Modulation of nasopharyngeal innate defenses by viral coinfection predisposes individuals to experimental pneumococcal carriage.
ABSTRACT: Increased nasopharyngeal colonization density has been associated with pneumonia. We used experimental human pneumococcal carriage to investigate whether upper respiratory tract viral infection predisposes individuals to carriage. A total of 101 healthy subjects were screened for respiratory virus before pneumococcal intranasal challenge. Virus was associated with increased odds of colonization (75% virus positive became colonized vs. 46% virus-negative subjects; P=0.02). Nasal Factor H (FH) levels were increased in virus-positive subjects and were associated with increased colonization density. Using an in vitro epithelial model we explored the impact of increased mucosal FH in the context of coinfection. Epithelial inflammation and FH binding resulted in increased pneumococcal adherence to the epithelium. Binding was partially blocked by antibodies targeting the FH-binding protein Pneumococcal surface protein C (PspC). PspC epitope mapping revealed individuals lacked antibodies against the FH binding region. We propose that FH binding to PspC in vivo masks this binding site, enabling FH to facilitate pneumococcal/epithelial attachment during viral infection despite the presence of anti-PspC antibodies. We propose that a PspC-based vaccine lacking binding to FH could reduce pneumococcal colonization, and may have enhanced protection in those with underlying viral infection.
Project description:Pneumococcal surface protein C (PspC) is an important candidate for a cost-effective vaccine with broad coverage against pneumococcal diseases. Previous studies have shown that Streptococcus pneumoniae is able to bind to both human factor H (FH), an inhibitor of complement alternative pathway, and human secretory IgA (sIgA) via PspC. PspC was classified into 11 groups based on variations of the gene. In this work, we used three PspC fragments from different groups (PspC3, PspC5, and PspC8) to immunize mice for the production of antibodies. Immunization with PspC3 induced antibodies that recognized the majority of the clinical isolates as analyzed by Western blotting of whole-cell extracts and flow cytometry of intact bacteria, while anti-PspC5 antibodies showed cross-reactivity with the paralogue pneumococcal surface protein A (PspA), and anti-PspC8 antibodies reacted only with the PspC8-expressing strain. Most of the isolates tested showed strong binding to FH and weaker interaction with sIgA. Preincubation with anti-PspC3 and anti-PspC5 IgG led to some inhibition of binding of FH, and preincubation with anti-PspC3 partially inhibited sIgA binding in Western blotting. The analysis of intact bacteria through flow cytometry showed only a small decrease in FH binding after incubation of strain D39 with anti-PspC3 IgG, and one clinical isolate showed inhibition of sIgA binding by anti-PspC3 IgG. We conclude that although anti-PspC3 antibodies were able to recognize PspC variants from the majority of the strains tested, partial inhibition of FH and sIgA binding through anti-PspC3 antibodies in vitro could be observed for only a restricted number of isolates.
Project description:Streptococcus pneumoniae evades C3-mediated opsonization and effector functions by expressing an immuno-protective polysaccharide capsule and Factor H (FH)-binding proteins. Here we use super-resolution microscopy, mutants and functional analysis to show how these two defense mechanisms are functionally and spatially coordinated on the bacterial cell surface. We show that the pneumococcal capsule is less abundant at the cell wall septum, providing C3/C3b entry to underlying nucleophilic targets. Evasion of C3b deposition at division septa and lateral amplification underneath the capsule requires localization of the FH-binding protein PspC at division sites. Most pneumococcal strains have one PspC protein, but successful lineages in colonization and disease may have two, PspC1 and PspC2, that we show affect virulence differently. We find that spatial localization of these FH-recruiting proteins relative to division septa and capsular layer is instrumental for pneumococci to resist complement-mediated opsonophagocytosis, formation of membrane-attack complexes, and for the function as adhesins.
Project description:Several Streptococcus pneumoniae proteins play a role in pathogenesis and are being investigated as vaccine targets. It is largely unknown whether naturally acquired antibodies reduce the risk of colonization with strains expressing a particular antigenic variant.Serum immunoglobulin G (IgG) titers to 28 pneumococcal protein antigens were measured among 242 individuals aged <6 months-78 years in Native American communities between 2007 and 2009. Nasopharyngeal swabs were collected >- 30 days after serum collection, and the antigen variant in each pneumococcal isolate was determined using genomic data. We assessed the association between preexisting variant-specific antibody titers and subsequent carriage of pneumococcus expressing a particular antigen variant.Antibody titers often increased across pediatric groups before decreasing among adults. Individuals with low titers against group 3 pneumococcal surface protein C (PspC) variants were more likely to be colonized with pneumococci expressing those variants. For other antigens, variant-specific IgG titers do not predict colonization.We observed an inverse association between variant-specific antibody concentration and homologous pneumococcal colonization for only 1 protein. Further assessment of antibody repertoires may elucidate the nature of antipneumococcal antibody-mediated mucosal immunity while informing vaccine development.
