Regions of Diversity 8, 9 and 13 contribute to Streptococcus pneumoniae virulence.

BACKGROUND: Streptococcus pneumoniae is the leading cause of community-acquired pneumonia. Previously, using comparative genomic analyses, 13 regions of genomic plasticity have been identified in the S. pneumoniae genome. These "Regions of Diversity" (RDs) accounted for half the genomic variation observed amongst all pneumococci tested, moreover, were determined to encode a variety of putative virulence factors. To date, genes within 5 RDs have been unequivocally demonstrated to contribute to S. pneumoniae virulence. It is unknown if the remaining RDs also contribute to virulence. RESULTS: Using allelic exchange, we created S. pneumoniae mutants that were deficient in RD2, 5, 7, 8, 9, 12 and 13. Mutants deficient in RD8, 9 and 13 were attenuated in a mouse model of disease. RD8 is 40,358 nucleotides in length and encodes 37 genes. Using a panel of isogenic mutants, we determined that RD8b3 is the operon within RD8 that is responsible for virulence. Mice infected with mutants deficient in RD8, RD8b3, RD9 and RD13 had significantly less bacteria in the blood two days after intranasal challenge and improved survival over time versus mice infected with wild type. In all instances mutants colonized the nasopharynx at levels equivalent to wild type. CONCLUSION: Genes within RD1, 3, 4, 6, and 10 have previously been shown to contribute to virulence. This study demonstrates that genes within RD8, 9 and 13 also contribute to virulence. The ability of mutants deficient in RD2, 5, 7, 8, 9, 12, and 13 to colonize the nasopharynx indicates that genes within these RDs are not required for asymptomatic carriage. Nonetheless, the observation that mutants deficient in RD8b3, 9 and 13 are attenuated indicates that genes within these loci are necessary for spread of the bacteria beyond the nasopharynx to normally sterile sites.

Statins protect against fulminant pneumococcal infection and cytolysin toxicity in a mouse model of sickle cell disease.

Sickle cell disease (SCD) is characterized by intravascular hemolysis and inflammation coupled to a 400-fold greater incidence of invasive pneumococcal infection resulting in fulminant, lethal pneumococcal sepsis. Mechanistically, invasive infection is facilitated by a proinflammatory state that enhances receptor-mediated endocytosis of pneumococci into epithelial and endothelial cells. As statins reduce chronic inflammation, in addition to their serum cholesterol-lowering effects, we hypothesized that statin therapy might improve the outcome of pneumococcal infection in SCD. In this study, we tested this hypothesis in an experimental SCD mouse model and found that statin therapy prolonged survival following pneumococcal challenge. The protective effect resulted in part from decreased platelet-activating factor receptor expression on endothelia and epithelia, which led to reduced bacterial invasion. An additional protective effect resulted from inhibition of host cell lysis by pneumococcal cholesterol-dependent cytotoxins (CDCs), including pneumolysin. We conclude therefore that statins may be of prophylactic benefit against invasive pneumococcal disease in patients with SCD and, more broadly, in settings of bacterial pathogenesis driven by receptor-mediated endocytosis and the CDC class of toxins produced by Gram-positive invasive bacteria.

Age-associated inflammation and toll-like receptor dysfunction prime the lungs for pneumococcal pneumonia.

