The Streptococcus pneumoniae capsule is vital for virulence and may inhibit complement activity and phagocytosis. However, there are only limited data on the mechanisms by which the capsule affects complement and the consequences for S. pneumoniae interactions with phagocytes. Using unencapsulated serotype 2 and 4 S. pneumoniae mutants, we have confirmed that the capsule has several effects on complement activity. The capsule impaired bacterial opsonization with C3b/iC3b by both the alternative and classical complement pathways and also inhibited conversion of C3b bound to the bacterial surface to iC3b. There was increased binding of the classical pathway mediators immunoglobulin G (IgG) and C-reactive protein (CRP) to unencapsulated S. pneumoniae, indicating that the capsule could inhibit classical pathway complement activity by masking antibody recognition of subcapsular antigens, as well as by inhibiting CRP binding. Cleavage of serum IgG by the enzyme IdeS reduced C3b/iC3b deposition on all of the strains, but there were still marked increases in C3b/iC3b deposition on unencapsulated TIGR4 and D39 strains compared to encapsulated strains, suggesting that the capsule inhibits both IgG-mediated and IgG-independent complement activity against S. pneumoniae. Unencapsulated strains were more susceptible to neutrophil phagocytosis after incubation in normal serum, normal serum treated with IdeS, complement-deficient serum, and complement-deficient serum treated with IdeS or in buffer alone, suggesting that the capsule inhibits phagocytosis mediated by Fc␥ receptors, complement receptors, and nonopsonic receptors. Overall, these data show that the S. pneumoniae capsule affects multiple aspects of complement-and neutrophil-mediated immunity, resulting in a profound inhibition of opsonophagocytosis.The Gram-positive pathogen Streptococcus pneumoniae is one of the most common causes of pneumonia, septicemia, and meningitis in children and adults in both industrialized and developing parts of the world (10). This large burden of disease is compounded by the increased incidence of S. pneumoniae infections associated with HIV and by increasing antibiotic resistance among clinical isolates, and there is a strong need to understand the molecular pathogenesis of S. pneumoniae infections to assist the development of new therapeutic targets. Probably the most important virulence factor for S. pneumoniae is the extracellular capsule, a layer consisting of chains of monosaccharides that surrounds the bacteria. For S. pneumoniae strains, there are 91 antigenically distinct capsular serotypes, dictated by the order and type of the monosaccharide units within the polysaccharide chain and by different side branches (5, 27). The importance of the S. pneumoniae capsule for virulence is demonstrated by the facts that (i) all clinical isolates causing invasive disease are encapsulated; (ii) loss of the capsule by either genetic mutation or enzymatic degradation dramatically reduces S. pneumoniae virulence in animal models of infection ...

Streptococcus pneumoniae strains vary considerably in the ability to cause invasive disease in humans, and this is partially associated with the capsular serotype. The S. pneumoniae capsule inhibits complement-and phagocyte-mediated immunity, and differences between serotypes in these effects on host immunity may cause some of the variation in virulence between strains. However, the considerable genetic differences between S. pneumoniae strains independent of the capsular serotype prevent an unambiguous assessment of the effects of the capsular serotype on immunity using clinical isolates. We have therefore used capsular serotype-switched TIGR4 mutant strains to investigate the effects of the capsular serotype on S. pneumoniae interactions with complement. Flow cytometry assays demonstrated large differences in C3b/iC3b deposition on opaque-phase variants of TIGR4(؊)؉4, ؉6A, ؉7F, and ؉23F strains even though the thicknesses of the capsule layers were similar. There was increased C3b/iC3b deposition on TIGR4(؊)؉6A and ؉23F strains compared to ؉7F and ؉4 strains, and these differences persisted even in serum depleted of immunoglobulin G. Neutrophil phagocytosis of the TIGR4(؊)؉6A and ؉23F strains was also increased, but only in the presence of complement, showing that the effects of the capsular serotype on C3b/iC3b deposition are functionally significant. In addition, the virulence of the TIGR4(؊)؉6A and ؉23F strains was reduced in a mouse model of sepsis. These data demonstrate that resistance to complement-mediated immunity can vary with the capsular serotype independently of antibody and of other genetic differences between strains. This might be one mechanism by which the capsular serotype can affect the relative invasiveness of different S. pneumoniae strains.

