Pneumo Virulence Factors

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The role of Streptococcus pneumoniae virulence factors in host respiratory colonization and disease
Aras Kadioglu*, Jeffrey N. Weiser, James C. Paton and Peter W. Andrew*
Abstract | Streptococcus pneumoniae is a Gram-positive bacterial pathogen that colonizes the mucosal surfaces of the host nasopharynx and upper airway. Through a combination of virulence-factor activity and an ability to evade the early components of the host immune response, this organism can spread from the upper respiratory tract to the sterile regions of the lower respiratory tract, which leads to pneumonia. In this Review, we describe how S. pneumoniae uses its armamentarium of virulence factors to colonize the upper and lower respiratory tracts of the host and cause disease.
Nasooropharynx
The parts of the pharynx that are above and below the palate, respectively.
Carrier state
The state of carrying an organism without manifestation of disease.
Herd immunity
Immunity induced by vaccination that protects a proportion of unvaccinated individuals.
*Department of Infection, Immunity & Inflammation, University of Leicester, Leicester LE1 9HN, United Kingdom. Department of Microbiology, School of Medicine, University of Pennsylvania, Pennsylvania 19104-6076, USA. School of Molecular and Biomedical Science, University of Adelaide, South Australia 5005, Australia. Correspondence to A.K. e-mail: ak13@leicester.ac.uk doi:10.1038/nrmicro1871
Streptococcus pneumoniae (often referred to as the pneumococcus) is the most common bacterial respiratory pathogen in the United Kingdom, and frequently causes community-acquired pneumonia, which can have mortality rates of more than 20% in patients with concurrent pneumococcal septicaemia1,2. Worldwide, the situation is worse; pneumococcal septicaemia is a major cause of infant mortality in developing countries, where it causes approximately 25% of all preventable deaths in children under the age of 5 and more than 1.2 million infant deaths per year3,4. In countries that have a high prevalence of HIV-1 infection, there has been a significant increase in the rate of pneumococcal pneumonia and associated bacteraemia, and this increase has been most marked in young adults. The pneumococcus resides on the mucosal surface of the upper respiratory tract. It can be readily cultured from the nasooropharynx of humans and, occasionally, other large mammals that live in association with humans. Although colonization at this site seems to be asymptomatic, if the organism gains access to the normally sterile parts of the airway a rapid inflammatory response ensues that results in disease. The most common manifestations of pneumococcal disease include acute otitis media, which involves infection of the middle-ear space, and pneumonia, which affects the terminal airways. Bacteraemic infection, which is associated with greater morbidity and mortality, typically occurs as a complication of pneumonia (bacteraemic pneumococcal pneumonia) or, less often, if the organism spreads directly from its niche in the pharynx
(occult bacteraemia). Thus, all pneumococcal disease begins with the establishment of colonization, that is, the creation of the carrier state5,6. None of the common forms of pneumococcal disease, however, promote pneumococcal transmission, which implies that the virulence characteristics of the pneumococcus are probably adaptations that increase its persistence within a host during colonization. Person-to-person spread is thought to occur through direct contact with the secretions of colonized individuals. Once acquired, an individual strain can be carried for weeks to months before its eventual clearance. However, it remains unclear how effective colonization is as an immunizing event. Colonization is most common in early childhood; most infants acquire one or many strains sequentially or simultaneously. The rates of carriage vary widely among the 91 known pneumococcal capsular serotypes, which express structurally and antigenically different capsular polysaccharides. The factors that are responsible for these differences, and the variations in serotype distribution between regions and over time, are not understood. After the first years of life, the rates of colonization decline to less than 10% in adult populations. The importance of carriage by young children as the main reservoir for this pathogen has been demonstrated by the recent widespread use of the pneumococcal capsular polysaccharide (CPS) conjugate vaccine (BOX 1). Reduced carriage of the organism in vaccinated children results in herd immunity, which can have an impact on the frequency of pneumococcal disease in those age groups that are not receiving the vaccine7.
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Box 1 | New approaches to pneumococcal vaccination
Current pneumococcal vaccines are exclusively targeted at the capsular polysaccharide (CPS) of Streptococcus pneumoniae, and these vaccines provide strictly serotype-specific protection. Polyvalent purified CPS vaccines were first licensed in the late 1970s, and the current 23-valent formulation is effective against approximately 90% of diseasecausing serotypes in the United States and Europe. Unfortunately, CPSs are T-cell-independent antigens and are therefore poorly immunogenic in young children, particularly for the five pneumococcal serotypes that most commonly cause invasive disease in children145. The poor immunogenicity of CPS antigens has been overcome by conjugation to protein carriers this converts them into T-cell-dependent antigens, which are considerably more immunogenic and a pneumococcal CPSprotein conjugate vaccine (PCV) is now licensed in several countries. However, protection is still serotype specific and, because of the high cost, the number of serotypes that are targeted has been reduced to seven. Disturbingly, studies of nasopharyngeal colonization with S. pneumoniae during trials of PCV showed that, although the carriage of vaccine serotypes was reduced, the vacated niche was promptly occupied by non-vaccine pneumococcal serotypes that are potentially capable of causing disease146. Similar trends were also observed after the general introduction of the conjugate vaccine in the United States147. Although PCV has dramatically reduced the incidence of invasive disease that is caused by vaccine serotypes in the United States, increases in the incidence of bacteraemia caused by non-included serotypes have also been reported148149. Thus, in the long term, the widespread introduction of PCV might merely alter the serotype distribution of invasive pneumococcal disease, rather than reducing its overall burden. In addition, the cost of PCV is high, and therefore it is unlikely that it will be used extensively in developing countries, where the death rate of children from invasive pneumococcal disease is highest. Clearly, there is an urgent need to develop alternative pneumococcal vaccines that do not suffer from these shortcomings. The most promising approach to date is to develop vaccines that are based on pneumococcal proteins that contribute to virulence and are common to all serotypes. Such vaccines need to be highly immunogenic and elicit immunological memory even in infants, who respond well to T-cell-dependent protein antigens. Ensuring high expression levels of antigenic proteins in recombinant bacteria will enable large-scale vaccine production at low cost, thereby producing vaccines that are more affordable, particularly for developing countries. Several candidate protein vaccine antigens have been identified, including non-toxic derivatives of pneumolysin, choline-binding proteins, such as PspA and CbpA, metal-binding lipoproteins, such as PsaA and PiaA, the poly-histidine triad proteins PhtB and PhtE and the neuraminidase NanA. Immunization with each of these proteins provided varying degrees of protection against challenge by one or more S. pneumoniae strains in mouse models of sepsis, pneumonia or carriage146, and there is now clear evidence that immunization with certain combinations of virulence proteins provides additive, or even synergistic, protection149151, the most effective to date being a combination of Ply, PspA and CbpA152,153.
Glycocalyx
Extracellular polysaccharide material that is excreted by epithelial cells and forms an outer layer on epithelial surfaces.
Mechanisms of colonization experimental colonization of adults has been used to investigate the host factors that affect susceptibility to acquisition of S. pneumoniae and its subsequent clearance8. These studies reveal that carriage induces the production of both mucosal and systemic immunoglobulin, which is mainly strain- and type-specific. In contrast to the high levels of serotype-specific anti-capsular antibody that are generated following administration of the conjugate vaccine, it is unclear whether the relatively small amounts that are induced by colonization are sufficient to enhance clearance. In this regard, the decline in carriage rates after childhood is observed widely among the different pneumococcal serotypes, which suggests that if exposure during prior colonization events leads to immunity it might not develop in a serotype-specific manner9. Immunogenic major cell-surface proteins, particularly pneumococcal surface protein A (PspA), also induce antibody, but this response is predominately directed at the more variable, exposed regions of PspA. The level of pre-existing cross-reactive immunoglobulin to PspA correlates with susceptibility to the establishment of pneumococcal carriage8. Animal models have also been used to define the host and bacterial factors that contribute to pneumococcal colonization (TaBlE 1). For example, isolates that were previously examined in human experimental colonization studies also colonized inbred adult mice when a similar inoculum was used for a similar duration.
These isolates also elicited a comparable immune response to CPS and PspA, thereby demonstrating the relevance of this animal model10. It should be noted, however, that mice can also be colonized by a wide spectrum of pneumococcal serotypes, although their susceptibility to disease by different strains varies greatly. Therefore, for certain pneumococcal serotypes, mice are also useful models to study the complications that arise from bacterial colonization, including pneumonia and bacteraemic infection. The experimental findings from mouse-colonization and airway-infection models will be summarized in the following sections. Interactions with host structures. Within minutes of entering the nasal cavity, S. pneumoniae cells encounter mucus secretions (FIG. 1a). The expression of a capsule reduces entrapment in the mucus, thereby allowing the pneumococcus to access the epithelial surfaces 11. Almost all pneumococcal CPSs are negatively charged, which could increase their repulsion of the sialic acidrich mucopolysaccharides that are found in mucus12. Initially, bacteria are detected within the glycocalyx layer that overlies the respiratory and olfactory epithelium11 (FIG. 1b). This might allow bacterial access to receptors on the apical surface of the epithelial cells that line the nasal spaces. Once at the epithelial surface, the expression of a thick capsule seems to be disadvantageous for the pneumococcus, because of its inhibitory effect on adherence. most pneumococcal isolates that have been investigated display phase variation between two forms
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Table 1 | Pneumococcal virulence factors and their main role in colonization and disease
Pneumococcal virulence factors and disease Upper-airway colonization
Capsule ChoP CbpA (also known as PspC) NanA, BgaA and StrH Hyl PavA Eno Prevents entrapment in the nasal mucus, thereby allowing access to epithelial surfaces. Also inhibits effective opsonophagocytosis. Binds to rPAF on the epithelial surface of the human nasopharynx. Binds to human secretory component on a polymeric Ig receptor during the first stage of translocation across the epithelium. Act sequentially to cleave terminal sugars from human glycoconjugates, which might reveal receptors for adherence. Breaks down hyaluronan-containing extracellular matrix components. Binds to fibronectin. Binds to plasminogen. Small antimicrobial peptide that targets members of the same species. Cytolytic toxin that also activates complement. An important determinant of virulence in in vivo models of disease. Wide range of effects on host immune components at sub-lytic concentrations. Prevents binding of C3 onto pneumococcal surface. Also binds lactoferrin. Digests the cell wall, which results in the release of Ply. Component of the ABC transport system, which is involved in resistance to oxidative stress. Component of the ABC transport system. Aid colonization by revealing receptors for adherence, modifying the surfaces of competing bacteria that are within the same niche and/or modifying the function of host clearance glycoproteins. Cleaves human IgA1.
main role in colonization
Competition in upper airway

