Factores de Virulencia Del Streptococcus Pneumoniae

 

Res. Microbiol. 151 (2000) 413–419  © 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S0923250800001753/REV

Virulence factors and the pathogenesis of disease caused by  St  Strr eptococcus ptococcus pne pneum umoni oniae ae *

Tim J. Mitchell

Division of Infection and Immunity, Institute of Biomedical and Life Sciences, Joseph Black Building, University of  Glasgow,, G12-8QQ Scotland, UK Glasgow Streptococcus pneumoniae   is Abstract –   Streptococcus

a major pathogen of man causing diseases such as pneumonia, meningitis and otitis media. The mechanisms by which this organism causes these diseases are still largely unknown. The use of molecular approaches to identifying and studying putative virulence factors in combination with the application of animal models has allowed some of the mechanisms of the disease process pro cess to be defined. © 2000 Édi Éditions tions sci scientifiq entifiques ues et médica médicales les Elsevi Elsevier er SAS  Stre  Str eptococ ptococcu cuss pn pne eumon umonii ae ae / / virulence factors / pathogenesis

1. Intr Introduct oduction ion Streptococcus pneumoniae  (the pneumococcus)

remains a major pathogen of man despite the advent of antibiotic therapy. therapy. The organism is the causative agent of several important diseases includ inc luding ing pneumo pneumonia nia,, mening meningiti itiss and oti otitis tis media. Even during infection with fully antibiotic susceptible strains of the organism there is an underlying mortality rate of 10% for pneumonia and up to 30% for meningitis [1]. Fatality rate ratess of 5–22 5–22% % repo report rted ed for for pn pneu eumo moco cocc ccal al pn pneu eu-monia have been suggested to be due to irreparable damage caused during the early phase of  the disease [2]. This situation is worsening due to the theoffail faantibiotic-resistant ilur uree of ch chem emot othe hera rapy py due due to thThere e appe appear ance strains [3].the isar-avaccine available for the prevention of pneumococcal disease but this has shortcomings in that it does not protect against all serotypes of the organism and it does not confer good protection in individuals at the extremes of age ( <  2 and > 60 years old). New conjugate vaccines based on capsular polysaccharides are in clinical trials  but these may have a limited life due to sero-

* Tel.: +44 (0)141 330 3749; fax: +44 (0)141 330 3727; [email protected]

type shifts made possible by horizontal gene transfer in this organism [4]. For this reason there is a pressing need for new therapeutic or prophylac proph ylactic tic measures measures again against st this organism organism.. One way to develop such measures is to understand the mechanism by which the organism causes disease. The availability of the (almost) comple com plete te genom genomee seq sequen uence ce of the or organ ganism ism allows us to probe the role of various genes in the pathogenesis of infection by construction of  isogenic mutants and use in animal models. The de defin finit itio ionn of th thee role ole of th thes esee gene geness in th thee disease process also involves an analysis of the host response to the organism. This review will summarise the role of some virulence factors in experimental infections andmight will also mention how these virulence factors be regulated. 2. Virulence factors and their regulation

Prior to the release in November 1997 of the genome sequence of the organism the number of putative virulence factors suggested for the pneumococcus was relatively small [5]. [ 5]. With the availability of the genome sequence we are now spoilt for choice in terms of genes to analyse and the emphasis has shifted from gene identification to functional studies. Here I will review some of the functional functional studies carried out with

 

414

T.J. Mitchell / Res. Microbiol. 151 (2000) 413–419

given in  table I . Pneumolysin has at least two  biological activities that contribute to virulence in animal models: i) lytic activity and ii) ability to activate complement. However, introduction of po poin intt mu muta tati tion ons, s, wh whic ich h bloc block k th thes esee tw two o acti activi vi-ties, results in an organism that is still more virulent than the pneumolysin-negative mutant [12]. This suggests that there is another activity of the toxin which contributes to virulence.

4. Surface Surface proteins proteins

by S. pneumoFigure 1. Some of the virulence factors produced by niae.

well-characterised virulence factors and discuss some of the data relating totranscriptional host response and the rolenewer of two-component regul regulato ators rs (TC (TCS) S) in vir virule ulence nce.. A sum summar mary y of  some of the putative virulence factors of the 1. pneumococcus is shown in figure in  figure 1.