Project description:PspC is one of three designations for a pneumococcal surface protein whose gene is present in approximately 75% of all Streptococcus pneumoniae strains. Under the name SpsA, the protein has been shown to bind secretory immunoglobulin A (S. Hammerschmidt, S. R. Talay, P. Brandtzaeg, and G. S. Chhatwal, Mol. Microbiol. 25:1113-1124, 1997). Under the name CbpA, the protein has been shown to interact with human epithelial and endothelial cells (C. Rosenow et al., Mol. Microbiol. 25:819-829, 1997). The gene is paralogous to the pspA gene in S. pneumoniae and was thus called pspC (A. Brooks-Walter, R. C. Tart, D. E. Briles, and S. K. Hollingshead, Abstracts of the 97th General Meeting of the American Society for Microbiology 1997). Sequence comparisons of five published and seven new alleles reveal that this gene has a mosaic structure, and modular domains have contributed to gene diversity during evolution. Two major clades exist: clade A alleles are larger and contain an extra module that is shared with many pspA alleles; clade B alleles are smaller and lack this pspA-like domain. All alleles have a proline-rich domain and a choline-binding repeat domain that show 0% divergence from similar domains in the PspA protein. Immunization of a rabbit with a recombinant clade B PspC molecule produced antiserum that cross-reacted with both PspC and PspA from 15 pneumococcal isolates. The cross-reactive antibodies afforded cross-protection in a mouse model system. Mice immunized with PspC were protected against challenge with a strain that expressed PspA but not PspC. The PspA- and PspC-cross-reactive antibodies were directed to the proline-rich domain present in both molecules.
Project description:PspC was found to bind human complement factor H (FH) by Western blot analysis of D39 (pspC(+)) and an isogenic mutant TRE108 (pspC). We confirmed that PspA does not bind FH, while purified PspC binds FH very strongly. The binding of FH to exponentially growing pneumococci varied among different isolates when analyzed by fluorescence activated cell sorting analysis.
Project description:Pneumococcal surface protein A (PspA) and PspC of Streptococcus pneumoniae are surface virulence proteins that interfere with complement deposition and elicit protective immune responses. The C-terminal halves of PspA and PspC have some structural similarity and contain highly cross-reactive proline-rich (PR) regions. In many PR regions of PspA and PspC, there exists an almost invariant nonproline block (NPB) of about 33 amino acids. Neither the PR regions nor their NPB exhibit the alpha-helical structure characteristic of much of the protection-eliciting N-terminal portions of PspA and PspC. Prior studies of PspA and PspC as immunogens focused primarily on the alpha-helical regions of these molecules that lack the PR and NPB regions. This report shows that immunization with recombinant PR (rPR) molecules and passive immunization with monoclonal antibodies reactive with either NPB or PR epitopes are protective against infection in mice. PR regions of both PspA and PspC were antibody accessible on the pneumococcal surface. Our results indicate that while PspA could serve as a target of these protective antibodies in invasive infections, PspC might not. When antibody responses to rPR immunogens were evaluated by using flow cytometry to measure antibody binding to live pneumococci, it was observed that the mice that survived subsequent challenge produced significantly higher levels of antibodies reactive with exposed PR epitopes than the mice that became moribund. Due to their conservation and cross-reactivity, the PR regions and NPB regions represent potential vaccine targets capable of eliciting cross-protection immunity against pneumococcal infection.
Project description:Streptococcus pneumoniae serotype 3 strains emerge frequently within clinical isolates of invasive diseases. Bacterial invasion into deeper tissues is associated with colonization and immune evasion mechanisms. Thus, pneumococci express a versatile repertoire of surface proteins sequestering and interacting specifically with components of the human extracellular matrix and serum. Hic, a PspC-like pneumococcal surface protein, possesses vitronectin and factor H binding activity. Here, we show that heterologously expressed Hic domains interact, similar to the classical PspC molecule, with human matricellular thrombospondin-1 (hTSP-1). Binding studies with isolated human thrombospondin-1 and various Hic domains suggest that the interaction between hTSP-1 and Hic differs from binding to vitronectin and factor H. Binding of Hic to hTSP-1 is inhibited by heparin and chondroitin sulfate A, indicating binding to the N-terminal globular domain or type I repeats of hTSP-1. Competitive inhibition experiments with other pneumococcal hTSP-1 adhesins demonstrated that PspC and PspC-like Hic recognize similar domains, whereas PavB and Hic can bind simultaneously to hTSP-1. In conclusion, Hic binds specifically hTSP-1; however, truncation in the N-terminal part of Hic decreases the binding activity, suggesting that the full length of the ?-helical regions of Hic is required for an optimal interaction.