The nasopharyngeal commensal bacteria Streptococcus pneumoniae is also a frequent cause of serious infections. Nasopharyngeal colonisation with S. pneumoniae inhibits subsequent re-colonisation by inducing Th17-cell adaptive responses, whereas vaccination prevents invasive infections by inducing antibodies to S. pneumoniae capsular polysaccharides. In contrast, protection against invasive infection after nasopharyngeal colonisation with mutant S. pneumoniae strains was associated with antibody responses to protein antigens. The role of colonisation-induced Th17-cell responses during subsequent invasive infections is unknown. Using mouse models, we show that previous colonisation with S. pneumoniae protects against subsequent lethal pneumonia mainly by preventing bacteraemia with a more modest effect on local control of infection within the lung. Previous colonisation resulted in CD4-dependent increased levels of Th17-cell cytokines during subsequent infectious challenge. However, mice depleted of CD4 cells prior to challenge remained protected against bacteraemia, whereas no protection was seen in antibody deficient mice and similar protection could be achieved through passive transfer of serum. Serum from colonised mice but not antibody deficient mice promoted phagocytosis of S. pneumoniae, and previously colonised mice were able to rapidly clear S. pneumoniae from the blood after intravenous inoculation. Thus, despite priming for a Th17-cell response during subsequent infection, the protective effects of prior colonisation in this model was not dependent on CD4 cells but on rapid clearance of bacteria from the blood by antibody-mediated phagocytosis. These data suggest that whilst nasopharyngeal colonisation induces a range of immune responses, the effective protective responses depend upon the site of subsequent infection.

Streptococcus pneumoniae infections induce inflammatory responses that contribute toward both disease pathogenesis and immunity, but the host–pathogen interactions that mediate these effects are poorly defined. We used the surface lipoprotein-deficient ∆lgt pneumococcal mutant strain to test the hypothesis that lipoproteins are key determinants of TLR-mediated immune responses to S. pneumoniae. We show using reporter assays that TLR2 signaling is dependent on pneumococcal lipoproteins, and that macrophage NF-κB activation and TNF-α release were reduced in response to the ∆lgt strain. Differences in TNF-α responses between Δlgt and wild-type bacteria were abrogated for macrophages from TLR2- but not TLR4-deficient mice. Transcriptional profiling of human macrophages revealed attenuated TLR2-associated responses to ∆lgt S. pneumoniae, comprising many NF-κB–regulated proinflammatory cytokine and chemokine genes. Importantly, non-TLR2–associated responses were preserved. Experiments using leukocytes from IL-1R–associated kinase-4–deficient patients and a mouse pneumonia model confirmed that proinflammatory responses were lipoprotein dependent. Our data suggest that leukocyte responses to bacterial lipoproteins are required for TLR2- and IL-1R–associated kinase-4–mediated inflammatory responses to S. pneumoniae.

e Different capsular serotypes of Streptococcus pneumoniae vary markedly in their ability to cause invasive infection, but the reasons why are not known. As immunity to S. pneumoniae infection is highly complement dependent, variations in sensitivity to complement between S. pneumoniae capsular serotypes could affect invasiveness. We have used 20 capsule-switched variants of strain TIGR4 to investigate whether differences in the binding of the alternative pathway inhibitor factor H (FH) could be one mechanism causing variations in complement resistance and invasive potential between capsular serotypes. Flow cytometry assays were used to assess complement factor binding and complement-dependent neutrophil association for the TIGR4 capsuleswitched strains. FH binding varied with the serotype and inversely correlated with the results of factor B binding, C3b/iC3b deposition, and neutrophil association. Differences between strains in FH binding were lost when assays were repeated with pspC mutant strains, and loss of PspC also reduced differences in C3b/iC3b deposition between strains. Median FH binding was high in capsule-switched mutant strains expressing more invasive serotypes, and a principal component analysis demonstrated a strong correlation between serotype invasiveness, high FH binding, and resistance to complement and neutrophil association. Further data obtained with 33 clinical strains also demonstrated that FH binding negatively correlated with C3b/iC3b deposition and that median FH binding was high in strains expressing more invasive serotypes. These data suggest that variations in complement resistance between S. pneumoniae strains and the association of a serotype with invasiveness could be related to capsular serotype effects on FH binding.