Bacteriocin (pneumocin) Ply
Respiratory-tract infection and pneumonia
PspA LytA PsaA PiaA and PiuA NanA and NanB
IgA
This list of virulence factors is not exhaustive and only selected examples are shown. For detailed descriptions of virulence factors see the main text. BgaA, -galactosidase; CbpA, choline-binding protein A; ChoP, phosphorylcholine; Eno, enolase; Hyl, hyaluronate lyase; IgA, IgA1 protease; IgA1, immunoglobulin A1; LytA, autolysin A; Nan, neuraminidase; PavA, pneumococcal adhesion and virulence A; PiaA, pneumococcal iron acquisition A; PiuA, pneumococcal iron uptake A; Ply, pneumolysin; PsaA, pneumococcal surface antigen A; PspA, pneumococcal surface protein A; StrH, -N-acetylglucosaminidase.
Phase variation
a molecular mechanism that leads to switching of the geneexpression state; for example, onoff expression.
Lipoteichoic acid
a teichoic acid species that is connected to membrane glycolipids. The stereochemistry of lTas and the biosynthetic origin of the glycerolphosphates are different from those of wall teichoic acids, which have glycerol-phosphate backbones.
Human secretory component

an epithelial glycoprotein that is required for the active transport of polymeric immunoglobulins across mucosal surfaces.
Glycoconjugate
a carbohydrate that is covalently linked to other chemical species.
that can be distinguished by their opaque or transparent colony morphologies (discussed below)13. During the initial stages of colonization, transparent variants that express a thinner capsule and possess other characteristics that promote binding to host tissues prevail over opaque variants13. By analysing human epithelial cells in culture, several receptorligand interactions have been proposed. The bacterial adhesins that are involved include phosphorylcholine (ChoP), which is a constituent of both cell-wall-associated acids and lipoteichoic acids14. ChoP is an unusual bacterial structural component that is a cell-surface feature of several other microorganisms that reside primarily in the upper respiratory tract, such as Haemophilus influenzae, Actinobacillus actinomycetemcomitans and commensal and pathogenic species of the genus Neisseria. As many ChoP-expressing species occupy a similar biological niche, and have a common set of genes (licAD) for choline uptake and incorporation, bacterial ChoP might be particularly important for host airway colonization15. In H. influenzae, ChoP expression enhances persistence in the upper respiratory tract16. In the pneumococcus, ChoP mediates bacterial adherence to the receptor for platelet-activating factor (rPAF) and
activates host cell signalling through this receptor. As the natural ligand for rPAF platelet-activating factor (PAF) also contains ChoP, the pneumococcus might mimic PAF to use its receptor, which is widely distributed on host tissues such as the epithelial surface of the human nasopharynx. Another adhesin is cholinebinding protein A (CbpA; also referred to as PspC or SpsA), which is non-covalently anchored to ChoP. CbpA binds to human secretory component, which is found on the polymeric immunoglobulin receptor, and secretory forms of immunoglobulin17,18. In addition, some isolates express a pilus-like structure that adheres to an unknown epithelial-cell receptor19,20. Other studies have provided evidence for adhesive interactions with host cell glycoconjugates. Pneumococcal adherence to epithelial cells from the human pharynx is inhibited by N-acetylglucosamine--(1,3)-galactose21. The pneumococcus is also one of many pathogens that have been reported to bind to N-acetylglucosamine-(1,4)-galactose, which is a constituent of some human glycosphingolipids22. In addition, S. pneumoniae produces three surface-associated exoglycosidases: a neuraminidase, NanA, a -galactosidase, BgaA, and a -N-acetylglucosaminidase, StrH23. These enzymes act