Project description:The pneumococcal capsular serotype is an important determinant of complement resistance and invasive disease potential, but other virulence factors have also been found to contribute. Pneumococcal surface protein C (PspC), a highly variable virulence protein that binds complement factor H to evade C3 opsonization, is divided into two subgroups: choline-bound subgroup I and LPxTG-anchored subgroup II. The prevalence of different PspC subgroups in invasive pneumococcal disease (IPD) and functional differences in complement evasion are unknown. The prevalence of PspC subgroups in IPD isolates was determined in a collection of 349 sequenced strains of <i>Streptococcus pneumoniae</i> isolated from adult patients. <i>pspC</i> deletion mutants and isogenic <i>pspC</i> switch mutants were constructed to study differences in factor H binding and complement evasion in relation to capsule thickness. Subgroup I <i>pspC</i> was far more prevalent in IPD isolates than subgroup II <i>pspC</i> The presence of capsule was associated with a greater ability of bound factor H to reduce complement opsonization. Pneumococcal subgroup I PspC bound significantly more factor H and showed more effective complement evasion than subgroup II PspC in isogenic encapsulated pneumococci. We conclude that variation in the PspC subgroups, independent of capsule serotypes, affects pneumococcal factor H binding and its ability to evade complement deposition.
Project description:The pneumococcal capsule is indispensable for pathogenesis in systemic infections; however, many pneumococcal diseases, including conjunctivitis, otitis media, and some systemic infections in immunocompromised patients, are caused by nonencapsulated Streptococcus pneumoniae (NESp). Null capsule clade 1 (NCC1), found in group 2 NESp, expresses pneumococcal surface protein K (PspK) and is becoming prevalent among pneumococcal organisms owing to the widespread use of pneumococcal conjugate vaccines. Despite its clinical importance, the molecular mechanisms underlying the prevalence of NCC1 have not been fully elucidated. Here, we investigated the role of the R3 domain of PspK in the epithelial cell adherence of NCC1. We found that the R3 domain of PspK mediated NCC1 adherence via its direct interaction with the epithelial surface protein annexin A2. Additionally, neutralization with purified recombinant PspK-R3 or rabbit anti-UD:R3 IgG inhibited binding of NESp to lung epithelial cells in vitro. Immunization with the 'repeat' domain of PspK-R3 or PspK-UD:R3 effectively elicited mucosal and systemic immune responses against PspK-R3 and provided protection against nasopharyngeal, lung, and middle ear colonization of NESp in mice. Additionally, we found that rabbit anti-UD:R3 IgG bound to PspC-R1 of the encapsulated TIGR4 strain and that UD:R3 immunization provided protection against nasopharyngeal and lung colonization of TIGR4 and deaths by TIGR4 and D39 in mice. Further studies using 68 pneumococcal clinical isolates showed that 79% of clinical isolates showed cross-reactivity to rabbit anti-UD:R3 IgG. About 87% of serotypes in the 13-valent pneumococcal conjugate vaccine (PCV) and 68% of non-vaccine serotypes were positive for cross-reactivity with rabbit anti-UD:R3 IgG. Thus, the R3 domain of PspK may be an effective vaccine candidate for both NESp and encapsulated Sp.
Project description:Pneumococcal serotypes are represented by a varying number of clonal lineages with different genetic contents, potentially affecting invasiveness. However, genetic variation within the same genetic lineage may be larger than anticipated.A total of 715 invasive and carriage isolates from children in the same region and during the same period were compared using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. Bacterial genome sequencing, functional assays, and in vivo virulence mice studies were performed.Clonal types of the same serotype but also intraclonal variants within clonal complexes (CCs) showed differences in invasive-disease potential. CC138, a common CC, was divided into several PFGE patterns, partly explained by number, location, and type of temperate bacteriophages. Whole-genome sequencing of 4 CC138 isolates representing PFGE clones with different invasive-disease potentials revealed intraclonal sequence variations of the virulence-associated proteins pneumococcal surface protein A (PspA) and pneumococcal choline-binding protein C (PspC). A carrier isolate lacking PcpA exhibited decreased virulence in mice, and there was a differential binding of human factor H, depending on invasiveness.Pneumococcal clonal types but also intraclonal variants exhibited different invasive-disease potentials in children. Intraclonal variants, reflecting different prophage contents, showed differences in major surface antigens. This suggests ongoing immune selection, such as that due to PspC-mediated complement resistance through varied human factor H binding, that may affect invasiveness in children.