Meyer. 1989. EMBO J. 8:2737.

10. Goodman, S.D., J J . Scocca. 1988. Proc. Natl. Acad. Sei. USA «5:6982. 11. Spratt, B. G., Q.-Y. Zhang, D. M. Jones A. Hutchinson, J. A. Brannigan, C. G. Dowson. 1989. Proc. Natl. Acad. Sei. USA 86:8988.

Structure, Function, and Regulation of the Iron-binding Protein, Fbp

S.A. Morse, S.A. B e r i s h , C.-Y, Chen, D.L. Trees D i v i s i o n of Sexually Transmitted Diseases Laboratory Research, Centers for Disease Control, A t l a n t a , Georgia 30333 T.A. Mietzner Department of Molecular Genetics and Biochemistry, U n i v e r s i t y of P i t t s b u r g h , Pittsburgh, Pennsylvania 15261 C.A. Genco, D, Kapczynski Dental Research Center, Emory University School of D e n t i s t r y , Atlanta, Georgia 30322

Introduction The predominant protein produced by gonococci and meningococci in response to an i r o n - l i m i t e d environment has been designated Fbp,

This protein has

an apparent molecular weight of ca. 37,000 Da when examined by SDS-PAGE (1) and i s synthesized during growth under a l l i r o n - r e s t r i c t e d conditions (1,2).

The presence of s p e c i f i c antibodies in the serum of patients with

gonococcal and meningococcal infections (3) and in the vaginal wash f l u i d s of women with uncomplicated infections or PID due to N e i s s e r i a gonorrhoeae (4) suggests that t h i s protein i s a l s o synthesized in v i v o . Broad r e a c t i v i t y with a f f i n i t y - p u r i f i e d polyclonal antibodies and monoclonal antibodies indicate that the structure of t h i s protein i s a l s o h i g h l y conserved ( 5 ) .

Fbp binds ca. 1 mol of Fe^"1" per mol of protein (5)

suggesting that i t may be involved in the a c q u i s i t i o n of iron by these microorganisms.

Results and D i s c u s s i o n We have cloned and sequenced the structural gene of the gonococcal Fbp

Neisseriae 1990 © 1991 by Walter de Gruyter& Co., Berlin • New York - Printed in Germany

454 (6),

The mature protein was found to c o n s i s t of 3Q8 amino acids with a

leader peptide of 22 amino acid residues.

The molecular weight based on

the consensus amino acid sequence was 33,571, and was l e s s than the estimated molecular weight obtained by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Using a s i m i l a r approach, we cloned and

sequenced fbp from a serogroup A s t r a i n of N^ m e n i n g i t i d i s ( 7 ) ,

Eight

base changes were noted; however, only two of them resulted in an amino acid change.

At amino acid 136, v a l i n e was replaced by alanine, and at

amino acid 146, i s o l e u c i n e was replaced by v a l i n e .

Thus, there was

greater than 99% s i m i l a r i t y between the gonococcal and meningococcal fbp at both the nulcleotide and amino acid l e v e l s .

D i g e s t i o n of genomic DNA

with r e s t r i c t i o n endonucleases and Southern h y b r i d i z a t i o n with an internal fbp probe indicated that there was only one copy of t h i s gene within the chromosome.

Tn order to more completely understand the regulation of t h i s protein by iron, the 5*1 flanking sequence was studied.

Using inverse polymerase

chain reaction, a fragment of DNA containing the 5" flanking sequence of fbp was amplified and sequenced (6).

This fragment contained a typical

ribosomal binding s i t e as well as degenerate -1Q and -35 regions.

An

examination of t h i s region revealed a 19-bp sequence with 63% homology to the consensus primary Fur binding sequence and a 15-bp sequence with 87% homology to the consensus secondary Fur binding sequence.

A gel

retardation assay (8) was used to confirm the i n t e r a c t i o n of Fur with the promoter region of fbp.

Fur bound to two s i t e s within a DNA fragment

encompassing -257 to the s t a r t condon ATG with h a l f maximal concentrations of 5 nM and 35 nM, r e s p e c t i v e l y .

A PCR-amplified Fur gene from

Escherichia c o l i did not hybridize to gonococcal genomic DNA under conditions of high stringency.

Thus, i t i s possible that gonococci

possess a DNA-binding protein that e x h i b i t s l i t t l e , i f any, DNA homology with f u r , but which binds to a s i m i l a r sequence(.s) in the promoter region of the Fbp gene.

Northern b l o t a n a l y s i s indicated that F b p - s p e c i f i c mRNA

was present only when gonococci were grown under i r o n - r e s t r i c t e d conditions.

This i s s i m i l a r to what has been described for promoters

regulated by Fur ( 9 ) .