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a 30 minutes b 1 day
c 3 days
d 14 days
Figure 1 | Nasal colonization by Streptococcus pneumoniae. Progression of nasal colonization of BALB/c mice with a Nature Reviews | Microbiology serotype 23F pneumococcal isolate. ad show the infection as it progresses from 30 minutes to 14 days after infection ( 40 magnification). Bacteria (red) were detected using serotype-specific antisera. Mouse tissue was stained using DAPI (4,6-diamidino-2-phenylindole; blue). The inset in c shows neutrophils stained (green) with an antibody to murine Ly6-G. Images courtesy of A. Roche, University of Pennsylvania, USA.
Basement membrane
a tissue structure that consists of a network of collagen fibres that attach the overlying epithelial layer to the connective tissue underneath.
sequentially to remove the terminal sugars that are found on many human glycoconjugates and, therefore, might unmask receptors for adherence, thereby affecting the function of glycosylated host clearance molecules and/or providing a nutrient source. Binding to extracellular matrix components could also promote bacterial adherence once the organism has gained access to basement membranes. The expression of a surface-attached hyaluronidase could facilitate bacterial spread through a matrix of the hyaluronan-containing polysaccharide component of connective tissues24. Pneumococcal adhesion and virulence A (PavA) and enolase are two pneumococcal surface adhesins that bind the extracellular-matrix components fibronectin and plasminogen, respectively 25,26. PavA is localized to the pneumococcal outer cell surface and has been shown to be important in virulence, as the absence of PavA significantly reduces systemic bacterial loads and increases host survival25. Pneumococcal enolase mutants also exhibit attenuation in a model of respiratory infection, which indicates that plasminogen binding has a role in respiratorydisease26.
The pneumococcal factors that enable host epithelial and tissue barriers to be breached during the progression from colonization to invasive infection are poorly understood. In both mouse models and in humans, opaque variants that express increased amounts of CPS and are more resistant to opsonophagocytic killing are selected for during the transition from the mucosal surface to the bloodstream27,28. Innate and adaptive immunity during colonization. Between 1 and 3 days after the initiation of colonization in mice, there is an influx of neutrophils and uptake of pneumococcal cells in the lumens of the nasal spaces11 (FIG. 1). The association of pneumococci with neutrophils resembles the host response to infection of normally sterile sites. However, this acute inflammatory response (or mild suppurative rhinitis) is ineffective at clearing the carrier state, and pneumococci persist on the epithelial surface, even after resolution of the neutrophilic infiltrate (FIG. 1d). Defined mutants that lack expression of the pore-forming cytotoxin pneumolysin show diminished neutrophil influx and persist for longer periods in a model of nasopharyngeal colonization29.

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epithelial cells in culture respond to the osmotic stress of pneumolysin-mediated pore formation by activating the p38 mitogen-activated protein kinase, which results in an increase in chemokines that attract neutrophils30. These effects of pneumolysin on the epithelial surface suggest that the pneumococcus might have evolved to promote the inflammatory response, perhaps because the resulting secretions increase the likelihood of transmission, even though this increased host response could accelerate the eventual clearance of the pathogen. Host-mediated killing of S. pneumoniae is generally thought to require opsonization by a serotypespecific antibody together with complement, followed by phagocytosis. Interestingly, the course of a colonization event is unaffected in mice that fail to generate a specific antibody, and neither the depletion of neutrophils nor complement impacts on initial colonization by the organism 10. By contrast, mice that lack Toll-like receptor 2 (TlR2), an initiator of the inflammatory responses that follow the recognition of lipoteichoic acid and/or lipoproteins, exhibit delayed pneumococcal clearance29. In addition, mice that do not express major histocompatibility complex class II also show prolonged carriage, which indicates an important role for CD4+ T cells rather than humoral immunity31 (BOX 2). The CD4+ T-cell-dependent effector functions that lead to loss of established colonization have yet to be identified. There is also evidence that pneumolysin stimulates inflammatory responses through TlR4 (REF. 32). Another factor that limits the effectiveness of the host humoral response on mucosal surfaces is bacterial expression of a secreted zinc metalloprotease that specifically targets human immunoglobulin A1 (IgA1), which constitutes more than 90% of the IgA in the human airway33. The cleavage of bound IgA1 produces bacterial surface antigens that are bound to Fab fragments, which prevents inflammation from being initiated through host recognition of the Fc region of the antibody. Because of the activity of the IgA1 protease, antibody-mediated clearance might occur only after sufficient amounts of other classes and subclasses of specific antibody have been generated. Indeed, the decrease in pneumococcal colonization following the use of the CPS conjugate vaccine could be attributable to its induction of high levels of IgG, which is not targeted by the IgA1 protease. The ability of S. pneumoniae to inactivate IgA1 in immunocompetent hosts could account for the generally unaltered incidence of infection that is observed in most IgA-deficient individuals. C-reactive protein (CRP) is another important component of the innate immune response against S. pneumoniae. CRP binds specifically to ChoP and, after binding, CRP interacts with complement component C1q to activate the classical pathway of complement. Transgenic mice that express the human crp gene are more resistant to pneumococcal infection34. moreover, at the concentrations found in the airway, CRP can block pneumococcal adherence through rPAF35. Impact of competition. It is estimated that more than 700 different microbial species can reside within the human pharynx36. Successful colonization probably requires mechanisms that counteract the presence of microbial competitors. There is evidence that in a mouse model of pneumococcal infection, co-colonization with another bacterial species that resides in the same niche can result in the clearance of S. pneumoniae through the induction of complement-mediated neutrophil killing37. This demonstrates that one microbial species can harness the innate immune response of its host to prevail over a competitor. A further implication is that the pneumococcus is resistant to the innate immune responses it stimulates, but is not necessarily resistant to the responses that are induced by its competitors. Pneumococcal strains also compete with each other. The increase in the prevalence of previously uncommon serotypes in populations in which the pneumococcal CPS conjugate vaccine is extensively used (a phenomenon that is referred to as serotype replacement) suggests that nonvaccine pneumococcal types are being out-competed by the serotypes that are present in the vaccine. One mechanism that could underlie this intra-species competition is the strain-specific activity of pneumococcal bacteriocins, which are known as pneumocins38. These small antimicrobial peptides, which are expressed by the blp locus and are under the control of a quorum-sensing pheromone (BlpC), target members of the same species that do not produce the cognate immunity protein. In a mouse model of colonization, differences of only a few amino acids in the bipartite pneumocin BlpmN are sufficient to dictate the outcome of competition between two isolates. Another consequence of the highly populated microbial environment in the human pharynx is the availability of exogenous DNA from closely related oral streptococcal species and co-colonizing pneumococci. These nucleic acids can be taken up by S. pneumoniae because of its natural competence and, subsequently, be used to increase its overall fitness. The acquisition of genes that encode altered penicillin-binding proteins, for example, has facilitated resistance to -lactam antibiotics, which is now a common problem in the treatment of pneumococcal infections39. The ability of the pneumococcus to take up DNA fragments and incorporate homologous sequences into its genome is only observed during aerobic growth. Indeed, this activity could also be a means of compensating for the high mutation rates that result from the oxidative lifestyle of this organism.
Complement
a part of the innate immune system that comprises serum proteins which can protect against infection.
Fab fragment
The region of an antibody that binds to an antigen.
Bacteriocin
a bacterially produced, small, heat-stable peptide that is active against other bacteria and to which the producer has a specific immunity mechanism. Bacteriocins can have a narrow or broad target spectrum.
Protein virulence factors Over the past 20 years, the importance of proteins for S. pneumoniae virulence has become clear. Research in this area has been stimulated by the realization that pneumococcal proteins represent a promising avenue for the development of vaccines that are common to all pneumococcal serotypes. Consequently, the number of potential virulence factors that have been characterized has increased greatly in recent years, and are too numerous to review in detail here. We will therefore discuss only a selection of the pneumococcal virulence factors that are promising candidates for use as vaccine antigens (FIG. 2).