BACKGROUND: Aging is associated with increased inflammation and risk of community-acquired pneumonia. Streptococcus pneumoniae co-opts the nuclear factor kappa B (NFkB)-regulated proteins polymeric immunoglobulin receptor (pIgR) and platelet-activating factor receptor (PAFr) to attach and invade cells. We sought to determine whether aging and chronic inflammation were associated with increased pIgR and PAFr levels in the lungs and increased susceptibility to S. pneumoniae infection. METHODS: Lung protein and messenger RNA levels were quantitated using Western blot and quantitative polymerase chain reaction. NFkB activation was measured by electrophoretic mobility shift assay. Cytokine levels were measured by cytometric bead analysis. To model chronic inflammation, mice were implanted with osmotic pumps that delivered tumor necrosis factor-alpha. RESULTS: Aged mice and those infused with tumor necrosis factor-alpha had increased levels of pIgR and PAFr in their lungs and were more susceptible to S. pneumoniae infection. During pneumonia, aged mice had reduced levels of pIgR and PAFr and less NFkB activation, despite greater bacterial burden. We determined that aged mice had decreased amounts of lung Toll-like receptors 1, 2, and 4 and reduced capacity to respond to S. pneumoniae with proinflammatory cytokine production. CONCLUSIONS: Aged mice and, potentially, elderly humans are more susceptible to pneumonia because of a priming effect of chronic inflammation and Toll-like receptor dysfunction.

Antibodies against PsrP, a novel Streptococcus pneumoniae adhesin, block adhesion and protect mice against pneumococcal challenge.

Pneumococcal serine-rich repeat protein (PsrP) is a putative adhesin encoded in the Streptococcus pneumoniae pathogenicity island psrP-secY2A2. Challenge of mice with serotype 4, strain TIGR4, and the isogenic mutants T4DeltapsrP and T4DeltapsrP-secY2A2 determined that PsrP was required for bacterial persistence in the lungs but not for colonization in the nasopharynx or replication in the bloodstream during sepsis. In vitro experiments corroborated this anatomical site-specific role; psrP mutants failed to bind to A549 and LA-4 lung cells, yet adhered normally to human nasopharyngeal epithelial cells and to cells from human and rodent capillary endothelial cell lines. We determined that the amino terminus of PsrP mediated adhesion. Microspheres coated with recombinant PsrP(SRR1-BR) (rPsrP(SRR1-BR)) adhered to A549 cells, and moreover, preincubation of cells with rPsrP(SRR1-BR) inhibited TIGR4 adhesion in vitro. Antibodies against rPsrP(SRR1-BR) also neutralized PsrP function; antiserum against rPsrP(SRR1-BR) blocked TIGR4 adhesion in vitro and, following passive immunization, it protected mice against challenge. We conclude that PsrP is an adhesin required for bacterial persistence in the lungs and that rPsrP(SRR1-BR) is a protective antigen.

Identification of a Candidate Streptococcus pneumoniae core genome and regions of diversity correlated with invasive pneumococcal disease.

Streptococcus pneumoniae is a leading cause of community-acquired pneumonia and gram-positive sepsis. While multiple virulence determinants have been identified, the combination of features that determines the propensity of an isolate to cause invasive pneumococcal disease (IPD) remains unknown. In this study, we determined the genetic composition of 42 invasive and 30 noninvasive clinical isolates of serotypes 6A, 6B, and 14 by comparative genomic hybridization. Comparison of the present/absent gene matrix (i.e., comparative genomic analysis [CGA]) identified a candidate core genome consisting of 1,553 genes (73% of the TIGR4 genome), 154 genes whose presence correlated with the ability to cause IPD, and 176 genes whose presence correlated with the noninvasive phenotype. Genes identified by CGA were cross-referenced with the published signature-tagged mutagenesis studies, which served to identify core and IPD-correlated genes required for in vivo passage. Among these, two pathogenicity islands, region of diversity 8a (RD8a), which encodes a neuraminidase and V-type sodium synthase, and RD10, which encodes PsrP, a protein homologous to the platelet adhesin GspB in Streptococcus gordonii, were identified. Mice infected with a PsrP mutant were delayed in the development of bacteremia and demonstrated reduced mortality versus wild-type-infected controls. Finally, the presence of seven RDs was determined to correlate with the noninvasive phenotype, a finding that suggests some RDs may contribute to asymptomatic colonization. In conclusion, RDs are unequally distributed between invasive and noninvasive isolates, RD8a and RD10 are correlated with the propensity of an isolate to cause IPD, and PsrP is required for full virulence in mice.