Presently, we cannot d i s t i n g u i s h between a

regulatory mechanism in which the binding protein functions as a repressor

455 or an a c t i v a t o r for t r a n s c r i p t i o n .

The only evidence supporting the

l a t t e r hypothesis i s that genes with degenerate -35 sequences such as fbp, often require an a c t i v a t o r that substitutes as part of a polymerase binding s i t e (10).

Since the -35 region in the 5' flanking sequence of

fbp is within the 19-bp Fur binding s i t e , i t i s p o s s i b l e that both a repressor and a c t i v a t o r may be involved in regulating the t r a n s c r i p t i o n of fbp. The presence of additional Fur binding sequences within the gonococcal genome could p o t e n t i a l l y be used to i d e n t i f y and clone other i r o n regulated genes.

In order to address t h i s p o s s i b i l i t y , an o l i g o n u c l i o t i d e

probe based upon the sequence of the 19-bp Fur binding s i t e in tJ. gonorrhoeae was synthesized, end-labeled and hybridized to gonococcal genomic DNA digested with various r e s t r i c t i o n endonucleases.

Southern

b l o t a n a l y s i s showed a s i n g l e hybrid band with each enzyme, the s i z e of which was consistent with the presence of a s i n g l e copy corresponding to the Fbp promoter (manuscript in preparation).

Furthermore, the s i z e of

the Fbp mRNA t r a n s c r i p t i d e n t i f i e d on Northern blots was only large enough to encode Fbp, suggesting that t h i s gene was not part of an iron-regulated operon (data not shown). The c e l l u l a r location of Fbp was studied in

c o l i by creating two f u s i o n

proteins that were under the control of the Lac Z promoter in pUC13.

PCR

was used to amplify the entire Fbp coding region with and without the leader peptide and the two amplified fragments were l i g a t e d into the Sma I s i t e to create in-frame t r a n s l a t i o n a l fusions with the alpha peptide of Lac Z.

The fusion containing the leader peptide was processed normally

r e s u l t i n g in a SDS-PAGE.

protein with an apparent molecular weight of 37,000 Da by

This protein was produced in large amounts (ca. 60 mg/liter of

culture) and was found in both the periplasm and cytoplasm of the c e l l . The presence of Fbp in the cytoplasm of these c e l l s was not unexpected since the quantity of Fbp synthesized probably exceeded the capacity of the c e l l to export i t .

The fusion protein without the leader peptide had

a higher apparent molecular weight due to i t s fusion with the alpha peptide of Lac Z and was confined to the cytoplasm. readily p u r i f i e d from

Both proteins were

c o l i by the same procedure used f o r p u r i f y i n g the

456 protein from gonococci and meningococci (.5),

Of p a r t i c u l a r i n t e r e s t was

the observation that

col i expressing the f u s i o n with the leader peptide 3+ were pink, which i s an i n d i c a t i o n of the a b i l i t y of FBP to bind Fe ; c e l l s expressing the fusion without the leader peptide were not pink. The function of Fbp i s presently unknown. 55 incubated with Fbp,

However, when gonococci were

Fe-human t r a n s f e r r i n , the iron became associated with

The t r a n s f e r of iron from t r a n s f e r r i n to Fbp was energy dependent;

however, we do not know whether i t occurred by d i r e c t transfer of the transferrin-bound iron or i f there were one or more intermediates involved.

Pulse-chase experiments 35determined that ca, 80% of the bound iron turned over per generation, S-Methionine-labeled Fbp did not turnover at the same rate suggesting that the iron was t r a n s i e n t l y bound to the Fbp,

These data are c o n s i s t a n t with a transport role for Fbp,

Evidence obtained from electron paramagnetic resonance studies (11) indicated that Fbp coordinated with iron in a manner s i m i l a r to the i r o n binding proteins t r a n s f e r r i n and l a c t o f e r r i n .

These proteins are

approximately twice as large as Fbp and have the capacity to bind 2 mol iron per mol protein.

I t i s of i n t e r e s t to note that the f o l d i n g motif

of each lobe of human l a c t o f e r r i n i s represented by an alpha/beta structure c o n s i s t i n g of an e l i p s o i d a l two-domain feature composed of 5 or 6 strands of noncontiguous beta sheet, covered on either side by connecting h e l i c e s ,

This folding motif i s a l s o conserved among the

bacterial periplasmic proteins that bind s u l f a t e , arabinose, galactose, and leucine/isoleucine/valine (12).

Computer generated algorithms

designed to predict the secondary structure from the primary sequence demonstrated that the consensus amino acid sequence of Fbp was composed of a l t e r n a t i n g alpha h e l i c a l and beta sheet r e g i o n s .

