Virulence factors and the pathogenesis of disease causedby

Streptococcus pneumoniae

Tim J. Mitchell

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

Abstract

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Streptococcus pneumoniae

is 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 largelyunknown. The use of molecular approaches to identifying and studying putative virulence factors incombination with the application of animal models has allowed some of the mechanisms of the diseaseprocess to be deﬁned. © 2000 Éditions scientiﬁques et médicales Elsevier SAS

Streptococcus pneumoniae

/ virulence factors / pathogenesis

1. Introduction

Streptococcus pneumoniae

(the pneumococcus)remains a major pathogen of man despite theadvent of antibiotic therapy. The organism is thecausative agent of several important diseasesincluding pneumonia, meningitis and otitismedia. Even during infection with fully antibi-otic susceptible strains of the organism there isan underlying mortality rate of 10% for pneu-monia and up to 30% for meningitis [1]. Fatalityrates of 5–22% reported for pneumococcal pneu-monia have been suggested to be due to irrepa-rable damage caused during the early phase of the disease [2]. This situation is worsening dueto the failure ofchemotherapy due to the appear-ance of antibiotic-resistant strains [3]. There is avaccine available for the prevention of pneumo-coccal disease but this has shortcomings in thatit does not protect against all serotypes of theorganism and it does not confer good protectionin individuals at the extremes of age (

2 and

60 years old). New conjugate vaccines basedon capsular polysaccharides are in clinical trialsbut these may have a limited life due to sero-type shifts made possible by horizontal genetransfer in this organism [4]. For this reasonthere is a pressing need for new therapeutic orprophylactic measures against this organism.One way to develop such measures is to under-stand the mechanism by which the organismcauses disease. The availability of the (almost)complete genome sequence of the organismallows us to probe the role of various genes inthe pathogenesis of infection by construction of isogenic mutants and use in animal models. Thedeﬁnition of the role of these genes in thedisease process also involves an analysis of thehost response to the organism. This review willsummarise the role of some virulence factors inexperimental infections and will also mentionhow these virulence factors might be regulated.

2. Virulence factors and their regulation

Prior to the release in November 1997 of thegenome sequence of the organism the numberof putative virulence factors suggested for thepneumococcus was relatively small [5]. With theavailability of the genome sequence we are nowspoilt for choice in terms of genes to analyseand the emphasis has shifted from gene identi-ﬁcation to functional studies. Here I will reviewsome of the functional studies carried out with

* Tel.: +44 (0)141 330 3749; fax: +44 (0)141 330 3727;T.Mitchell@bio.gla.ac.uk

well-characterised virulence factors and discusssome of the newer data relating to host responseand the role of two-component transcriptionalregulators (TCS) in virulence. A summary of some of the putative virulence factors of thepneumococcus is shown in

ﬁgure 1

3. Pneumolysin

Much work has been carried out on the roleof this protein in infection models. The genewas originally cloned in 1983 [6, 7] and theavailability of this gene allowed the construc-tion of de

ﬁ

ned pneumolysin-negative knock-outs and isogenic replacement mutants [8

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10].These mutants have been used in several labo-ratories to probe the role of the toxin. The toxinitself has been the subject of a detailed structurefunction analysis and a three-dimensional struc-ture has been proposed based on the structureof the related toxin perfringolysin O. Functionaldomains have been mapped onto this structure[11]. By altering these domains andre-introducing the gene into the pneumococcusit has been possible to probe the role of indi-vidual amino acids in the pathogenesis of infec-tion [8]. A summary of the effects of the muta-tions and the possible role of pneumolysin isgiven in

table I

. Pneumolysin has at least twobiological activities that contribute to virulencein animal models: i) lytic activity and ii) abilityto activate complement. However, introductionofpoint mutations,which block these two activi-ties, results in an organism that is still morevirulent than the pneumolysin-negative mutant[12]. This suggests that there is another activityof the toxin which contributes to virulence.