3. Pneum Pneumolysi olysin n

The surface of the pneumococcus is decorated with a range of surface proteins that may play a ro role le in pat pathog hogene enesis sis.. The These se pr prote oteins ins may be either anchored to the cell wall via the Grampositive attachment motif (LPXTG) or may be noncovalently attached via an interaction with choline (choline-binding proteins, Cbps). One of  the neuraminidase enzymes and hyaluronidase produced by the pneumococcus spend part of  their time as cell surface proteins as judged by the presence of the LPXTG motif and cell localisation satio n exper experiment imentss [13] [13].. Choli Choline-bi ne-bindin nding g pro pro-teins share common repeat elements associated with this property and include PspA, LytA, and CbpA which will be described here. 4.1. Pneumococca Pneumococcall surface protein A (PspA)

Much work has been carried out on the role of this protein in infection models. The gene wass orig wa origin inal ally ly clon cloned ed in 19 1983 83 [6 [6,, 7] an and d th thee availability of this gene allowed the constructi tion on of defined pneu pneumoly molysin-n sin-negati egative ve knoc knockkouts and isogenic replacement mutants [8 –10]. These mutants have been used in several laboratories to probe the role of the toxin. The toxin itself has been the subject of a detailed structure function analysis and a three-dimensio three-dimensional nal structure has been proposed based on the structure of the related toxin perfringolysin O. Functional domains have been mapped onto this structure [11]. By altering these domains and re-introducing the gene into the pneumococcus it has been possible to probe the role of individual amino acids in the pathogenesis of infection [8]. A summary of the effects of the mutations and the possible role of pneumolysin is

The mechanism mechanism of actio action n of PspA is not fully understood, but the protein is present on the surface of all pneumococci and is required for full viulence [14, 15]. It has been reported that PspA is a lactoferrin-binding protein by Strepto[16]. PspA also inhibits complement activation by  Streptococcus pneumoniae [17]. pneumoniae  [17]. The amino-terminal half  of the protein contains a highly charged coil – coil domain and it is variations in this sequence that generate the heterogeneity observed in this molecu mol ecule. le. The regio region n still still con contai tains ns su suff ffici icient ent conserved epitopes to allow vaccination with a single PspA type to confer protective immunity to a range of other PspA types [18]. 4.2. Choline-binding Choline-binding protein protein (CbpA)

CbpA is present on the surface of the pneumococcus and reacts with pooled convalescent

 

T.J. Mitchell / Res. Microbiol. 151 (2000) 413–419

415

Mutations ions of pneumoly pneumolysin sin and their effec effects ts on the virule virulence nce of  S. Table I. Mutat   S. pneumoniae.

Mutation Null

Complement activity abolished

Biological effects Respiratory tract Reduced virulence Less inflammation during pneumonia Delayed invasion of bloodstream from lungs Less Less ti tigh ghtt-ju jun nctio ction n sep epar arat atio ion n in hu huma man n or orga gan n cu cult ltur ures es No TNF production in alveoli Bacteraemia model Chronic infection rather than acute In Indu duce cess prot protec ecti tion on fr from om subs subseq eque uent nt chal challe leng ngee wit with h wil wild d ty type pe Eye infection model Mediates inflammation  Meningitis No overall effect on inflammation Mediates hearing loss Otitis media No role Respiratory tract Reduced virulence Im Impo porrtan tant at lat later tim imes es of infe infect ctio ion n (lob (lobar ar pne pneumon umonia ia)) Protec Pro tects ts small small num number berss of bacter bacteria ia ear early ly (br (bronc onchoho-pne pneumo umonia nia)) Bacteraemia model No effect Eye infection infection model Mediates inflammation

Respiratory tract Lytic activity abolished Reduced virulence Important at early times of infection Mediates permeability change in epithelium

serum from patients with bacteraemic pneumococcal pneumonia [19]. If the gene for CbpA is disrupted by insertion duplication mutagenesis the mutant pneumococci have a reduced ability to bind bind to cyto cytoki kine ne-a -act ctiv ivat ated ed type type II pn pneu eu-mocytes and endothelial cells in vitro suggesting that this protein plays a role in adherence. This is supported by the fact that the mutant organism also shows decreased ability to bind to gl glyc yco oco conj nju ugates ates co con ntain tainin ing g la lact ctoo-NNneotetraose and sialic acids, the putative receptorss for pne tor pneum umoco ococci cci on suc such h act activa ivated ted cel cells. ls. Moreover, the mutant organism is less able to colonise the nasopharynx nasopharynx of infant ra rats. ts. Another pneumococcal surface protein designated SpsA, whic wh ich h bind bindss to th thee se secr cret etor ory y comp compon onen entt of  secretor secr etory y immu immunog noglobu lobulin lin A, has been desc described ribed [20]. [20 ]. Seq Sequen uence ce ana analys lysis is sho shows ws tha thatt Sps SpsA A and CbpA are variants of the same protein. CbpA therefore has at least two possible functions in