D i r e c t experimental

evidence for t h i s p o s s i b i l i t y has been obtained from c i r c u l a r dichroism studies of the p u r i f i e d Fbp and indicated that i t exhibits an alpha h e l i c a l content of ca. 32% and a beta sheet content of 27%,

These values

were s i m i l a r to the values deduced from the X-ray c r y s t a l l o g r a p h i c data from several periplasmic binding proteins (12), supporting the p o s s i b i l i t y that Fbp represents a member of t h i s c l a s s of proteins.

Convincing

457 evidence supporting t h i s contention awaits the r e s u l t s of X-ray crystal lographic a n a l y s i s currently underway. Our current hypothesis i s that Fbp functions within the periplasm of N_. gonorrhoeae and N^, m e n i n g i t i d i s to transport iron from the outer membrane to the cytoplasmic membrane.

The evidence that supports t h i s r o l e in iron

a c q u i s i t i o n i s that Fbp synthesis i s t i g h t l y regulated by i r o n , the Fbp binds a s i n g l e mole of Fe3 + per mole of protein, i t has s t r u c t u r a l c h a r a c t e r i s t i c s that are common to a c l a s s of proteins which function in transport, and i t i s a transient acceptor of i r o n .

References 1.

Mietzner, T,A,, G.H, Luginbuhl, E, Sandstrom, S.A. Morse.

1984,

I n f e c t . Immun. 45:410. 2.

West, S,E,H,, P.F. S p a r l i n g ,

3.

Fohn, M.J,, T,A, Mietzner, T.W, Hubbard, S,A, Morse, E,W. Hook, I I I . 1987,

4.

1987,

I n f e c t , Immun.

47:388.

I n f e c t , Immun. 55:3065.

Morse, S , A , , C,J. Lammel, W.0, S c h a l l a , T.A, Mietzner, G,F. Brooks. 1989.

Vaccines for sexually transmitted diseases (A. Meheus and R,E.

Spier eds), Butterworths p, 45, 5.

Mietzner, T,A,, R.C, Barnes, Y.A, JeanLouis, W.M. Shafer, S.A, Morse, 1986,

6.

I n f e c t . Immun, 51:60,

B e r i s h , S . A , , T.A, Mietzner, L.W, Mayer, C,A. Genco, B,P, Holloway, S.A, Morse.

7.

1990,

J. Exp, Med, 171:1535,

B e r i s h , S , A . , D.R, Kapczynski, S.A. Morse,

1990,

Nucleic Acids Res,

18:4596. 8.

Bagg, A,, J.B, Neilands,

1987.

Biochemistry 26:5471.

9.

Bagg, A , , J,B, Neilands,

1987.

M i c r o b i o l , Rev, 5^:509.

10. Bujard, H,

1990,

Trends Biochem. S c i , 5:274,

458 11.

Morse, S.A., C.-Y. Chen, A. LeFaou, T.A. Mietzner.

1988.

Infect. Dis. 10(suppl.2):S306. 12.

Quiocho, F.A.

1990.

Phil. Trans. R. Soc. Lond. B 326:1.

Rev.

Heterogeneity

of Iron-Regulated Meningococcal

70 kDa and 98

kDa OMPs

A. Pettersson, E. Scheper, J. Tommassen Department of Molecular Cell Biology, University of Utrecht, The Netherlands B. Kuipers, J. T. Poolman Laboratory of Bacterial Vaccines, National Institute for Public Health and Environmental Protection, Bilthoven, The Netherlands

Introduction When

grown

expresses

under a

iron

number

of

limitation

Neisseria

additional

outer

meningitidis

membrane

proteins

(OMPs) (Fig. 1). Some of these proteins are thought to play a role in the uptake of iron from lactoferrin and transferrin (1,2,3) . These

OMPs

are

studied

to

assess

their

vaccine

potential. Different meningococcal strains express OMPs with different

molecular weights. Most

(kilodalton)

common,

strains express a 70 kDa

iron-regulated

OMP

(4) and an OMP

of

about 98 kDa. In this study the antigenic variability of the immunodominant, surface exposed regions of these two proteins was investigated. We also demonstrate that neither of these proteins is the transferrin receptor.

Results Mice

were

bacteria

immunized grown

in

a

with

outer membrane

fermentor

under

preparations

iron

limitation

from (5) .