4. Surface proteins

The surface of the pneumococcus is decoratedwith a range of surface proteins that may play arole in pathogenesis. These proteins may beeither anchored to the cell wall via the Gram-positive attachment motif (LPXTG) or may benoncovalently attached via an interaction withcholine (choline-binding proteins, Cbps). One of the neuraminidase enzymes and hyaluronidaseproduced by the pneumococcus spend part of their time as cell surface proteins as judged bythe presence of the LPXTG motif and cell locali-sation experiments [13]. Choline-binding pro-teins share common repeat elements associatedwith this property and include PspA, LytA, andCbpA which will be described here.

4.1. Pneumococcal surface protein A (PspA)

The mechanism of action of PspA is not fullyunderstood, but the protein is present on thesurface of all pneumococci and is required forfull viulence [14, 15]. It has been reported thatPspA is a lactoferrin-binding protein [16]. PspAalso inhibits complement activation by

Strepto-coccus pneumoniae

[17]. The amino-terminal half of the protein contains a highly charged coil

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coil domain and it is variations in this sequencethat generate the heterogeneity observed in thismolecule. The region still contains sufficientconserved epitopes to allow vaccination with asingle PspA type to confer protective immunityto a range of other PspA types [18].

4.2. Choline-binding protein (CbpA)

CbpA is present on the surface of the pneu-mococcus and reacts with pooled convalescent

Figure 1

. Some of the virulence factors produced by

S. pneumo-niae.

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

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419

serum from patients with bacteraemic pneumo-coccal pneumonia [19]. If the gene for CbpA isdisrupted by insertion duplication mutagenesisthe mutant pneumococci have a reduced abilityto bind to cytokine-activated type II pneu-mocytes and endothelial cells in vitro suggest-ing that this protein plays a role in adherence.This is supported by the fact that the mutantorganism also shows decreased ability to bindto glycoconjugates containing lacto-N-neotetraose and sialic acids, the putative recep-tors for pneumococci on such activated cells.Moreover, the mutant organism is less able tocolonise the nasopharynx of infant rats.Anotherpneumococcal surface protein designated SpsA,which binds to the secretory component of secretory immunoglobulin A,has been described[20]. Sequence analysis shows that SpsA andCbpA are variants of the same protein. CbpAtherefore has at least two possible functions inthe pathogenesis ofpneumococcal disease.Inter-action with IgA secretory component may inter-fere with the protective function of IgA and mayalso promote adherence directly [20].The mecha-nism by which CbpA mediates attachment tocytokine-activated lung cells is independent of its ability to bind to the secretory component asthis protein was not present in the assays of binding to these cells in vitro.

4.3. Pneumococcal surface adhesin A (PsaA)

PsaA was originally reported as a surfaceadhesin based on sequence homology with otherstreptococcal adhesins [21]. Pneumococci thatdo not express PsaA are virtually avirulent inanimal models of disease and show reducedability to adhere to type II pneumocytes in vitro[22]. The

psaA

gene is part of an operon consist-ing of three genes [23]. The operon has thefeatures of an ATP binding cassette (ABC) trans-

Table I

. Mutations of pneumolysin and their effects on the virulence of

S. pneumoniae.

Mutation Biological effects Reference

Respiratory tract

Null Reduced virulence [9]Less in

ﬂ

ammation during pneumonia [27]Delayed invasion of bloodstream from lungs [27]Less tight-junction separation in human organ cultures [35]No TNF production in alveoli Our unpublished data

Bacteraemia model

Chronic infection rather than acute [36]Induces protection from subsequent challenge with wild type [36]Eye infection modelMediates in

ﬂ

ammation [37]

Meningitis

No overall effect on in

ﬂ

ammation [38]Mediates hearing loss [31]

Otitis media

No role [39]

Respiratory tract

Complement activityabolishedReduced virulence [40]Important at later times of infection (lobar pneumonia) [40]Protects small numbers of bacteria early (broncho-pneumonia) [8]

Bacteraemia model

No effect [41]

Eye infection model

Mediates in

ﬂ

ammation [42]

Respiratory tract