The contribution of pneumolysin to the pathogenicity of Streptococcus pneumoniae

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38 Ben-Jacob,E. et al. (1995) Nature 373, 566-567 39 Budrene,E.O. and Berg,H.C. (1991) Nature 349, 630-633 40 Budrene,E.O. and Berg,H.C. (1995) Nature 376, 49-53 41 Dunny,G.M.et al. (1978) Proc. Natl Acad. Sci. USA 75, 3479-3483 42 Clewell,D.B. (1981) Microbiol. Rev. 45,409-436 43 Clewell,D.B. (1993) Cell 73, 9-12 44 Wirth,R. (1994) Eur. J. Biochem. 222, 235-246 45 Dunny,G.M. (1995)J. Bacteriol. 177, 871-876 46 Tanimoto,K., An, F.Y. and Clewell,D.B. (1993)J. Bacteriol. 175, 5260-5264 47 Ruhfel,R.E., Manias,D.A. and Dunny,G.M. (1993)J. Bacteriol. 175, 5253-5259 48 Fujimoto,S. et al. (1995)J. Bacteriol. 177, 5574-5581 49 Clewell,D.B.et al. (1990) Plasmid 24, 156-161 50 Nakayama,J. etal. (1994)J. Bacteriol. 176, 7405-7408 51 Firth,N. et al. (1994)J. Bacteriol. 176, 5871-5873 52 Clewell,D.B.et al. (1985)J. Bacteriol. 162, 1212-1220 53 Ember,J.A. and Hugli,T.A. (1989)Am.J. Pathol. 134, 797-805 54 Galli,D. and Wirth, R. (1991)J. Bacteriol. 173, 3029-3033 55 Hirt, H. et al. (1993) Eur. J. B iochem. 211,711-716 56 Galli,D., Lottspeicb,F. and Wirth, R. (1990) Mol. Microbiol. 4, 895-904 57 Galli,D., Friesenegger,A. and Wirth, R. (1992) Mol. Microbiol. 6, 1297-1308

58 Kao,S-M.et al. (1991)J. Bacteriol. 173, 7650-7664 59 Galli,D., Wirth, R. and Wanner,G. (1989) Arch. Microbiol. 151,486-490 60 Wanner,G. et al. (1989) Arch. Microbiol. 151,491-497 61 Kreft,B. et al. (1992) Infect. Immun. 60, 25-30 62 Weaver,K.E.and Clewell,D.B. (1998)J. Bacteriol. 170, 4343-4352 63 Muscholl,A. et al. (1993) Eur. J. Biochem. 214, 333-338 64 Tanimoto,K. and Clewell,D.B. (1993)J. Bacteriol. 175, 1008-1018 65 Chung,J.W. and Dunny,G.M. (1992) Proc. Natl Acad. Sci. USA 89, 9020-9024 66 Chung,J.W.and Dunny,G.M.(1995)J. Bacteriol. 177, 2118-2124 67 Allison,C. et al. (1993) MoI. MicrobioI. 8, 53-60 68 LeRoith,D. et al. (1980)J. Biol. Chem. 256, 6533-6536 69 Chatterjee,A. et al. (1995) Appl. Environ. Microbiol. 61, 1959-1967 70 Bassler,B.L.and Silverman,M.R. (1995) in Two-component Signal Transduction (Hoch,J.A. and Silhavy,T.J., eds), pp. 431-445, AmericanSocietyfor Microbiology Note added in proof An outstanding comparative discussion of regulatory functions in the P. fischeri and V. harveyi quorumsensingsystemscan be foundin Ref. 70.