Hybridoma cell lines were selected and cloned on the basis of

Neisseriae 1990 © 1991 by Walter de Gruyter & Co., Berlin • New York - Printed in Germany

reaction in ELISAs and Western blot with membranes of bacteria grown under iron limitation, but not with membranes from bacteria grown with excess iron. The strains used for immunization were H44/7 6 (B:15:P1.7, 16 ) and 2 996 (B:2b:P1.2) (Fig. 1) . Ten different monoclonal antibodies against the 70 kDa OMP of H44/76 were obtained. Nine of them recognized linear epitopes, and one, mn70K4, a conformational epitope (data not shown). In competition ELISAs the antibodies were found to recognize at least five different epitopes. Six of them were bactericidal in tests using human serum from an immunoglobulin-deficient patient. Two monoclonal antibodies against the 70 kDa OMP of strain 2996 were obtained. Both of them were bactericidal.

-Fe

+Fe

-Fe

+Fe

94 K 67 K 43 K

1 2 3/4

30 K

MW

H44/76

2996

Figure 1. SDS-PAGE analysis of OMPs from strains H44/76 (B:15:P1.7,16) and 2996 (B:2b:P1.2) after growth to a stationary phase in 40 1 fermentor cultures with and without 150 |AM Fe (N03) 3. The figure is from ref. 5.

461

The ability of the monoclonals to recognize the 70 kDa protein of

wild

type

meningococcal

strains

(6) was

experiments were done as whole-cell ELISAs

tested.

These

(7) of cells grown

on GC agar plates. Strains H44/76 and 2996 produce the 70 kDa OMP under these conditions.

The results obtained with

four

antibodies are listed in Table 1. The other antibodies gave similar results. The antibodies recognize only a very limited number of strains. One monoclonal against the 70 kDa OMP of strain

H44/76

binds

to

5 of

74

strains.

The

monoclonals

against the 70 kDa OMP of 2996 bind to 3 and 4 of 74 strains, respectively.

Therefore,

the

immunodominant

cell-surface

exposed regions of the 70 kDa OMP appear to be antigenically variable. Table 1. Characterization of monoclonal antibodies Name

mn7 0Kl mn70K7 A1A-A2 4F3-E5 A4F-A7 2G9-H5

OMP recognized

70 kDa-H44/76 It

70kDa-2996 It

98kDa-2 99 6 ii

Bactericidal

Strains

titer 3

recognized13

6400

0(5)

800

5(74)

+

4 (74)

+

3(74)

-

42 (74)

—

44 (74)

a

The bactericidal titer is the dilution of the antibody, which kills half of the meningococcal cells. -, no bactericidal activity. +, bactericidal activity but titer not determined. b The number of strains recognized by the mAbs, and, between bracketts, the number of strains tested, are indicated. Similarily, two monoclonals against the 98 kDa OMP of strain 2996 were obtained. Neither of them was bactericidal

(Table

1). These antibodies, however, showed cross-reactivity. of them bind to more than 50 % of the strains tested.

Both

462 The ability of the monoclonal antibodies to block the binding of human transferrin to whole cells was studied. The assay was done as a whole-cell ELISA with peroxidase-labelled human transferrin and increasing concentrations of monoclonal antibodies. None of the antibodies was able to block the binding of human transferrin. This indicates, that neither the 70 kDa OMP nor the 98kDa OMP is a transferrin receptor. The actual transferrin receptor was identified in a Western blot, performed with OMPs and whole cells from strains 2996 and H44/76. The assay with peroxidated human transferrin was done as described (2). No binding to the 70 kDa or 98 kDa OMP was observed (data not shown). In both strains the transferrin bound to a protein of about 80 kDa.

Discussion The results with regard to the 70 kDa OMP were unexpected, since a common, antigenically stable OMP is described in the literature (4,8). The explanation for this discrepancy is probably the methodology used to select for monoclonal antibodies. Monoclonal antibodies reacting with outer membrane complexes from meningococci grown without, but not with Fe(N03)3, were selected. In this way, only monoclonals against cell-surface exposed epitopes were selected. Neither the 70 kDa OMP nor the 98 kDa OMP seems to be the transferrin receptor. The results regarding the 70 kDa OMP are in accordance with a recent report (9), where the 70 kDa OMP was shown not to be the transferrin receptor. The receptor showed variability in molecular weight. In both strains used in this study, it appears to have a molecular weight of 80 kDa.

463

Acknowledgement s We would like to thank M. van Tol and J. Labadie, Academic Hospital Leiden, The Netherlands, for providing the human serum for bactericidal tests.

References 1.

Schryvers, A. B., B. C. Lee. 1989. Can. J. Microbiol. 35:409-415.

2.

Schryvers, A. B., L. J. Morris. 1988. Mol. Microbiol. 2:281-288.

3.

Schryvers, A. B., L. J. Morris. 1988. Infect. Immun. 55:1144-1149.

4.

Dyer, D. W., E. P. West, W. McKenna, S. A. Thompson, P. F. Sparling. 1988. Infect. Immun. 56:977-983.

5.