Lipoproteins are an important class of surface associated proteins that have diverse roles and frequently are involved in the virulence of bacterial pathogens. As prolipoproteins are attached to the cell membrane by a single enzyme, prolipoprotein diacylglyceryl transferase (Lgt), deletion of the corresponding gene potentially allows the characterisation of the overall importance of lipoproteins for specific bacterial functions. We have used a Δ lgt mutant strain of Streptococcus pneumoniae to investigate the effects of loss of lipoprotein attachment on cation acquisition, growth in media containing specific carbon sources, and virulence in different infection models. Immunoblots of triton X-114 extracts, flow cytometry and immuno-fluorescence microscopy confirmed the Δ lgt mutant had markedly reduced lipoprotein expression on the cell surface. The Δ lgt mutant had reduced growth in cation depleted medium, increased sensitivity to oxidative stress, reduced zinc uptake, and reduced intracellular levels of several cations. Doubling time of the Δ lgt mutant was also increased slightly when grown in medium with glucose, raffinose and maltotriose as sole carbon sources. These multiple defects in cation and sugar ABC transporter function for the Δ lgt mutant were associated with only slightly delayed growth in complete medium. However the Δ lgt mutant had significantly reduced growth in blood or bronchoalveolar lavage fluid and a marked impairment in virulence in mouse models of nasopharyngeal colonisation, sepsis and pneumonia. These data suggest that for S. pneumoniae loss of surface localisation of lipoproteins has widespread effects on ABC transporter functions that collectively prevent the Δ lgt mutant from establishing invasive infection.

We examined the contribution of serotype on Streptococcus pneumoniae adhesion and virulence during respiratory tract infection using a panel of isogenic TIGR4 (serotype 4) mutants expressing the capsule types 6A (+6A), 7F (+7F) and 23F (+23F) as well as a deleted and restored serotype 4 (+4) control strain. Immunoblots, bacterial capture assays with immobilized antibody, and measurement of mean fluorescent intensity by flow cytometry following incubation of bacteria with antibody, all determined that the surface accessibility, but not total protein levels, of the virulence determinants Pneumococcal surface protein A (PspA), Choline binding protein A (CbpA), and Pneumococcal serine-rich repeat protein (PsrP) changed with serotype. In vitro, bacterial adhesion to Detroit 562 pharyngeal or A549 lung epithelial cells was modestly but significantly altered for +6A, +7F and +23F. In a mouse model of nasopharyngeal colonization, the number of +6A, +7F, and +23F pneumococci in the nasopharynx was reduced 10 to 100-fold versus +4; notably, only mice challenged with +4 developed bacteremia. Intratracheal challenge of mice confirmed that capsule switch strains were highly attenuated for virulence. Compared to +4, the +6A, +7F, and +23F strains were rapidly cleared from the lungs and were not detected in the blood. In mice challenged intraperitoneally, a marked reduction in bacterial blood titers was observed for those challenged with +6A and +7F versus +4 and +23F was undetectable. These findings show that serotype impacts the accessibility of surface adhesins and, in particular, affects virulence within the respiratory tract. They highlight the complex interplay between capsule and protein virulence determinants.

Avirulent strains of a bacterial pathogen could be useful tools for investigating immunological responses to infection and potentially effective vaccines. We have therefore constructed an auxotrophic TIGR4 ⌬pab strain of Streptococcus pneumoniae by deleting the pabB gene Sp_0665. The TIGR4 ⌬pab strain grew well in complete medium but was unable to grow in serum unless it was supplemented with para-aminobenzoic acid (PABA). The TIGR4 ⌬pab strain was markedly attenuated in virulence in mouse models of S. pneumoniae nasopharyngeal colonization, pneumonia, and sepsis. Supplementing mouse drinking water with PABA largely restored the virulence of TIGR4 ⌬pab. An additional ⌬pab strain constructed in the D39 capsular serotype 2 background was also avirulent in a sepsis model. Systemic inoculation of mice with TIGR4 ⌬pab induced antibody responses to S. pneumoniae protein antigens, including PpmA, PsaA, pneumolysin, and CbpD, but not capsular polysaccharide. Flow cytometry demonstrated that IgG in sera from TIGR4 ⌬pab-vaccinated mice bound to the surface of TIGR4 and D39 bacteria but not to a capsular serotype 3 strain, strain 0100993. Mice vaccinated with the TIGR4 ⌬pab or D39 ⌬pab strain by intraperitoneal inoculation were protected from developing septicemia when challenged with the homologous S. pneumoniae strain. Vaccination with the TIGR4 ⌬pab strain provided only weak or no protection against heterologous challenge with the D39 or 0100993 strain but did strongly protect against a TIGR4 capsular-switch strain expressing a serotype 2 capsule. The failure of cross-protection after systemic vaccination with ⌬pab bacteria suggests that parenteral administration of a live attenuated vaccine is not an attractive approach for preventing S. pneumoniae infection.