The contribution of pneumolysin to the pathogenicity of

Streptococcuspneumoniae James C. Paton The antiphagocytic polysaccharide capsule charides themselves (of which treptococcus pneumoniae there are over 80 serotypes) are of Streptococcus pneurnoniae has long is a human pathogen.of completely nontoxic and cannot been been considered the principal malor importance, causing alone account for death from virulence determinant of this organism. invasive diseases such as pneupneumococcal infection. Host However, there is growing evidence that monia, meningitis and bacterthe toxin pneumolysin plays an important inflammatory responses to cellaemia, as well as otitis media wall components undoubtedly role in the pathogenesis of pneumococcal and sinusitis. Morbidity and contribute to pathogenesis, but disease and may thus be a significant mortality from pneumococcal so too does direct attack on host disease remain high, even in additional target for vaccine development. cells and tissues by the toxin countries where antimicrobial J.C. Paton is in the Molecular Microbiology Unit, pneumolysin. therapy is readily available. Women's and Children's Hospital, North Adelaide, The growing problem of drugSA 5006, Australia. tek +61 8 204 6302, Structure and mode of action resistant pneumococci, coupled fax: +61 8 204 6051, e-mail: [email protected] Pneumolysin is a potent toxin with the suboptimal clinical efproduced by virtually all clinificacy of purified pneumococcal cal isolates of S. p n e u m o n i a e 1. This 53 kDa protein is polysaccharide vaccines in high-risk groups (particularly located in the cytoplasm, but is released when pneuyoung children), has highlighted the need to understand mococci undergo spontaneous autolysis. Pneumolysin the mechanisms underlying pneumococcal disease better. is a member of a family of structurally related toxins Such information is an essential prerequisite for the called the thiol-activated cytolysins, which are prodevelopment of improved preventative strategies. duced by representatives of several Gram-positive Although the antiphagocytic capsule of S. p n e u genera 2. Their mode of action involves interaction with m o n i a e is essential for virulence, the capsular polysac-

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cholesterol in target-cell membranes and insertion into the lipid bilayer, followed by oligomerization to form transmembrane pores, which bring about cell lysis 3. Erythrocytes are particularly susceptible to pneumolysin but the toxin can theoretically interact with any cell that has cholesterol in its plasma membrane. Pneumolysin is a bifunctional toxin and, in addition to its cytotoxic properties, it is capable of directly activating the classical complement pathway in the absence of specific antibody, with a concomitant reduction in serum opsonic activity4. This is mediated by its ability to bind directly to the Fc region of human immunoglobulin G (Ref. 5). The gene encoding pneumolysin has been cloned and sequenced, and structure-function analysis has revealed several interesting features6. First, a domain towards the carboxyl terminus of the toxin (residues 427-437), which includes the only cysteine residue, is critical for cytotoxicity. This domain is highly conserved among other members of the thiol-activated cytolysin family (such as streptolysin O and listeriolysin O, which are produced by Streptococcus pyogenes and Listeria monocytogenes, respectively). Several single amino acid substitutions within this region (for example, cysteine 428 to glycine, or tryptophan 433 to phenylalanine) reduce the cytotoxic activity of pneumolysin by as much as 99.9%. However, these mutations did not block binding of the toxin to target-cell membranes or oligomerization of toxin molecules7. Cell binding was, however, blocked by deletion of the six carboxy-terminal residues or by a single amino acid substitution in this region (proline 462 to serine) 8, and oligomerization was abrogated by mutation of histidine 367 to arginineL Thus, it appears that several different toxin domains are involved in the generation of cytolysis. A separate region of pneumolysin, which has a degree of amino acid similarity to human C-reactive protein, appears to be responsible for both immunoglobulin binding and complement activation, and a mutation within this domain (aspartate 385 to asparagine) significantly reduced both these properties s. Effects of purified pneumolysin on cells and tissues In vitro studies have demonstrated that highly purified

pneumolysin has a variety of detrimental effects on cells and tissues (reviewed in Ref. 1). The multiplicity of susceptible cell types and toxic effects is undoubtedly a reflection of the ubiquity of cholesterol in mammalian cell membranes and the importance of functional integrity of the plasma membrane to many cellular processes. Studies in the early 1980s indicated that sublytic doses of pneumolysin inhibit the migration, respiratory burst and bactericidal activity of human polymorphonuclear leukocytes and macrophages, and block proliferative responses and immunoglobulin production by lymphocytesL Pneumolysin has been shown to disrupt the surface integrity and slow ciliary beating of human nasal epithelium maintained in organ cuhure 9. More recently, direct cytotoxicity for respiratory endothelial and epithelial cells has been reported m,ll. Thus, pneumolysin may function in pathogenesis by inter-