A. Pettersson, B. Kuipers, M. Pelzer, E. Verhagen, R. H. Tiesjema, J. Tommassen, J. T. Poolman. 1990. Infect. Immun. 58:3036-3041.

6.

Abdillahi, H., J. T. Poolman. 1988. Microb. Pathogen. 4:27-32.

7.

Abdillahi, H., J. T. Poolman. 1987. FEMS Microbiol. Lett. 48:367-371.

8.

Black, J. R., D. W. Dyer, M. K. Thompson, P. F. Sparling. 1986. Infect. Immun. 54:710-713.

9.

Ala'Aldeen, D. A., H. A. Davies, R. A. Wall, S. P. Borriello. 1990. FEMS Microbiol. Lett. 69:37-42.

Repeated elements in silent pil sequences found upstream of the class 1 meningococcal pilE gene.

Wendy Potts, Helen 0'Sullivan and Jon Saunders Department of Genetics and Microbiology, University of Liverpool, Liverpool L69 3BX, UK.

Introduction The majority of meningococcal isolates produce class I pili which are structurally similar to gonococcal pili, react with monoclonal antibodies SMI and SM2 and have subunit M^ of 17-23K (1,2,3). In contrast, some strains of Neisseria meningitidis produce class II pili which do not react with these Mabs and have pilin subunit M

of 15-17K (3,A). The genomes of

all meningococcal isolates, regardless of whether they elicit class I or class II pili, contain sequences homologous to the gonococcal pilin expression locus pilE (3,5). Class I-producing meningococci contain a single pilE locus and a number of partial silent pilS sequences (6), whereas the genomes of class II-producers contain only silent sequences that are truncated to various extents (3,5). Pilus antigenic and phase variation in Neisseria gonorrhoeae, and presumably also in N.meningitidis, is effected by recombination events that result in the replacement of sequences at pilE by sequence information from the pilS loci (7,8). Pilus variation is largely dependent on the recA analogue of N.gonorrhoeae (9) which suggests that recombination between wholly or partially homologous sequences in pilE and pilS loci are involved. A series of repeated sequences (recombination sequences or RS) have been identified in pilS loci of N.gonorrhoeae (8). We have examined pil sequences in N.meningitidis and determined a number of similarities and differences between the RS-like elements present and the RS elements in gonococci.

Neisseriae 1 9 9 0 © 1991 by Walter d e Gruyter & Co., Berlin • New York - Printed in Germany

466 Results and Discussion All DNA sequences homologous to the gonococcal pilE pilin expression gene (6) were located on contiguous DNA fragments representing a total of 8.Akbp of the genome of the class I pilus-producing strain C311 of N.meningitidis (4). The meningococcal pilE gene lies on a 1.7kbp HindIII-Sau3A fragment that is located towards the 3' end of this region of the meningococcal genome. A silent locus, pilS2 located on a contiguous Sau3A-HindIII fragment maps approximately 750 bp upstream of pilE and all the remaining pilS loci are located on an adjacent 5.3kbp Sau3A fragment. The region lying upstream of the pilE gene of N.meningitidis C311 was sequenced. The pilS2 locus of strain C311 contains a coherent open reading frame for part of a single copy of a variant pilin sequence that is truncated at its 5'-terminus, starting at a position equivalent to codon 35 of mature pilin. Although the overall structure of gonococcal and class I meningococcal pil sequences is similar, there are numerous differences in the form of base substitutions and insertions when the sequences are compared which would be consistent with differences in the respective pilE genes of the two organisms. The configuration

and

sequence of so called RS-elements

also varies in silent pil sequences of gonococci and class I meningococcal pilus-producers. There is a complex arrangement of repeat sequences, some overlapping, in the region flanking the class I meningococcal pilS2 copy. Certain tracts of DNA containing RS elements show homologies with sequences in the intergenic region between copies 2 and 3 of the pilSl locus of N.gonorrhoeae (8). Four meningococcal repeat elements observed in the pilS2 region were similar in sequence, but longer at 53bp, than the gonococcal RS2 (49bp) element (Table 1). This silent sequence lacks a termination codon and is bounded at its 3' terminus by an element that is closely related in sequence (but longer at 43bp) to the gonococcal recombination sequence RSI (39/A0bp)(8). The meningococcal pilS2 locus also contains a configuration of tandemly repeated DNA sequences, for example sequences X and a (Table 1), that is not present in previously sequenced pilin genes from gonococci.