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Box 2 | CD4+ T cells and pneumococcal disease
Research into the host immune response to pneumococcal disease has focused primarily on the role of humoral components of innate and acquired immunity and the cellular aspects of innate immunity. However, an appreciation of the involvement of CD4+ T cells in the clinical setting, in which HIV-1-infected patients who have lower CD4+ T-cell counts are significantly more likely to be persistent pneumococcal carriers than non-HIV-1-infected patients and patients with AIDS frequently develop severe pneumococcal infections, has led to more detailed investigations of the role of these cells154,155. Recently, the role of CD4+ T cells in pneumococcal disease has been revisited using mouse models of pneumococcal disease. To summarize, this work showed that in lungs of mice that are infected intranasally with Streptococcus pneumoniae, CD4+ T cells contribute to early host resistance to infection, as shown by an early rapid increase in T-cell infiltration to areas that are subject to increased pneumococcal invasion. In addition, an increased host susceptibility to infection that was due to significantly increased bacterial loads both in lungs and blood was observed in CD4+-deficient mice44,57. Interestingly, infections that used pneumolysin-deficient isogenic pneumococcal mutants resulted in a total lack of T-cell infiltration into infected lungs, which suggests that pneumolysin was responsible for the pattern of T-cell infiltration57. Studies that used in vitro chemotaxis assays with purified human CD4+ T cells have shown that the CD4+ T-cell migratory process is indeed dependent on the presence of pneumolysin, as pneumolysin-deficient pneumococci failed to stimulate CD4+ T-cell chemotaxis. Sub-lytic concentrations of purified pneumolysin or pneumolysin-positive S. pneumoniae were able to successfully stimulate CD4+ T-cell chemotaxis44. Protection studies in mice have recently confirmed the role of CD4+ T cells as part of an antibody-independent immune response to pneumococcal infection31. It was demonstrated that intranasal immunization with live pneumococci (or a killed, non-encapsulated whole-cell vaccine) against subsequent pneumococcal intranasal challenge, protected antibody-deficient mice, but not CD4+ T-cell-deficient mice. Other researchers have also shown that CD4+ T cells are required for efficient clearance of nasopharyngeal pneumococcal colonization in naive mice, as CD4+ T-cell-deficient mice failed to clear pathogen colonization29. The inability of CD4+ T-cell-deficient mice to clear nasopharyngeal colonization was explained by the lack of induction of a T helper 1 (TH1) response, which was previously shown to have a protective role in the response to pneumococcal disease in humans156. In addition, it has been demonstrated that pneumolysin promotes CD4+ T-cell-dependent clearance of colonization29. Recent studies have also found that CD4+ T-cell proliferative responses to pneumolysin are significantly higher in children who do not have detectable pneumococci in their nasopharynx, which suggests that natural CD4+ T-cell immunity to pneumococcal protein antigens could modulate nasopharyngeal carriage157. Indeed, previous work had shown that mucosal antibody production against pneumococcal proteins, such as pneumolysin and CbpA, was T-cell dependent158. Although it is unclear whether this T-cell immunity cleared existing bacterial carriage or prevented new colonization, it is worth considering that future vaccines should include conserved pneumococcal protein antigens that are capable of inducing CD4+ T-cell immunity.
Pneumolysin. many studies have shown that pneumolysin is a potent, wide-ranging virulence factor. It is found in virtually all pneumococcal isolates, and its amino acid sequence is well conserved, although a small number of variants have been described40,41. Pneumolysin is a member of the family of cholesterol-dependent cytolysins that are synthesized by Gram-positive bacteria. Pneumolysin is produced as a 52 kDa soluble protein that oligomerizes in the membrane of target cells to form a large ring-shaped transmembrane pore. The pore is 260 in diameter and is composed of approximately 40 monomer subunits. During its conversion from a soluble monomer to a membrane-inserted oligomer, pneumolysin undergoes a series of spectacular structural changes42. The oligomers are thought to be responsible for the cytolytic activity of the toxin and the plethora of cellmodulatory activities that are evident at sub-lytic concentrations. These activities include: inhibition of ciliary beating on respiratory epithelium and brain ependyma; inhibition of the phagocyte respiratory burst; and induction of cytokine synthesis and CD4+ T-cell activation and chemotaxis43,44. Site-directed mutagenesis has shown that pneumolysin activates the classical complement pathway independently of its cell-modulatory activity45. Both the cell-modulatory and complement-activation activities of pneumolysin have been shown to contribute to pneumococcal virulence in murine models of
pneumonia4648; however, it is noteworthy that strains of a serotype 1 clone that produce a non-cytolytic pneumolysin have been isolated from cases of invasive pneumococcal disease41. Interestingly, S. pneumoniae that expressed a mutated pneumolysin protein which lacked haemolytic and complement-activating activity was shown to be more virulent than a pneumococcus in which the pneumolysin gene was deleted47. This indicated that pneumolysin has an additional, unidentified function. It was suggested that this activity was an interaction with TlR4, because a pneumococcal mutant that produced pneumolysin without haemolytic activity was reported to activate TlR4-dependent responses32. In addition, Baba and colleagues 49 reported that a non-cytolytic pneumolysin stimulated the production of interferon-. This could also be an unidentified function, although S. pneumoniae strains that express this form of pneumolysin were no more virulent than the pneumococcus in which the pneumolysin gene had been knocked out (A.K. and P.W.A., unpublished observations). There is substantial evidence that pneumolysin is crucial for pneumococcal virulence in pneumonia4648,50,51, although its relative importance can vary from strain to strain. For example, Alexander et al.52 found that mice that had been immunized with pneumolysin were protected significantly from nine pneumococcal
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ATP-binding cassette transporter
LytA Pneumolysin Capsule Cell wall Cell membrane Metalbinding proteins
a recent review of the role and action of pneumolysin in pneumococcal diseases65. S. pneumoniae is also a natural pathogen of horses66. Interestingly, all equine isolates are serotype 3 (REF. 66), and have a major deletion within the gene that encodes pneumolysin, as well as the gene that encodes the autolysin lytA (discussed below)67.
PsaA PiaA PiuA
Eno PavA
Sortases PspA Hyl
PspC Cholinebinding proteins
Pneumococcal cell-surface proteins Cell-surface proteins have attracted considerable attention because of their potential as vaccine antigens that can stimulate the production of opsonic antibodies. Three major groups of pneumococcal cell-surface proteins have been identified: choline-binding proteins, lipoproteins and proteins that are covalently linked to the bacterial cell wall by a carboxy (C)-terminal sortase (lPXTG; in which X denotes any amino acid) motif.
Choline-binding proteins. As mentioned previously, pneumococci express ChoP as a component of their cell-wall teichoic acids and membrane-bound lipoteichoic acids. ChoP anchors a group of proteins, the cholinebinding proteins, to the cell wall. most of these proteins have repeat sequences of approximately 20 amino acids that mediate attachment of the proteins to the cell surface through phosphorylcholine. The amino (N)-terminal sequences vary widely, and are the sites of the specific activities of the different proteins 68. S. pneumoniae encodes 1015 choline-binding proteins69, including PspA, PspC and lytA. PspA. PspA has three structural domains, and its N-terminal region is composed of repeated -helices that protrude from the bacterial cell surface70. Its highly electronegative properties are thought to inhibit complement binding70. Between the N terminus and the C-terminal choline-binding region is a proline-rich region of 6080 amino acids that probably confer flexibility70. PspA is a highly variable molecule that, based on the sequences of the N termini, can be grouped into three families that, in turn, can be subdivided into six different clades71. PspA interferes with the fixation of complement component C3 on the pneumococcal cell surface, and thus inhibits complement-mediated opsonization. PspA is also a lactoferrin-binding protein and, through this activity, is thought to protect the bacterium from the bactericidal activity of apolactoferrin. PspA-knockout mutants are more sensitive to killing by apolactoferrin, and anti-PspA antibodies enhance the bactericidal activity of apolactoferrin72,73. There are conflicting data, however, on the in vivo effect of deleting the pspA gene. Although studies of serotype 3 (REFs 74,75) and 4 (REF. 20) pneumococci showed that PspA is required for in vivo growth, similar experiments that used a loss-of-function mutant of a serotype 2 strain failed to show an effect54,76,77. An investigation by Abeyta and colleagues 77 suggested that the contribution of PspA to virulence might depend on the properties of the pneumococcal polysaccharide capsule.
LPXTG-anchored neuraminindase proteins
Figure 2 | Pneumococcal virulence factors. Streptococcus pneumoniae virulence is multi-faceted. Important pneumococcal virulence factors include: the capsule; the cell wall; choline-binding proteins; pneumococcal surface proteins A and C (PspA and PspC); the LPXTG-anchored neuraminidase proteins; hyaluronate lyase (Hyl); pneumococcal adhesion and virulence A (PavA); enolase (Eno); pneumolysin; autolysin A (LytA); and the metal-binding proteins pneumococcal surface antigen A (PsaA), pneumococcal iron acquisition A (PiaA) and pneumococcal iron uptake A (PiuA).
Nature Reviews | Microbiology
Opsonic antibody
a bacteria-binding (opsonizing) antibody, such as IgG and Iga, that interacts with Fc receptors on phagocytic cells, which leads to an increased uptake of bacteria.
Teichoic acid
a cell-envelope glycopolymer that is composed of many identical sugar-phosphate repeating units, which are usually modified with d-alanine and additional sugars.
Apolactoferrin
The iron-depleted form of the glycoprotein lactoferrin.
strains, but no protection was afforded against a tenth strain. It is likely that the strain-specific nature of this pneumococcal virulence factor will also hold true for many other, and perhaps all, pneumococcal virulence factors. In a mouse model of acute pneumonia, pneumolysin was shown to be essential for the survival of S. pneumoniae in both the upper and lower respiratory tracts53,54. There is also good evidence that pneumolysin is required for bacterial spread from the lungs to the bloodstream5356. In bacteraemic infection, if pneumolysin is expressed, high numbers of pneumococci are found in the bloodstream, and the host succumbs to infection54,56; however, in the absence of pneumolysin, high pneumococcal numbers are tolerated in the blood without overt disease symptoms57, and chronic bacteraemia can develop58. For models of meningitis, however, there is more debate on the importance of pneumolysin. In contrast to a report that suggested only a limited role for pneumolysin in S. pneumoniae meningitis59, subsequent studies found that pneumolysin was a key determinant of pneumococcal virulence in this disease 60,61. Furthermore, pneumolysin has also been shown to be required for pneumococcal-induced deafness in meningitis62 and for pneumococcal-induced damage to the brain ependyma63,64. Additional evidence that supports the role of pneumolysin in meningitis was discussed in