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fering with both phagocytic and ciliary clearance of pneumococci, by blocking humoral immune responses and by aiding penetration of host tissues. Recent evidence also suggests that pneumolysin may contribute to sensorineural hearing loss, which is a common sequela of pneumococcal otitis media and meningitis. Perfusion of pneumolysin through the scala tympani produced widespread electrophysiological and morphological damage to the guinea-pig cochlea (particularly the hair cells) ~2.Whilst the precise mechanism is uncertain, it has been suggested that much of the damage could be mediated by induction of excess production of nitric oxide 13. In meningitis, pneumolysin released in the subarachnoid space may gain access to the cochlea directly via the cochlear aqueduct. The round-window membrane forms the major barrier between the middle ear and the cochlea, but Engel et al. TM have recently demonstrated that exposure to the related thiol-activated toxin streptolysin O severely perturbs this barrier, allowing free access of solutes and macromolecules (including the toxin itself) to the cochlea. They postulate that pneumolysin perturbs the round-window membrane in an analogous manner during pneumococcal otitis media. Pneumolysin as a mediator of inflammation

Induction of inflammation is undoubtedly a key component of the pathogenesis of pneumococcal disease, and the role of cell-wall degradation products, particularly teichoic acid, in generating this response is well documentedis. However, several pieces of evidence suggest that pneumolysin is also capable of direct induction of inflammatory responses. The capacity of pneumolysin to activate complement directly could result in the generation of chemotactic and anaphylotoxic peptides C3a and C5a. As has been shown for streptolysin 0 (Ref. 16), activation also occurs with membrane-fixed toxin, and this may result in direct complementmediated attack on host cells. Houldsworth et al.17 have reported that treatment of human peripheral blood monocytes, or a monocyte cell line, with very low doses of pneumolysin (<1 ng ml -~) resulted in release of greater levels of both tumour necrosis factor o~and interleukin 1[3 (IL- 1 [~) than occurred in the presence of 50 ng mllipopolysaccharide. Both these pro-inflammatory cytokines are important mediators of inflammation and tissue damage in pneumococcal meningitis. This finding is in agreement with earlier studies that demonstrated similar secondary cellular reactions, including release of IL-118, after treatment with other bacterial pore-forming toxins 3. Rubins et al. TM have also demonstrated that pneumolysin is a potent activator of phospholipase A in pulmonary endothelial cells. The authors postulated that this might result in release of cytotoxic products such as free fatty acids and lysophosphatides, as well as inflammatory mediators such as arachidonic acid. Collectively, the above studies are consistent with the previous findings of Feldman et a1.19 who demonstrated that injection of purified pneumolysin into the apical lobe bronchus of rats induced a severe lobar pneumonia, indistinguishable histologically from that seen when virulent pneumococci were injected. Interestingly, the

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histological changes were of a lesser magnitude when mutated pneumolysin derivatives, lacking either the cytotoxic or complement-activation properties, were administered. This was the first in v i v o evidence that both properties of the toxin contribute to pathogenesis. Studies with pneumolysin-negative pneumococci Access to the cloned pneumolysin gene and the availability of sequence and structure-function data have permitted a direct asessment of the contribution of pneumolysin as a whole, and its separate cytotoxic and complement-activation properties, to pathogenesis in several model systems. Complete inactivation of the pneumolysin gene (by insertion-duplication mutagenesis) in either a type 2 or a type 3 pneumococcus reduced virulence for mice challenged by both the intranasal and intraperitoneal routes, as judged by median survival time and LDs0 (Refs 20,21). Additional studies by Rayner et al. 22 demonstrated that the same pneumolysin-negative type 2 strain (PLN-A) caused significantly less damage to human respiratory mucosa in organ culture than its otherwise isogenic parental strain (D39). The most notable difference was that, unlike D39, PLN-A failed to cause separation of tight junctions between epithelial cells and failed to adhere to the separated edges, a factor that may be important for the invasion process. Canvin et al. 23 demonstrated that intranasal challenge of mice with PLN-A rather than D 3 9 resulted in a less severe inflammatory response, a reduced rate of multiplication within the lung and a delayed onset of bacteraemia. Pneumolysin also appears to have a role after bacteraemia has developed. Benton et al. 24 have reported that intravenous challenge of mice with PLN-A resulted in a chronic bacteraemia, with numbers of pneumococci in the blood remaining at, or below, 107 colonyforming units (c.f.u.)m1-1 for a week in some mice. Intravenous administration of an identical dose of D3 9, however, resulted in fulminant infection; mice invariably died within 28 h, at which time there were approximately 109-1010 c.f.u, m1-1 blood. They concluded that during the first few hours of bacteraemia, pneumolysin plays a critical role by preventing the generation of inflammation-based immunity, thereby permitting continued exponential net growth of pneumococci. Rubins et al. 2s used endotracheal instillation of PLN-A and D39 to examine the contribution of pneumolysin to the early stages of pneumococcal pneumonia in mice. PLN-A had a reduced capacity to injure the alveolar capillary barrier and a reduced capacity to multiply within the lungs. PLN-A was also less able than D 3 9 to penetrate from the alveoli to the interstitium of the lung and to invade the bloodstream. When purified pneumolysin was co-instilled with PLN-A, the pattern of multiplication in the lung was comparable to D39. Experiments involving co-instillation of mutated pneumolysin derivatives lacking either cytotoxic activity or complement activation indicated that the former property accelerated bacterial growth and invasion into lung tissue during the initial period after infection, while the latter appeared to facilitate persistence of pneumococci in the alveoli at later times.