Reference [9] [27] [27] [3 [35] 5] Our unpublished data [36] [36 [36]] [37] [38] [31] [39] [40] [4 [40] 0] [8] [41] [42] [40] [40] [40]

the pat pathog hogene enesis sis of pn pneum eumoco ococca ccall disea disease. se. Int Inter er-action with IgA secretory compo component nent may interfere with the protective protective function of IgA and may also als o pr prom omote ote adhe adhere renc ncee dire direct ctly ly [20 [20]. ]. Th Thee mec mechahanism by which CbpA mediates attachment to cytokine-activated lung cells is independent of  its ability to bind to the secretory component as this protein was not present in the assays of   binding to these cells in vitro. 4.3. Pneumococcal Pneumococcal surface surface adhesin A (PsaA)

PsaA was ori PsaA origin ginall ally y repor reported ted as a sur surfac facee adhesi adh esin n bas based ed on seq sequen uence ce hom homolo ology gy wit with h oth other er streptococ strep tococcal cal adhes adhesins ins [21] [21].. Pneum Pneumococc ococcii that do not express PsaA are virtually avirulent in animal ani mal mod models els of dis diseas easee and sho show w red reduce uced d ability to adhere to type II pneumocytes in vitro [22]. The psaA The  psaA gene  gene is part of an operon consistin ing g of th thre reee ge gene ness [23] [23].. Th Thee op oper eron on ha hass th thee features of an ATP binding cassette (ABC) trans-

 

416

T.J. Mitchell / Res. Microbiol. 151 (2000) 413–419

port system that appears to be a manganese transporter. PsaA has been crystallised and its thre threee-di dime mens nsio iona nall stru struct ctur uree dete determ rmin ined ed to 2-angstrom resolution [24], which showed that PsaA Psa A has a nov novel el str struct uctur uree for an ABCABC-typ typee  binding protein. The PsaA protein may be an adhe ad hesi sin n or it ma may y reflec ectt a re requ quir irem emen entt fo forr manganese either as a growth factor in vivo or a regulator of adhesin expression.

5. Pneum Pneumococc ococcal al enzymes invol involved ved in pathogenesis 5.1. Autolysin Autolysin

Autolysin Autoly sin is a cel cell-w l-wall all-d -degr egradi ading ng enz enzyme yme found in the cell envelope of the pneumococcus. Thee en Th enzy zyme me is no norm rmal ally ly in inac acti tive ve bu butt un unde derr conditions in which biosynthesis stops, such as nutrient starvation or penicillin treatment, the enzyme is activated and causes autolysis of the  bacterial cell. Such autolysis releases degradation products of the cell wall, including peptidogl do glyc ycan an an and d te teic icho hoic ic ac acid id.. Th Thes esee ce cell ll wa wall ll degradation products can cause inflammation. The action of autolysin also releases intracellular constituents including toxins such as pneumolysin. The role of autolysin in virulence has been demo demons nstr trat ated ed by us usee of au auto toly lysi sinn-ne nega gati tive ve mutants muta nts const construct ructed ed by insert insertion-d ion-duplic uplication ation muta mu tage gene nesi siss [25, [25, 26]. 26]. Su Such ch mu muta tant ntss ar aree le less ss virule vir ulent nt tha than n the their ir wil wildd-typ typee par parent ents. s. Whe When n in inst stil ille led d in into to th thee mo mous usee lu lung ng th thee au auto toly lysi sinnnegative mutant was rapidly cleared from the lu lung ng an and d di did d no nott ca caus usee pn pneu eumo moni niaa [2 [27] 7].. No inflammatory response was seen in the lungs of  animal ani malss inf infect ected ed wit with h the aut autoly olysin sin-ne -negat gative ive mutant. Autolysin probably contributes to virulence  by allowing the release of other active components of the pneumococcus such as pneumolysin. 5.2. Neuraminidas Neuraminidases es