467 Table 1. Recombination-Enhancing Sequences in pilS Loci of Gonococci and Meningococci. N.gonorrhoeae

RSI

51-TGCATAAAAACACCACGCGCCGATTTCAAACACTTCCAAA-31

N.meningitidis RSI-like 51-TGCACAGAAACACCAAGCGGCCGATTTCCAATTCATTTTCCAAG-31 N.gonorrhoeae

RS2 51-GGAATCCGGAACGCAAAATCTAAAGAAACCGTTTTACCCGATAAGTTTC-31

N.meningitidis RS2-like 5'-GGGAATCCGAGAACGTAAAATCTCAAGAAACCGTTTTCCCGATAAGTTTCCGT-3' N.gonorrhoeae/N.meningitidis RS3 N.gonorrhoeae/N.meningitidis RS4 N.meningitidis tandem repeat a N.meningitidis tandem repeat X

5'-ATTCCC-31 or 5"-GGGAAT-3' 51-TAAAATTTCA-31 5'-CATTCCCACAAAAACAGCAACCTAAAA-3' 51-CCTAAAACTTAAAATTTCA-31

As in gonococci, it is likely that the RS-like sequences in meningococcal class I pil loci arise from, or are required for, the processes of gene conversion or deletion repair that are required to drive pilus antigenic and phase variation (7,8,10,11). There has clearly been some sequence divergence in these regions of the two pathogenic Neisseria species. Gonococci and meningococci also contain strongly homologous sequences downstream of pilE in the so called Smal-Clal (N.gonorrhoeae) or pseudo Smal-Clal (N.meningitidis) repeat region (5,7,12). In the case of meningococci this region exhibits strong homology to the hin site for site-specific (inversion) recombination of flagellin expression in Salmonella typhimurium (5). It is however not clear whether any of these sequences are substrates for specific enzymes involved in the machinery of recombination or simply provide short homologies for base-pairing between the DNA molecules participating in the transactions involved.

468 Acknowledgements This research was supported by grants from The Wellcome Trust and The Meningitis Trust and by MRC postgraduate studentships to H.O'S and W.P.

References 1.

Diaz, J.-L., M. Virji, and J.E. Heckels. 1984. FEMS Microbiol. Let. 21, 181-184.

2.

Stephens, D.S., A.M. Whitney, J. Rothbard and G.K. Schoolnik. 1985. J. Exp. Med. 161, 1539-1553.

3.

Virji, M., J.E. Heckels, W.J. Potts, C.A. Hart and J.R. Saunders. 1989. J. Gen. Microbiol. 136, 3259-3251.

4.

Perry, A.C.F. C.A. Hart, I.J. Nicolson, J.E. Heckels and J.R. Saunders. 1987. J. Gen. Microbiol. 133, 1409-1418.

5.

Perry, A.C.F., I.J. Nicolson and J.R. Saunders. 1988. J. Bacteriol. 170, 1691-1697.

6.

Potts, W.J. and J.R. Saunders. 1988. Mol. Microbiol. 2, 647-653.

7.

Segal, E., P. Hagblom, H.S. Seifert and M. So. 1986. Proc. Nat. Acad. Sci. USA. 83, 2177-2181.

8.

Haas, R., and T.F. Meyer. 1986. Cell 44, 107-115.

9.

Koomey, M., E.C. Gotschlich, K. Robbins, S. Bergstrom and J. Swanson. 1987. Genetics 1T7. 391-398.

10. Hill, S.A., S.G. Morrison and J. Swanson, J. 1990. Molec. Microbiol. 4, 1341-1352. 11. Scocca, J.J. 1990. Mol. Microbiol. 4, 321-327. 12. Perry, A.C.F., I.J. Nicolson and J.R. Saunders. 1987. Gene 60, 85-92.

Iron acquisition of systemic and n o n s y s t e m i c m e n i n g o c o c c a l isolates Teije O. R0d and Kjell B0vre Kaptein W. Wilhelmsen og Frues Bakteriologiske Institutt, University of Oslo, Rikshospitalet, N 0027 Oslo 1, Norway

Introduction Iron is indispensable for bacterial metabolism. However, the efficacy of iron assimilating mechanisms may vary considerably between bacterial species as well as between closely related strains (1,2). Experimental evidence, particularly from research on Neisseria (3, 4, 5) suggests a correlation between virulence and the capacity to acquire iron. A simple test was used (T. O. R0d, O. Spanne, B. Bjorvatn, in preparation), based on the ability of iron chelators to inhibit meningococci when grown on agar media. It is assumed that iron chelators, when added to a well in agar medium, form gradients of iron deprivation in the medium.