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Recently, a series of S. p n e u m o n i a e D39 derivatives have been constructed in which the wild-type pneumolysin gene has been replaced by mutated genes encoding toxins with a series of defined point mutations affecting either or both of the cytotoxic and complementactivation properties 26. Comparison of the virulence of these strains for mice demonstrated that, in the intraperitoneal challenge model, the cytotoxic property of the toxin is more important than its capacity to activate complement. A strain producing complementactivation-deficient pneumolysin was fully virulent. Strains in which pneumolysin cytotoxic activity was undetectable were significantly less virulent than those expressing the wild-type toxin but, interestingly, a D39 derivative producing a form of pneumolysin that retained only about 0.1% cytotoxic activity had intermediate virulence. This implies that the amount of pneumolysin required to achieve maximal in vivo effect may be very small indeed, which might account for the apparent lack of correlation between pneumolysin production and virulence in clinical isolates 26. Recently, the same set of D 3 9 derivatives has been used to confirm distinct roles for both properties of the toxin in the pathogenesis of pneumococcal pneumonia, using the mouse intratracheal challenge model 27. Strains producing pneumolysin lacking either property were less virulent than the wild type. Cytotoxic activity was required for damage to the alveolar capillary barrier and for optimal bacterial multiplication in the alveoli and lung tissue during the first 6 h of infection. However, the complement-activation property was associated with increased numbers of pneumococci in lung tissue and the blood 24 h after infection. Johnson et a l ? have also recently reported a role for the complement-activation property of pneumolysin in the pathogenesis of pneumococcal ocular infection. Deletion of the pneumolysin gene had been previously shown to reduce virulence in a rabbit intracorneal challenge model. Full virulence was reconstituted by transformation with a plasmid carrying a copy of the wild-type pneumolysin gene, but was only partially restored when a pneumolysin gene carrying a point mutation affecting complement activation was used. Pneumolysin as a vaccine antigen It has been known for some time that existing pneumococcal capsular polysaccharide vaccines are poorly immunogenic in several high-risk groups (such as children). Furthermore, anti-polysaccharide antibodies confer a strictly serotype-specific protection and not all serotypes are covered by existing formulations. The former difficulty is being addressed by conjugating the polysaccharides to protein carriers such as tetanus or diphtheria toxoids. Such conjugate antigens induce T-cell-dependent immune responses and are highly immunogenic in young children. However, there are concerns that high levels of anti-carrier antibodies caused by repeated use of these proteins in multivalent conjugate formulations, as well as in routine childhood immunization programmes, could result in suppression of immune responses to the conjugates.

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Questions for future research • How can the apparent direct involvement of pneumolysin in pathogenesis be reconciled with the fact that it is a cytoplasmic protein, released only when pneumococci undergo autolysis? • Why is it that the complement-activation property of pneumolysin contributes to pathogenesis of lung infection, but has no apparent impact on virulence in an intraperitoneal challenge model? • What is the relative importance of the direct cytotoxicity of pneumc~ lysin and its secondary effects (such as induction of inflammatory cytokine release) in the pathogenesis of pneumococcal disease?