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PspC. PspC is a multifunctional cell-surface protein that is known by several other names that reflect its different activities. For example, it is also known as choline-binding protein A (CbpA), because it was the predominant entity to be purified by ChoP-affinity chromatography78. A pspC-knockout mutant binds less well to epithelial cells and sialic acid in vitro, and shows reduced nasopharyngeal colonization compared with the wild type78. PspC also binds the polymeric immunoglobulin receptor that normally transports secretory IgA; hence it is also called SpsA (secretory pneumococcal surface protein A, as mentioned above)79,80. This activity could be the first stage of translocation across the respiratory epithelium, which is consistent with the reduced virulence of a pspC mutant in a mouse model of pneumonia48. Iannelli et al.81 reported that PspC mutants of serotype 2 and 3 S. pneumoniae were less virulent in a sepsis model. Additional studies have supported this finding82. However, Orihuela et al.54 failed to find any effect on sepsis or pneumonia using a pspA mutant of the same serotype 2 strain, although an impairment in pathogen survival in the nasopharynx was observed. An additional property of PspC is its ability to bind to factor H83,84, which has been shown to prevent formation of C3b though the alternative complement pathway, and thus prevent pneumococcal opsonization82. PspC proteins are highly polymorphic54, and can be divided into two structural groups. In addition to the PspC molecule that binds to phosphorylcholine, the variant that was first described as Hic83 is anchored to the bacterial cell wall using the classical lPXTG motif. However, both the Hic variant and the classic choline-binding form of the protein are able to sequester factor H. PspC also binds complement component C3 (REF. 85). LytA. lytA is an amidase that cleaves the N-acetylmuramoyl-l-alanine bond of pneumococcal peptidoglycan86. The autolytic action of this enzyme leads to the cell lysis that is typical of pneumococcal cells growing in batch culture, but this enzyme also participates in cellwall growth and turnover. lytA-negative S. pneumoniae mutants were shown to have reduced virulence in murine models of pneumonia and bacteraemia51,54,76,87. The contribution of lytA to virulence is thought to be mediated, in part, by its function in the release of pneumolysin and inflammatory peptidoglycan and teichoic acids from lysed bacterial cells. Originally, PsaA was proposed to be a pneumococcal adhesin because of its sequence similarities to putative adhesins from other streptococci93. Subsequent investigations seemed to support this assertion. psaA pneumococcal mutants were deficient in binding to mammalian cells in vitro90,94 and, more recently, it was reported that anti-PsaA antibodies inhibited pneumococcal adherence95. However, PsaA is actually the divalent metal-ion-binding lipoprotein component of an ATP-binding cassette (ABC) transport system that has specificity for manganese96,97. Structural analysis of PsaA shows a potential divalent metal-ion-binding site that can accommodate zinc or manganese ions98, and psaA mutants require manganese for normal growth96. Therefore, the observed effects on bacterial adherence are presumably a result of the pleiotropic impact of a deficiency in manganese transport on the expression of other pneumococcal genes, including adhesins. This proposal is consistent with structural studies which indicate that PsaA is unlikely to protrude beyond the pneumococcal cell wall68,98. Furthermore, psaA is transcribed as part of the psaBCA operon, and if other genes of the operon (psaB or psaC) are mutated, comparable deficiencies in bacterial adherence are observed99. A more important outcome of the psaA mutant studies, however, is that manganese uptake seems to be essential for pneumococcal resistance to oxidative stress, which can result from the production of hydrogen peroxide during pneumococcal metabolism, as well as the generation of reactive oxygen species during the host innate immune response. It is this property that probably accounts for the avirulence of psaA mutants in mouse models of colonization and invasive disease97,100. PiaA and PiuA. Three ABC transporter operons that mediate iron uptake, pia, piu and pit, have been described in S. pneumoniae101,102. each operon comprises genes that encode a metal-binding protein, a membrane permease and an ATPase, but the pia and piu systems seem to be particularly important102. PiaA and PiuA are the lipoprotein metal-binding components of these systems, and immunization with these proteins is protective103. There seems to be redundancy in the iron-uptake systems, however, as only a piupia double-mutant strain had significantly reduced growth in an iron-deficient medium and, although a single mutation in pia or piu decreased virulence in models of pneumonia and bacteraemia, the double mutant was attenuated to a significantly greater extent102.
Factor H
a component of the alternative complement pathway that is involved in the regulation of complement activation.
Divalent metal-ion-binding lipoproteins Between 42 and 45 pneumococcal cell-surface lipoproteins have been described to date69. These include the peptide isomerases PpmA and SlrA, which have been shown to be involved in S. pneumoniae virulence88,89. The metal-binding lipoproteins pneumococcal surface antigen A (PsaA), pneumococcal iron acquisition A (PiaA) and pneumococcal iron uptake A (PiuA) are additional cell-surface lipoproteins that have been shown to be essential for pneumococcal virulence.
PsaA. Deleting psaA abolishes virulence in murine models of pneumonia, bacteraemia and colonization9092.
LPXTG-anchored proteins The best-characterized mechanism of anchoring proteins to the peptidoglycan of Gram-positive bacteria is through the sortase transpeptidase that recognizes the amino-acid sequence lPXTG in surface-located proteins. Some pneumococcal strains encode a single sortase gene, whereas in others multiple sortase-like genes are predicted69. Where multiple sortase genes are present, it is proposed that StrA is responsible for anchoring most lXPTG-containing proteins and the other sortase proteins mediate the anchoring of a subset of surface