A var variet iety y of evi eviden dence ce sug sugges gests ts tha thatt neu neura ramin miniidase enzymes play a role in pathogenesis. His-

tochemica tochem icall exa examin minati ation on of mic micee dyi dying ng aft after er intraperitoneal administration of partially purified ne neur uram amin inid idas asee ha hass indi indica cate ted d ma mark rked ed decreases in the sialic acid content of the liver and kidney when compared to control animals [28]. Coma and bacteraemia occur signi ficantly more often in patients with pneumococcal menin ingi giti tiss wh when en th thee conc concen entr trat atio ion n of NN-ac acet etyl yl neuram neu ramini inicc aci acid d in the cer cerebr ebrosp ospina inall   fluid uid is elevated [29]. The pneumococcus makes at least two enzymes with neuraminase activity (NanA an and d Na NanB nB)) an and d th thee gene geness for for th thes esee prot protei eins ns havee bee hav been n clo cloned ned [13 [13,, 30] 30].. Avai vailab labili ility ty of the genes allowed the construction of an isogenic mutant of the pneumococcus which does not express nanA [31]. This mutant w was as less virulent in an animal model of pneumonia (TJM, unpublished data). The number of mutant organisms in the lung remained the same over 3 days of  in infe fect ctio ion n wh whil ilee the the wi wild ld-t -typ ypee orga organi nism smss increased by This 4 logs duringthat 2 days when the animals died. suggests neuraminidase allows the pneumococcus to survive and replica cate te in the the lung lung.. Na NanA nA doe doess no nott ap appe pear ar to contribute to inflammation or hearing loss in animal models of meningitis [31]. The role of  NanB Nan B in the pat pathog hogene enesis sis of pne pneumo umonia nia still still remains to be defined. 5.3. Hyaluronidas Hyaluronidase e

The pn The pneu eumo moco cocc ccus us prod produc uces es th thee enzy enzyme me hyaluronidase, hyaluronid ase, which degrades hyaluronic acid, a component of connective tissue. It has therefore been suggested that hyaluronidase allows greater access of organisms to the tissue and may ma y also also play play a role role in th thee tran transl sloc ocat atio ion n of  pneumococci between tissues, from the lung to the blood for example. As well as playing a role in tissue integrity hyaluronic acid also plays a role ole in the the gen eneerati ratio on of the the inflammatory response. resp onse. Pro Product duction ion of hyal hyaluron uronidase idase coul could d al also so affe affect ct th thee inflamm ammato atory ry res respon ponse se in the lung. The gen genee for pne pneumo umococ coccal cal hya hyalur luroni onidas dasee has been cloned [32]. This sequence has been used use d to con constr struct uct a hya hyalur luroni onidas dase-n e-nega egativ tivee mutant muta nt by insert insertionion-dupl duplicati ication on mutag mutagenesi enesiss (TJM, unpublished). Interruption of the hyalu-

 

T.J. Mitchell / Res. Microbiol. 151 (2000) 413–419

417

two-componen mponentt systems (TCS) of  Streptococcus Table II. The two-co   Streptococcus pneumoniae*.

TCS System 1 2 3 4 5 6 7 8 9 10 11 12 13

Possible function

Reference

Unknown Unknown Unknown Homo Homolo logy gy sugg sugges ests ts phos phosph phat atee sens sensin ing. g. Rece Recent nt stud study y sugg sugges ests ts not not invo involv lved ed in [43] phosphate metabolism Penicillin tolerance. Competence [44] Virul irulen ence ce.. Null Null mu muta tant nt sh show owss redu reduce ced d viru virule lenc ncee in muri murine ne pn pneu eumo moni niaa mode modell Our Our un unpu publ blis ishe hed d da data ta Unknown Virul irulen ence ce.. Null Null mu muta tant nt sh show owss redu reduce ced d viru virule lenc ncee in muri murine ne pn pneu eumo moni niaa mode modell Our Our un unpu publ blis ishe hed d da data ta Unknown Vancomycin tolerance [45] Unknown Competence [46] Peptide (quorum?) sensing Our unpublished data

* Table is based on [34].

ronidase gene caused a reduction in virulence in a pne pneumo umonia nia mod model. el. Du Durin ring g pne pneumo umonia nia the hyaluronidase-negative mutant of the pneumo-

pneumococcus [34]. These are summarised in table II . The role of these systems in virulence ha hass no nott ye yett be been en full fully y st stud udie ied. d. An Anal alys ysis is of 

coccus in theparent bloodstream at the same time asappeared the wild-type but only reached levels of 103/mL, whereas the wild type reached 108/mL. Direct instillation of pneumococci into the blood showed no difference in the ability of  the mutant and wild type to survive. Hyaluronidase is therefore involved in the invasion of  the bloodstream.