Materials and Methods Systemic meningococcal isolates (from blood or cerebrospinal fluid) and corresponding clinical data for 67 patients were collected in Norway from December 1981 to April 1982 (6; referred to as MO). Nonsystemic i.e.throat isolates of meningococci were collected from 108 healthy carriers in Troms0, Norway from May to July 1984 (7; referred to as BT). The 175 meningococcal isolates were serogrouped, serotyped and their sulfonamide susceptibility determined in previous investigations (7, 8,9). The categorization in clinical disease entities of the 67 patients was according to original reports (6, 8), with slightly modified nomenclature (10) as specified under Results. Inhibition zones around diffusing depots of iron chelators (see below) were studied on agar media with 36 g GC agar base (Difco laboratories, Detroit, MI, U.S.A.), 2.5 g glucose and 1 ml E4-solution (11) per liter, and containing different fractions of human blood, type O in 5% of its original concentration: (a) intact blood cells, isolated by 2x sedimentation and washing in isotonic saline, (b) blood cells as above, lysed by addition of distilled water and 2x freezing and thawing, (c) plasma, isolated by sedimentation of the cells and collection of the supernatant, and (d) no blood component (control agar plates). Cultural passages were performed on a heated hemoglobin agar medium (basic composition as above) containing 10 g bovine hemoglobin (BBL Microbiology Systems, Becton Dickinson and Co, Cockeysville, MD, U.S.A.) per liter. This medium was also used in tests of inhibition zones at different atmospheric conditions. The meningococci were lyophilized or stored frozen at -70 °C in brain heart infusion (Oxoid Ltd, Basingstoke, Hampshire, England) supplemented with 10% glycerol, before

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470 r e c u l t i v a t i o n o n h e a t e d h e m o g l o b i n agar. F i v e m l of a 5*10 5 b a c t e r i a / m l s u s p e n s i o n of t h e r e l e v a n t isolates w a s p o u r e d over an agar plate, dried, and w e l l s w e r e p u n c h e d o u t of t h e 4 m m t h i c k a g a r l a y e r a n d filled with 5 0 |il of 100 m g / m l trivalent iron c h e l a t o r D e s f e r a l ( d e s f e r r i o x a m i n B m e s y l a t e ; C i b a - G e i g y A G , Basel, S w i t z e r l a n d ) or bivalent iron c h e l a t o r b a t h o p h e n a n t h r o l i n e d i s u l f o n a t e (Sigma C h e m i c a l C o m p a n y , St L o u i s , M O , U . S . A . ) b e f o r e i n c u b a t i o n o f t h e p l a t e (standard tests on m e d i a (a)-(d), a e r o b i c a l l y with 5 % C 0 2 ) . T h e d i a m e t e r of the resulting z o n e s of bacterial growth inhibition w a s m e a s u r e d with a caliper. In cases of d i f f u s e zone demarcation, the diameter w a s measured at the e x t r e m e z o n e periphery. S u b c u l t i v a t i o n of f o u r selected isolates, the systemic isolates M O 0 0 1 ( s e r o g r o u p B , n o n t y p a b l e ) a n d M O 0 7 5 ( s e r o g r o u p B, serotype -:P1.16), a n d the n o n s y s t e m i c isolates B T 7 1 8 ( s e r o g r o u p B , n o n t y p a b l e ) and B T 7 2 2 ( s e r o g r o u p B, n o n t y p a b l e ) w a s p e r f o r m e d in o r d e r to a d a p t to d i f f e r e n t a t m o s p h e r i c conditions. T h e isolates w e r e s u b c u l t i v a t e d 5 t i m e s a e r o b i c a l l y in air with or without 5% C 0 2 , or anaerobically, and the size of t h e inhibition z o n e s o n heated h e m o g l o b i n agar was then m e a s u r e d at these conditions. T h e G a s P a c k

Plus

s y s t e m ( B B L M i c r o b i o l o g y Systems, Becton Dickinson and Co., Cockeysville, E n g l a n d ) w a s used f o r t h e a n a e r o b i c incubations: H 2 and C 0 2 w a s d e v e l o p e d , the reaction b e t w e e n H 2 a n d atmospheric 0 2 w a s catalyzed by palladium, with formation of H 2 0 and anaerobic conditions. All incubations w e r e p e r f o r m e d for 24 h at 35 °C, in a h u m i d atmosphere. S t a t i s t i c a l a n a l y s e s w e r e p e r f o r m e d by u s i n g t h e p r o g r a m m e D a t a D e s k , O d e s t a C o r p o r a t i o n , N o r t h b r o o k , IL 6 0 0 6 2 , U . S . A . M e a n s w e r e c o m p a r e d by u s i n g t-tests a n d analysis of variance. Correlations were studied using Spearman rank correlation test.