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proteins, perhaps in response to specific environmental cues20. StrA has been reported to have a role in pneumococcal colonization, pneumonia and bacteraemia, and in adhesion to nasopharyngeal cells104106. It is estimated that up to 20 S. pneumoniae proteins are anchored by an lPXTG motif 69, including the neuraminidases. Neuraminidases. Neuraminidases, also known as sialidases, cleave terminal sialic acid residues from glycoproteins, glycolipids and cell-surface oligosaccharides. A recent study showed that neuraminidases can remove sialic acid from soluble proteins, such as lactoferrin, IgA2 and secretory component107. S. pneumoniae encodes at least three neuraminidase genes: nanA, nanB and nanC. All strains encode nanA, and most also encode nanB; however, only approximately 50% of strains encode nanC108. Neuraminidases are secreted from the cell, but only NanA contains the lPXTG sequence, which suggests that these enzymes have different functions in vivo. This hypothesis is supported by the observation that NanB has a much lower acidic pH optimum than NanA109. experiments that used loss-of-function mutants in a mouse model of acute pneumonia have shown that NanA and NanB are important for pneumococcal survival in the respiratory tract and bloodstream110. These experiments also indicated that the two enzymes have differing roles; the NanA mutant was rapidly cleared from the respiratory tract, but the NanB mutant persisted, although the number of organisms did not increase110. However, other work that used a mouse nasopharyngeal-colonization model showed no reduction in the ability of a NanA mutant to colonize the nasopharynx over a longer period23. Although there is no direct experimental evidence of a biological role for NanC, an analysis of the distribution of nanC among S. pneumoniae isolates suggested a tissue-specific role nanC was more common in isolates from cerebrospinal fluid than in carriage isolates108. to induce pneumococcal biofilm formation in vitro, and the pattern of pneumococcal-gene expression in biofilms paralleled that observed in the lungs, whereas the gene-expression profile in the bloodstream echoed that observed in planktonic culture in vitro111.
C-type lectin
a carbohydrate-binding protein that is found in a wide range of animals. C-type lectins share a highly conserved calcium-dependent carbohydrate-recognition domain that is used to distinguish them from other animal lectins.
Tissue specificity of virulence It is clear that the contribution of each virulence factor varies, individually and collectively, according to the in vivo location of the bacterium. Using reversetranscription PCR, a recent study observed two patterns of in vivo gene expression by S. pneumoniae: one pattern was characteristic of pneumococci in the bloodstream and the other of pneumococci in the lungs and brain111. In the bloodstream, for example, the expression of ply (which encodes pneumolysin) and pspA was increased, whereas in lung and brain tissue the expression of nanA and nanB, and the competence genes comA, comE and comX, was higher. The involvement of the competence system in virulence was confirmed by administering competence-stimulating peptide (CSP) and using a competence-negative S. pneumoniae mutant. CSP modulated virulence, but in a tissue-specific manner. Thus, the administration of CSP enhanced virulence in pneumonia models, and a comD mutant had reduced virulence. These observations contrast directly with those of bacteraemia, in which the administration of CSP decreases virulence. Finally, CSP was also shown
The pneumococcal capsule Structure, function and role in virulence. The polysaccharide capsule forms the outermost layer of S. pneumoniae cells, and is approximately 200400 nm thick112. With the exception of serotype 3, and possibly others, the capsule is covalently attached to the outer surface of the cell-wall peptidoglycan113. A total of 90 structurally and serologically distinct CPS types have been recognized to date114. Capsule production is indispensable for pneumococcal virulence and is strongly anti-phagocytic in non-immune hosts115 Although non-encapsulated strains have been associated with superficial infections, such as conjunctivitis116,117, clinical isolates from other sterile sites are encapsulated, and spontaneous non-encapsulated derivatives of these strains are largely avirulent. most CPS serotypes are highly charged at physiological pH, and this can directly interfere with interactions with phagocytes118. The capsule also forms an inert shield that seems to prevent either the Fc region of IgG or complement component iC3b, which is bound to deeper cell-surface structures (for example, teichoic acids and cell-surface proteins), from interacting with their relevant receptors on phagocytic cells119,120. more recent data suggest that the capsule can also reduce the total amount of complement that is deposited on the bacterial surface 121. It also reduces the trapping of pneumococcal cells in neutrophil extracellular traps122. The virulence of S. pneumoniae is related to the thickness of the capsule in a particular strain and serotype123. However, pneumococci from the different CPS serotypes differ markedly in their capacity to cause disease115. This presumably reflects their relative capacity to resist phagocytosis, as well as differences in their ability to elicit a humoral immune response. For example, Hostetter124 reported serotype-dependent differences in both the amount and site of covalently bound C3b on the surface of opsonized pneumococcal cells, as well as differences in the degradative processing of bound C3b to iC3b and C3d. Recent studies in mice also suggest an additional potential contributing factor. SIGN-R1, a C-type lectin that is expressed on macrophages in the marginal zone of the spleen was shown to mediate the uptake of both purified CPS and S. pneumoniae125. The spleen is known to play a crucial part in the clearance of blood-borne pneumococci, and mice that are deficient in SIGN-R1 are hypersensitive to invasive pneumococcal disease 126. C-type lectins, such as SIGN-R1 and its human homologues, exhibit oligosaccharide specificity, and differences in their affinity for individual CPS pneumococcal types would undoubtedly influence phagocytosis and bacterial clearance. SIGN-R1 could also influence the presentation of CPS antigens to the immune system, thereby affecting the hosts capacity to mount an antibody response. Finally, Fernebro et al.127 reported that the capsule provides a degree of resistance to spontaneous or antibiotic-induced autolysis, thereby