mutations in anof inmice vivosuggested model involving systemic challenge that none of  thee 11 syst th system emss test tested ed (two (two of th thee mu muta tati tion onss weree let wer lethal hal)) had any effec effectt on vir virule ulence nce.. We have introduced these mutations into a type 2 strain of the pneumococcus and used them to evaluate the role played by these systems in a pneumonia model. Our preliminary data suggest that at least two of the systems are essential fo forr viru virule lenc ncee in this this mo mode del. l. Th Thee pa path thwa ways ys involved appear to be specific for pulmonary infection routes. The signals being sensed and the genes being regulated by these pathways are currently being evaluated. Preliminary data

5.4. Superoxide Superoxide dismutase dismutase (SOD)

The pn The pneu eumo moco cocc ccus us cont contai ains ns tw two o type typess of  SOD (MnSOD and FeSOD). Inactivation of the gene for MnSOD (sodA) (sodA)   showed that MnSOD plays role inmodels the virulence the pneumococcus in aanimal [33]. Theofpattern of in flammation in lungs infected with the mutant was different from that seen with wild-type organisms. After infection with the mutant neutrophils were packed around bronchioles in contrast to the wild-type infection where neutrophils were more diffusely localised.

6. Two-component signal transduction systems Analysis of the genome sequence released by TIGR shows that there are 13 TCS systems in the

suggest the systems may regulate the expression or activity of surface proteins.

References [1] Lee C.-J., Banks S.D., S.D., Lee J.P., Virulence, immun immunity ity,, and vaccine related to   Streptococcus pneumoniae, Crit. Rev. Microbiol. 18 (2) (1991) 89–114. [2] Austrian R., Pneumococcal infections, in: Germanier R. (Ed.), BacBacterial Vaccines, Academic Press, London, 1984, pp. 257–288. [3] Spika J.S., Facklam R.R., Pikaytis Pikaytis B.D., B.D., Oxtoby M.J., Party P.S.W P.S.W., Antimicrobial resistance of  Streptococcus  Streptococcus pneumoniae  in the United States, 1979–1987, J. Infect. Dis. 163 (1991) 1273–1278. [4] Nesin Nesin M., Ramirez Ramirez M., Tomasz Tomasz A., Capsula Capsularr transformatio transformation n of a multidrug-resistant  Streptococcus pneumoniae in vivo, J. Infect. Dis. 177 (1998) 707–713.

 

418

T.J. Mitchell / Res. Microbiol. 151 (2000) 413–419

[5] Paton J.C., Andr Andrew ew P.W P.W., Boulnois Boulnois G.J., G.J., Mitchell Mitchell T.J., Molecular Molecular analysis of the pathogenicity of  Streptococcus  Streptococcus pneumoniae:  The role of pne pneumo umococ coccalprote calproteins ins,, Ann Annu. u. Re Revv. Mic Micro robio biol. l. 47 (1993) (1993) 89 – 115. 115. [6] Wa Walke lkerr J.A., J.A., Allen R.L., Falmagne P., P., Johnson M.K., Boulnois G.J., Molecular Molecu lar cloning, cloning, chara characteri cterizatio zation, n, and comple complete te nucleotide nucleotide sequence sequen ce of the gene for pneumolysin pneumolysin,, the sulfhydryl-a sulfhydryl-activa ctivated ted toxin toxin of   Streptoc Infe fect ct.. Immu Immun. n. 55 (198 (1987) 7) Streptococcus occus pneumoniae pneumoniae, In 1184 –  1184  – 1189. 1189. [7] Pat Paton on J.C J.C., ., Ber Berry ry A.M., A.M., Loc Lock k R.A R.A., ., Hansma Hansman n D., Mannin Manningg P.A., .A., Cloningg and expression Clonin expression in   Escherichia Escherichia coli   of the the   Streptococcus

[23] [23] Dintil DintilhacA., hacA., Alloin Alloingg G.,GranadelC., G.,GranadelC., Cla Claver verys ys J.P., Compet Competenc ence e and virulence virul ence of Streptococ Streptococcus cus pneumoniae: Adcand PsaA PsaA mut mutant antss exh exhibi ibitt a requirement for Zn and Mn resulting from inactivation of putative ABC metal permeases, Mol. Microbiol. 25 (1997) 727–739. [24] Lawrence M.C., Pilling P.A., Epa V.C., V.C., Berry A.M., Ogunniyi A.D., Paton J.C., The crytal structure structure of pneumococcal surface surface antigen antigen PsaA PsaA revea reveals ls a met metalal-bin bindin dingg sit site e anda nov novel el struct structur ure e fora put putati ative ve ABC-type binding protein, Structure 6 (1998) 1553–1561. [25] Berry A.M. A.M.,, Lock R.A., Hansman Hansman D., Paton Paton J.C., Contribution Contribution of  autolysin to virulence of  Streptococcus   Streptococcus pneumoniae, Infect. Immun.