The inclusion of pneumolysin as a component of pneumococcal vaccines may prove advantageous for several reasons. First, pneumolysin has been shown to be a protective immunogen in its own right. Moreover, it is produced by virtually all clinical isolates of S. p n e u m o n i a e and antigenic variation has not been reported 1. Potential problems of toxicity have been overcome by the use of site-directed mutagenesis and these genetically engineered pneumolysin toxoids have been shown to provide a degree of protection against pneumococci regardless of serotype 29. Second, pneumolysin has shown great promise as a carrier for the otherwise poorly immunogenic polysaccharides in conjugate formulations. Immunization of mice with a conjugate of a nontoxic pneumolysin derivative and type 19F polysaccharide elicited a strong and boostable antibody response to both protein and polysaccharide moieties, and provided infant mice with a high degree of protection against challenge 3°,31. Similar results have also been reported recently for conjugates of pneumolysin with type 18C polysaccharide 32. Clearly, such antigens have the potential to evoke a significant anti-capsular response, as well as an anti-virulence-protein response in humans, including young children, thereby conferring comprehensive protection against pneumococcal disease. Acknowledgements

I thank all my colleagueswho havecontributedto the work described in thisreview,particularlyTimMitchell,PeterAndrewandJeffRubins,

for fruitfulcollaborations. I am also gratefulto the National Health and Medical Research Council of Australia for ongoing financial support. References

1 Paton,J.C. et al. (1993) Annu. Rev. Microbiol. 47, 89-115 2 Alouf,J.E. and Goeffroy,C. (1991) in Sourcebook of Bacterial Toxins (Alouf,J.E. and Freer,J.H., eds),pp. 147-186, AcademicPress 3 Bhakdi,S. and Tranum-Jensen,J. (1988) Prog. Allergy 40, 1-43 4 Paton,J.C. et al. (1984) Infect. Imrnun. 43, 1085-1087 5 Mitchell,T.J. et al. (1991)Mol. Microbiol. 5, 1883-1888 6 Boulnois,G.J. et al. (1991) Mol. Microbiol, 5, 2611-2616 7 Saunders,F.K.et al. (1989) Infect. Immun. 57, 2547-2552 8 Owens,R.H. et al. (1994)FEMSMicrobiol. Lett. 121,217-221 9 Feldman,C. et al. (1990) Microb. Pathog. 9, 275-284 10 Rubins,J.B. et al. (1992) Infect. Immun. 60, 1740-1746 11 Rubins,J.B. et al. (1993)Infect. Immun. 61, 1352-1358 12 Comis,S.D.et al. (1993)Acta Oto-laryngol. 113, 152-159 13 Amaee,F.R.,Comis,S.D.and Osborne, M.P. (1995) Acta Oto-laryngol. 115, 386-39l 14 Engel,F. et al. (1995) Infect. Immun. 63, 1305-1310 15 Tuomanen,E. et al. (1985)J. Infect. Dis. 151,859-868 16 Bhakdi,S. and Tranum-Jensen,J. (1985) Infect. lmmun. 48, 713-719 17 Houldsworth,S., Andrew,P.W. and Mitchell,T.J. (1994) Infect. Immun. 62, 1501-1503 18 Rubins,].B. et al. (1994) Infect. Immun. 62, 3829-3836 19 Feldman,C. et al. (1991) Am. J. Respir. Cell. Mol. Biol. 5, 416-423 20 Berry,A.M. et al. (1989) Infect. Imrnun. 57, 2037-2042 21 Berry,A.M., Paton,J.C. and Hansman,D. (1992) Microb. Pathog. 12, 87-93 22 Rayner,C.F.J.et al. (1995) Infect. Immun. 63, 442-447 23 Canvin,J.R. etal. (1995)J. Infect. Dis. 172, 119-123 24 Benton,K.A.,Everson,M.P. and Briles,D.E. (1995) Infect. lmmun. 63,448-455 25 Rubins,J.B. et al. (1995)J. Clin. Invest. 95, 142-150 26 Berry,A.M. et al. (1995) Infect. Immun. 63, 1969-1974 27 Rubins,J.B. et al. Am. J. Respir. Cell. Mol. Biol. (in press) 28 Johnson,M.K.et al. (1995) Curr. Eye Res. 14, 281-284 29 Alexander,J.E. et al. (1994) Infect. Immun. 62, 5683-5688 30 Paton,J.C. et al. (1991) Infect. Immun. 59, 2297-2304 31 Lee,C-J.et al. (1994) Vaccine 12, 875-878 32 Kuo,J. et al. (1995) Infect. Immun. 63, 2706-2713

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