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Clonal type
a pneumococcal clonal lineage is determined by sequence analysis of a set of housekeeping genes, as opposed to its capsular serotype.
contributing to antibiotic tolerance in clinical isolates. Interestingly, this capacity also varied significantly between capsular serotypes. Although isogenic S. pneumoniae expresses different CPS serotypes that exhibit marked differences in murine virulence, non-capsular factors are also clearly important128,129. molecular epidemiological analysis has demonstrated that properties which are associated with a particular clonal type, in addition to capsular serotype, influence the potential of S. pneumoniae to cause invasive disease in humans. The contribution of host factors was demonstrated in a subsequent study in which isolates that had high human-invasion potential exhibited significantly different virulence and disease kinetics in BAlB/c mice compared with C57Bl/6 mice130. Regulation of CPS production. The transition from nasopharyngeal colonization to invasive disease is clearly a watershed in the relationship between S. pneumoniae and its host, and involves a major switch in the expression of important virulence determinants as the pathogen adapts to its altered microenvironment. maximal expression of capsule is essential for systemic virulence, but the extent of exposure of other important pneumococcal surface structures, such as adhesins, is also influenced by capsular thickness. Non-encapsulated pneumococci exhibit greater adherence to human respiratory epithelial cells (A549) in vitro compared with isogenic derivatives that express either serotype 3 or 19F capsules131. Furthermore, previously encapsulated pneumococci
Ligase? +
Wzx flippase
Wzy capsular polysaccharide polymerase
Figure 3 | Steps in Streptococcus pneumoniae capsule biosynthesis. Capsule Nature Reviews | Microbiology biosynthesis is regulated by the cps locus, which encodes specific glycosyltransferases that assemble the oligosaccharide repeats on the cytoplasmic face of the membrane, and a flippase (Wzx), which transports the repeat units to the external surface of the membrane. At this location, the repeat units are polymerized by Wzy to form highmolecular-weight capsular polysaccharides, which are then, presumably, ligated to the cell wall. The cps locus also encodes enzymes for the synthesis of activated sugar precursors, as well as conserved genes that are involved in regulation.
seem to express little CPS during intimate contact with respiratory epithelial cells in vitro or in vivo132. The capacity to regulate CPS production at the transcriptional, translational or post-translational level is important for the survival of S. pneumoniae in different host environments. To date, no transcriptional-control elements have been identified in association with the pneumococcal 70 cps promoter133. However, there is some evidence to suggest that the level of expression of the cps locus differs between the transparent and opaque phase variants, as more CpsD was detectable by western immunoblotting in the opaque phase variants28. The level of S. pneumoniae cps mRNA, relative to 16S ribosomal RNA, that was isolated from the blood of infected mice was shown to be approximately fourfold higher compared with the levels in S. pneumoniae that had been grown in vitro134. However, significant differences in cps mRNA levels could not be detected between pneumococci that were isolated from the nasopharynx, blood or lungs of infected mice135. The first four genes of the cps locus (cpsAD) are common to all pneumococcal serotypes, with the exception of serotypes 3 and 37. The proteins that are encoded by these genes, CpsAD, have been shown to affect the level of CPS expression136. The central region of the cps locus comprises genes that encode specific glycosyl transferases that assemble the serotype-specific oligosacchariderepeat unit on a lipid carrier. This region also includes a flippase (Wzx) that transports the repeat unit to the external face of the membrane and a polymerase (Wzy) that links the units together. The final region of the locus comprises genes that encode the synthesis of activated sugar precursors (FIG. 3). Interestingly, a CpsA homologue in group B streptococci seems to function as a transcriptional activator137; however, to date, the cpsA gene has not been shown to impact on cps transcription in S. pneumoniae. Nevertheless, cpsA-deletion mutants produced reduced levels of CPS138140. CPS biosynthesis of all but two pneumococcal serotypes has been shown to be dependent upon a regulatory system that is determined by CpsB, CpsC and CpsD. CpsB is a manganese-dependent phosphotyrosine-protein phosphatase, CpsC is a membrane protein that is related to polysaccharide co-polymerases and CpsD is an autophosphorylating protein-tyrosine kinase136,138,139. CpsC is required for CpsD tyrosine autophosphorylation; a cpsC-deletion mutant is rough, and CpsD does not become phosphorylated. mutation of the cpsD gene to inactivate the ATP-binding site eliminated CPS production. It has also been shown that CpsB is required to dephosphorylate CpsD; in cpsB-deletion mutants, the proportion of CpsD that is phosphorylated increases dramatically, and there is a significant decrease in the amount of CPS that is produced. These observations suggest that the non-phosphorylated form of CpsD is active in CPS biosynthesis136,138,139. Recently, a novel role for CpsC in the attachment of CPS to the pneumococcal cell wall was also identified141. Therefore, CpsB, CpsC and CpsD function together to regulate CPS assembly, export and attachment to the cell wall by tyrosine phosphorylation of CpsD (FIG. 4).
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a b
3 1 Cell wall Cell wall P CpsC CpsC P ATP Wzx Wzy P CpsD ATP CpsD 2 P Wzx Wzy CpsB CpsC CpsC Ligase
production by controlling the availability of precursors or co-factors could be one of the regulatory mechanisms that is used by S. pneumoniae.
P P
Ligase
P P
P P
CpsD P
CpsD P
4 CpsB
Figure 4 | model showing the regulation of capsular polysaccharide (cPS) production by tyrosine phosphorylation of cpsD. a | CpsC, CpsD and|ATP interact to Nature Reviews Microbiology promote CPS biosynthesis by the polysaccharide polymerase (step 1). CpsD autophosphorylates, which causes a change in protein interactions and slows CPS biosynthesis (step 2). b | The CPS polymer is then transferred to the putative CPS cell-wall ligase, and is ligated to the cell wall (step 3). Finally, CpsB dephosphorylates CpsD, thereby allowing the cycle to be repeated (step 4).