gene encoding pneumolysin, pneumolysin, Infect. Immun. 54 (1) pneumoniae (1986) 50 – 55. 5 5. Alexa Alexander nder J.E., Berry A.M., Paton J.C., Rubins J.B., Andrew Andrew P.W P.W., Mitchell T.J., Amino acid changes affecting the activity of pneumolysin alter the behaviour of pneumococci in pneumonia, Microb. Pathog.. 24 (3) (1998) 167 – 174. Pathog 174. Berry A.M., Yother Yother J., Briles D.E., D.E., Hansman D., D., Paton J.C., Reduced virulence virul ence of a defined defined pneumolysin pneumolysin-nega -negativemutant tivemutant of Streptococcus pneumoniae, Infect. Immun. 57 (7) (1989) 2037–2042. Berry A.M., Alexander J.E., Mitchell T. T.J., J., Andrew P P.W .W.,., Hansman D., Paton J.C., Effect of defined point mutations in the pneumolysin gene on the virulence of  Streptococcus   Streptococcus pneumoniae, Infect. Immun. 63 (1995) 1969–1974. Rossjo Rossjohn hn J., Gilbert R.J.C., Crane D., Morgan P P.J., .J., Mitchell Mitchell T.J., Rowe A.J., Andrew P.W., Paton J.C., Tweten R.K., Parker M.W., The molecular molecu lar mechanism of pneumolysin pneumolysin,, a virulence virulence factor from 449–461. Streptococcus pneumoniae, J. Mol. Biol. 284 (1998) 449–461. Berry A.M., Ogunniy Ogunniyii A.D., Mill Miller er D.C., Paton J.C., Comparative Comparative virule virulence nce of   Stre Streptoco ptococcus ccus pneumoniae pneumoniae   strains strains with insertioninsertionduplication, point, and deletion mutations in the pneumolysin gene,

57 (8) (1989) 2324–2330. 2324–2330. [26] Berry A.M., Pato Paton n J.C., Hansman D., Ef Effect fect of insertional insertional inactivainactivatio tion n of the genes enc encodi oding ng pne pneumol umolysi ysin n and autolysi autolysin n on the virulence virul ence of  Streptococcus   Streptococcus pneumoniae  type 3, Microb. Pathog. 12 (1992) 87–93. [27] Canvin J.R., M Marvin arvin A.P., A.P., Sivakumaran M., M., Paton J.C., Boulnois G.J. G.J.,, Andrew P.W., Mitchell T.J., The role of pneumolysin and autolysin in the pathology of pneumonia and septicemia in mice infected with a type 2 pneumococcus, J. Infect. Dis. 172 (1995) 119–123. [28] Kell Kellyy R., Greiff Greiff D D.,., Toxicity Toxicity of pneumococcal neuraminidase, Infect. Immun. 2 (1) (1970) 115–117. [29] [29] O’Tool oole e R.D., R.D., Goo Goode de L., Howe C., Neuram Neuramini inidas dase e act activi ivity ty in bacterial meningitis, J. Clin. Invest. 50 (1971) 979–985. [30] Berry A.M., Lock R.A., Paton J.C., Cloning Cloning and characterization on nanB, a second  Streptococcus pneumoniae  neuraminidase gene, and purification of the nanB enzyme from recombinant  Escherichia coli ,  J. Bacteriol. 178 (1996) 4854–4860. 4854–486 0. [31] Winte Winterr A.J. A.J.,, Comis S.D., S.D., Osborne M.P., M.P., Ta Tarlow rlow M.J., Stephe Stephen n J., Andrew P.W., Hill J., Mitchell T.J., A role for pneumolysin but not