Results S y s t e m i c v s n o n s y s t e m i c isolates. S y s t e m i c i s o l a t e s had s i g n i f i c a n t l y s m a l l e r m e a n inhibition z o n e than n o n s y s t e m i c isolates when tested on intact blood cell- or p l a s m a m e d i a . A l s o , there w a s a particularly p r o n o u n c e d variation of trivalent iron c h e l a t o r r e s p o n s e f o r systemic isolates on intact blood cell m e d i u m (Table 1, Figure 1). Z o n e size and other strain characteristics. Possible connections between zone size and other strain characteristics were investigated within the collection of systemic isolates, and a m o n g the n o n s y s t e m i c isolates, respectively. N o significant d i f f e r e n c e ( P > 0 . 0 1 ) w a s found between

serotypable and n o n t y p a b l e

isolates, or b e t w e e n

sulfonamide-resistant

and -sensitive isolates. A m o n g n o n s y s t e m i c isolates, no significant d i f f e r e n c e ( P > 0 . 0 1 ) w a s f o u n d between serogroupable and nongroupable isolates. T h e r e w a s a general correlation between zone sizes of bivalent and trivalent chelator. A particularly high correlation was registered on the control m e d i u m (Table 2). T h e r e s u l t s o b t a i n e d in a s e p a r a t e study (T. O. R o d , O. S p a n n e , B. B j o r v a t n , in p r e p a r a t i o n ) , using the trivalent iron chelator Desferal and b o v i n e heated h e m o g l o b i n , w e r e c o m p a r e d to the results on the media containing intact blood cells, lysed blood cells, p l a s m a , or no blood c o m p o n e n t (control agar plates) (Table 3). I r o n l i m i t a t i o n at d i f f e r e n t a t m o s p h e r i c c o n d i t i o n s . At a n a e r o b i c

conditions

t h e isolates tested could g r o w fairly well on m e d i u m containing lysed blood cells and on the

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F i g u r e 1. Inhibition of m e n i n g o c o c c a l growth by iron chelators. (Systemic (N=67) and nonsystemic (N=108) isolates were grown on agar m e d i u m containing intact human blood cells and tested on their ability to grow around d i f f u s i n g d e p o t s of D e s f e r a l (A) or bathophenanthroline disulfonate (B). Isolates were also grown on medium containing human plasma and tested against Desferal (C) or bathophenanthroline disulfonate (D). T h e diameter of the resulting inhibition zone was measured. From the same study as Table 1.

472 Table 1. Meningococcal sensitivity to iron deprivation. Systemic isolates (N=67) compared to nonsystemic (healthy carrier) isolates (N=108), grown aerobically, with 5% CC^. Agar medium

Intact blood cells (a) Lysed blood cells (b) Plasma (c) Control medium (d)

Iron chelator Des. Bat. Des. Bat. Des. Bat. Des. Bat.

Systemic isolates S.D. zone 34.6 6.68 34.4 2.63 21.1 4.76 23.0 1.16 2.54 36.5 22.6 1.39 46.6 5.98 5.94 36.4

Nonsystemic isolates S.D. zone 3.62 41.5 3.20 35.9 5.65 20.5 2.02 22.9 2.12 38.8 25.2 2.46 47.6 5.13 36.6 4.72

Probability of equal m e a n s 0.0000 0.0014 0.4541 0.9560 0.0000 0.0000 0.4897 0.8698

Isolates were tested on their ability to grow around diffusing depots of the trivalent iron chelator D e s f e r a l (Des.) or the bivalent iron chelator b a t h o p h e n a n t h r o l i n e d i s u l f o n a t e (Bat.). T h e resulting inhibition z o n e s w e r e studied o n agar m e d i a (a)-(d) as specified in text. T h e diameter of the inhibition zone and its standard deviation ( S . D . ) are given in m m . T h e m e a n s of the two groups of isolates were compared, testing the hypothesis thai their m e a n s w e r e equal. T h e results correspond to those illustrated in Figure 1.

Table 2. Meningococcal sensitivity to iron deprivation. Correlation between results obtained by using the trivalent iron chelator Desferal and the bivalent iron chelator bathophenanthroline disulfonate. From the same study as Table 1. Agar medium Intact blood cells Lysed blood cells Plasma Control medium

Systemic isolates 0.191 0.430 0.223 0.938

Nonsystemic isolates 0.442 0.200 0.466 0.846

C o r r e l a t i o n s w e r e studied using S p e a r m a n r a n k corr e l a t i o n test. S y s t e m i c i s o l a t e s ( N = 6 7 ) and n o n systemic isolates ( N = 1 0 8 ) were tested. From the same study as T a b l e 1.

T a b l e 4. M e n i n g o c o c c a l s e n s i t i v i t y to iron deprivation. Selected isolates adapted to growth at different atmospheric conditions, and studied on heated hemoglobin medium. Isolate

MO MO BT BT

001 075 718 722

Desferal inhib. zone Air + C 0 2 An. 18.6 11.9