Interestingly, Regm, a homologue of the staphylococcal catabolite control protein CcpA, which is involved in the regulation of sugar-metabolism pathways, has been shown to affect transcription of the cps locus, which suggests that a carbon source might also influence capsular expression142. Indeed, two proteins that are involved in sugar metabolism have been shown to affect CPS production. Pgm is the phosphoglucomutase that catalyses the conversion of glucose-6-phosphate to glucose-1-phosphate, and GalU is a glucose-1-phosphate uridylyltransferase that catalyses the formation of uridine diphosphateglucose (UDP-Glc) from glucose-1-phosphate. mutants of S. pneumoniae in which either the galU or pgm gene was disrupted produced almost no CPS and exhibited growth defects137,143. Additionally, pneumococcal strains in which the pgm gene had defined point mutations that significantly reduced but did not eliminate enzymatic activity still produced reduced amounts of CPS, even though the mutants no longer exhibited growth defects144. Both Pgm and GalU are required for the synthesis of UDP-Glc, which is a precursor for the biosynthesis of all 90 pneumococcal CPS types, as well as other cellular structures, such as teichoic acid. Thus, limiting the supply of this precursor would be expected to impact heavily upon CPS production in the pneumococcus. Indirect modulation of CPS
Conclusions The pneumococcus has proven to be a particularly formidable foe, and can evade elimination by serum therapy, chemotherapy, multiple classes of antibiotic and, more recently, conjugate polysaccharide vaccines. For each of these approaches, there was considerable initial confidence that Sir William Oslers captain of the men of death had finally been dealt a fatal blow. The adaptability of the organism in response to an array of selective pressures, however, has allowed it to retain its capacity to colonize its human host without decreasing its fitness to the point of diminished virulence. The ability of S. pneumoniae to adapt stems from the remarkable plasticity and heterogeneity of its genome. Whole-genome sequencing of large numbers of pneumococcal isolates is now revealing both the extent of strain-to-strain variation in gene content and also the size of the S. pneumoniae pan-genome, as well as other streptococci that provide an additional reservoir of genetic information. For the most part, the initial focus of investigations of S. pneumoniae was limited to the roles of its various capsule serotypes and the mechanisms that underpin its natural competence for the uptake of DNA. As outlined in this Review, advances in molecular biology and bacterial genetics have not only facilitated a fuller understanding of the biology of its capsule and competence, but have also allowed characterization of the many attributes that contribute to bacteriahost interactions. In addition, representative models of pneumococcal infection are now being applied in genetically modified mice to define the host factors and other aspects of the immune response that contribute to colonization and disease. These studies have, for example, emphasized the importance of CD4+ T cells, rather than antibody responses, in the mediation of immunity to S. pneumoniae (BOX 2). The complexity of the bacteria and host factors that have been illuminated by these studies has also provided insight into novel strategies for antimicrobials and a next generation of more broadly effective pneumococcal vaccines that will use combinations of protein antigens (BOX 1). Despite our progress, key aspects of pneumococcal biology, such as the bacteria and host factors that impact on pathogen transmission, remain largely unexplored. It is still premature, therefore, to assume that the pneumococcus has yielded all of its most important secrets.
1. 2.
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Bergmann, S., Rohde, M., Chhatwal, G. & Hammerschmidt, S. -Enolase of Streptococcus pneumoniae is a plasmin(ogen)-binding protein displayed on the bacterial cell surface. Mol. Microbiol. 40, 12731287 (2001). 27. Kim, J. O. et al. Relationship between cell-surface carbohydrates and intrastrain variation on opsonophagocytosis of Streptococcus pneumoniae. Infect. Immun. 67, 23272333 (1999). 28. Weiser, J. et al. Changes in availability of oxygen accentuate differences in capsular polysaccharide expression by phenotypic variants and clinical isolates of Streptococcus pneumoniae. Infect. Immun. 69, 54305439 (2001). van Rossum, A., Lysenko, E. & Weiser, J. Host and bacterial factors contributing to the clearance of colonisation by Streptococcus pneumoniae in a murine model. Infect. Immun. 73, 77187726 (2005). Ratner, A. et al. Epithelial cells are sensitive detectors of bacterial pore-forming toxins. J. Biol. Chem. 281, 1299412998 (2006). Malley, R. et al. CD4+ T cells mediate antibodyindependent acquired immunity to pneumococcal colonisation. Proc. Natl Acad. Sci. USA 102, 48484853 (2005). Malley, R. et al. Recognition of pneumolysin by Tolllike receptor 4 confers resistance to pneumococcal infection. Proc. Natl Acad. Sci. USA 100, 19661971 (2003). Demonstrates that the pneumococcal toxin pneumolysin triggers inflammatory responses in host macrophages by interacting with TLR4. Such signalling is crucial for the innate immune response to the pneumococcus, and is a paradigm of the fine balance between the protective and deleterious effects of innate inflammatory responses to mucosal pathogens. Wani, J., Gilbert, J., Plaut, A. & Weiser, J. Identification, cloning and sequencing of the immunoglobulin A1 protease gene of Streptococcus pneumoniae. Infect. Immun. 64, 39673974 (1996). Szalai, A. J., Briles, D. E. & Volanakis, J. E. Human C-reactive protein is protective against fatal Streptococcus pneumoniae infection in transgenic mice. J. 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REVIEWS
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DATABASES
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=gene comA | comD | comE | cpsB | galU | nanA | nanB | pgm | psaA | psaB | psaC | pspA | pspC Entrez Genome Project: http://www.ncbi.nlm.nih.gov/ entrez/query.fcgi?db=genomeprj Actinobacillus actinomycetemcomitans | Haemophilus influenzae | Streptococcus pneumoniae Entrez Protein: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=protein BgaA | CcpA | CpsC | CpsD | CRP | LytA | PiaA | StrH
FURTHER INFORMATION
Aras Kadioglus homepage: http://www.le.ac.uk/iii/staff/ ak13.html Jeffrey N. Weisers homepage: http://www.med.upenn.edu/ micro/faculty/weiser.html
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REVIEWS

288 | APRIl 2008 | VOlUme 6

NATURe ReVIeWS | microbiology

main role in colonization

Competition in upper airway

Respiratory-tract infection and pneumonia

PspA LytA PsaA PiaA and PiuA NanA and NanB

Human secretory component

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NATURe ReVIeWS | microbiology

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LytA Pneumolysin Capsule Cell wall Cell membrane Metalbinding proteins

PsaA PiaA PiuA

Sortases PspA Hyl

PspC Cholinebinding proteins

LPXTG-anchored neuraminindase proteins

Nature Reviews | Microbiology

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NATURe ReVIeWS | microbiology

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Wzy capsular polysaccharide polymerase

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68. 69. 70.

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