Infect. Immun. 67 (2) (1999) 981–985. [13] Camara M., Boulnois G.J., Andrew P P.W .W.,., Mitchell T.J., T.J., A neuraminidase from  Streptococcus pneumoniae  has the features of a surface protein, Infect. Immun. 62 (1994) 3688–3695. [14] McDaniel L.S., Y Yother other J., Vijayakumar M., Mcgarry L., Guild W.R., W.R., Briles D.E., Use of insertional inactivation to facilitate studies of  biological biolog ical properties properties of pneumococcal pneumococcal surface prot protein-a ein-a (pspa), (pspa),  J. Exp. Med. 165 (1987) 381–394. 38 1–394. [15] Crain M.J., M.J., II W.D .D.W .W.,., Turner J.S., Yother J., Talki alkington ngton D.F., .F., McDaniel McDan iel L.S., L.S., Gra Grayy B.M. B.M.,, Bril Briles es D.E., Pneumococcalsurface Pneumococcalsurface prot protein ein A (PspA) is serologically highly variable and is expressed by all clinically important capsular serotypes of  Streptococcus  Streptococcus pneumoniae, Infect Immun. 58 (1990) 3293–3299. [16] Hammerschmidt S., Bethe G., Remane P P.H., .H., Chhatwal G.S., IdentiIdentification of pneumococcal surface protein A as a lactoferrin-binding protein prot ein of   Streptococcus Streptococcus pneumoniae, Infect Infect.. Imm Immun. un. 67 (1999) (1999) 1683–1687. [17] Tu A.-H.T A.-H.T., Fulgram R.L., McCrory M.A., Briles D.E., D.E., Szalai Szalai A.J., Pneumococcal surface protein A inhibits complement activation by Streptococcus pneumoniae, Infect. Immun. (67) (1999) 4720–4724. [18] [18] Tart R.C. R.C.,, McD McDani aniel el L.S., L.S., Ral Ralph ph B.A., B.A., Briles Briles D.E., .E., Truncat runcated ed streptococcus-pneumoniae pspa molecules elicit cross-protective immunity against pneumococcal challenge in mice, J. Infect. Dis. 173 (1996) 380–386. [19] [19] Ros Roseno enow w C., Rya Ryan n P., Weiser eiser J.N J.N., ., Jo Johns hnson on S.,Fontan S.,Fontan P., Ortqvi Ortqvist st A., Masure Masur e H.R., Contribu Contribution tion of novel choline-bindi choline-binding ng proteins proteins to adherence, colonization and immunogenicity of  Streptococcus  Streptococcus pneumoniae, Mol. Microbiol. 25 (1997) 819–829. [20] Hammerschmidt S., T Talay alay S.R., Bra Brandtzaeg ndtzaeg P., P., Chhatwal G.S., SpsA, a novel pneumoco pneumococcal ccal surface protein with specific specific bindi binding ng to secretoryImmunogl secr etoryImmunoglobuli obulin n A andsecretorycomponent andsecretorycomponent,, Mol.Microbiol. 25 (1997) 1113–1124. [21] Sampson J.S., O’Connor SS.P .P.,., Stinson A.R., Tharpe J.A., Russell H., Cloning and nucleotide sequence analysis of  psaA  psaA, the Streptococcus pneumoniae   gene encoding a 37-kilodalton protein holologous to previously reported  Streptococcus  sp. adhesins, Infect. Immun. 62 (1994) 319–324. [2 [22] 2] Be Berr rryy A.M. A.M.,, Pa Pato ton n J.C., J.C., Se Seque quenc nce e he hete tero roge gene neit ityy of ps psaa aa,, a 37 37-k -kil ilod odal alto ton n put putat ativ ive e ad adhe hesi sin n es esse sent ntia iall fo forr viru virule lenc nce e of  streptococcus-pneumoniae, Infect. Immun. 64 (1996) 5255–5262.

neuraminidase in the hearing loss and cochlear damage induced by experiment exper imental al pneumococca pneumococcall meningiti meningitiss in guinea guinea pigs, pigs, Infe Infect. ct. Immun. Immun. 65 (1997) 4411–4418. Berry A.M., Lock R.A., Thomas S.M., Rajan D.P., Hansman D., Paton J.C., Cloning and nucleotide sequence of the  Streptococcus pneumoniae hy hyalu aluron ronida idase se gene gene andpurifi andpurificat cation ion of theenzymefrom recombinant Escherichia coli , Infect. Immun. 62 (1994) 1101–1108. Yesilkaya H., Kadioglu A., Gingles N., Alexander J.E., Mitchell T T.J., .J., Andrew P.W P.W.,., Role of manganese containing superoxide dismutase (MnSOD) in oxidative stress and virulence of  Streptococcus  Streptococcus pneumoniae, Infect. Immun. 68 (2000) 2819–2826. Lange R., W Wagner agner C., Saizi Saizieu eu A.D., A.D., Flint N., Molnos J., Steig Steiger er M., Caspers Caspe rs P., Kambe Kamberr M., Keck Keck W., Amr Amrein ein K.E., Domain Domain organisati organisation on and molecular characterization of 13 two-component sytems identified by genome sequencing of   S. pneumoniae, Gene (1999), in press.. Ra Rayne ynerr C., Jac Jackso kson n A.D., A.D., Rut Rutman man A., Dewar A., Mit Mitche chell ll T.J. .J.,, Andrew P.W P.W.,., Cole P.J., Wilson R., Interaction of pneumolysinsufficient and -deficient isogenic variants of  Streptococcus   Streptococcus pneumo-

[8]

[9] [10]

[11]

[12]

[32]

[33]

[34]

[35] [35]

[36]

[37]

[38]

[39]

[40]

  with human respirato respiratory ry mucosa mucosa,, Infect. Infect. Immun. Immun. 63 (1995) niae 442–447. Benton K.A., Everson M.P., Brile Briless D.E., A pneumolysin pneumolysin-nega -negative tive mutantt of   Streptococcus mutan chronic bacteremia bacteremia Streptococcus pneumoniae   causes chronic rather than acute sepsis in mice, Infect. Immun. 63 (1995) 448–455. Johnson M.K., Hobde Hobden n J.A., Hagenah Hagenah M., O’Callaghan O’Callaghan R.J., Hill Hill J.M., Chen S., The role of pneumolysin in ocular infections with   Streptococcus pneumoniae, Curr. Eye Res. 9 (1990) 1107–1114. Friedland I.R. I.R.,, Paris M. M.M., M., Hicke Hickeyy S., Shelt Shelton on S., Olsen K., Paton J.C., J.C., McCracken G.H., The limited role of pneumolysin in the pathogenesis of pneumococcal meningitis, J. Infect. Dis. 172 (1995) 805–809. Sato K., Quarte Quarteyy M.K., Liebeler Liebeler C.L., Le C.T., C.T., Giebink G.S G.S.,., Roles of  autolysin and pneumolysin in middle ear inflammation caused by a type 3  Streptococcus pneumoniae strain in the chinchilla otitis media model, Infect. Immun. 64 (1996) 1140–1145. Rubin Rubinss J.B., Charboneau Charboneau D., Fasching Fasching C., Berry A.M., Paton J.C., Alexander J.E., Andrew P.W P.W.,., Mitchell T.J., Janoff E.N., Distinct role for pneumo pneumolysin lysin’’s cytoto cytotoxic xic and complement complement activitie activitiess in the pathogenesis of pneumococcal pneumonia, p neumonia, Am. J. Respir. Crit. Care Med. 153 (1996) 1339–1346.

 

T.J. Mitchell / Res. Microbiol. 151 (2000) 413–419 [41] [41] Ben BentonK.A, tonK.A, Pat Paton on J.C J.C., ., Bri BrilesD. lesD.E.,The E.,The hem hemoly olyticand ticand comple complemen menttactivating properties of pneumolysin do not contribute individually to virulence in a pneumococcal bacteramia model, Microb. Pathog. 23 (1997) 201 –  201 – 209. 209. [42] Johns Johnson on M.K., Callega Callegan n M.C., Enge Engell L.S., O’ O’Callaghan R.J., Hill J.M., Hobden J.A., Boulnois G.J., Andrew P.W., Mitchell T.J., Growth and virulence of a complement-activation-negative mutant of   Streptococcus pneumoniae  in the rabbit cornea, Curr. Eye Res. 14 (1995) 281 – 285. 285. [43] Novak R., Cauw Cauwels els A., Charpentier E., E., T Tuomanen uomanen E., Identi Identifi fication of a  Streptococcus encoding proteins of an pneumoniae ABC phosph phosphate ate tran transporter sporter  gene and alocus two-compone two-c omponent nt regu regulatory latory system, J. Bacteriol. 181 (4) (1999) 1126 –  1126 – 1133. 1133.

419

[44] [44] Guen Guenzi zi E. E.,, Gasc Gasc A.M. A.M.,, Si Sica card rd M.A. M.A.,, Hack Hacken enbe beck ck R., R., A twotwocomponent signal transducing system is involved in competence and penicillin susceptibility in laboratory mutants of  Streptococcus pneumoniae, Mol. Microbiol. 12 (1994) 505 –  505 – 515. 515. [45] Novak R., Braun J.S J.S.,., Charpentier E., Tuomanen E., Penicillin toler toler-ance genes of  Streptococcus   Streptococcus pneumoniae:  the ABC-type manganese permease complex Psa, Mol. Microbiol. 29 (5) (1998) 1285 –  1285 – 1296. 1296. [46] Pestova E.V E.V.,., Havarstein L.S., L.S., Morrison D.A., D.A., Regulation of competence for genetic transformation  by an  Streptococcus pneumoniae autoinduced peptide pheromone in and a two-component regulatory system, Mol. Microbiol. 21 (1996) 853 –  853 – 862. 862.