Peritoneal challenge modulates expression of pneumococcal surface protein C during bacteremia in mice.

Differential expression of pneumococcal virulence proteins has been demonstrated. We previously demonstrated challenge route-dependent differences in pneumococcal surface protein C (PspC) expression during bacteremia. In this study, we investigated differences in PspC expression during the transition of pneumococci from the peritoneum to the blood. Time course analysis of PspC expression using flow cytometry demonstrated that Streptococcus pneumoniae D39 collected from blood expressed significantly more PspC than did D39 collected from the peritoneum of intraperitoneally (i.p.)-infected mice. Various challenge models were then used to determine whether host responses originating from the peritoneum can influence PspC expressed by pneumococci in the blood. Using heat-inactivated D39 (HI-D39) and sterile peritoneal dialysis fluid (PDF), we investigated whether stimulation of peritoneal responses can influence PspC expression. Injection of mice i.p. with HI-D39 or PDF immediately prior to intravenous (i.v.) infection with D39 caused a significant increase in PspC expressed by D39 in the blood. Finally, we used cytokine array analysis to investigate specific inflammatory mediators that may result in differential PspC expression. Of the 96 inflammatory cytokines assayed, D39 i.p. challenge led to increased expression of 33 cytokines in serum; whereas D39 i.v. challenge led to increased expression of 15 and decreased expression of 11 cytokines relative to serum of the uninfected control. These results indicate that PspC is differentially regulated during growth in vivo and that the level of expression varies depending on the host environment.

Factor H binding to PspC of Streptococcus pneumoniae increases adherence to human cell lines in vitro and enhances invasion of mouse lungs in vivo.

Pneumococcal surface protein C (PspC) binds to both human secretory immunoglobulin A (sIgA) and complement factor H (FH). FH, a regulator of the alternative pathway of complement, can also mediate adherence of different host cells. Since PspC contributes to adherence and invasion of host cells, we hypothesized that the interaction of PspC with FH may also mediate adherence of pneumococci to human cells. In this study, we investigated FH- and sIgA-mediated pneumococcal adherence to human cell lines in vitro. Adherence assays demonstrated that preincubation of Streptococcus pneumoniae D39 with FH increased adherence to human umbilical vein endothelial cells (HUVEC) 5-fold and to lung epithelial cells (SK-MES-1) 18-fold, relative to that of D39 without FH on the surface. The presence of sIgA enhanced adherence to SK-MES-1 6-fold and to pharyngeal epithelial cells (Detroit 562) 14-fold. Furthermore, sIgA had an additive effect on adherence to HUVEC; specifically, preincubation of D39 with both FH and sIgA led to a 21-fold increase in adherence. Finally, using a mouse model, we examined the significance of the FH-PspC interaction in pneumococcal nasal colonization and lung invasion. Mice intranasally infected with D39 preincubated with FH had increased bacteremia and lung invasion, but they had similar levels of nasopharyngeal colonization compared to that of mice challenged with D39 without FH.

Characterization of Streptococcus pneumoniae isolated from children with otitis media.

Streptococcus pneumoniae is the main causative agent of acute otitis media in children. Serotype-based vaccines have provided some protection against otitis media, but not as much as anticipated, demonstrating the need for alternative vaccine options. Pneumococcal otitis media isolates were obtained from children 5 years old or younger from hospitals around Mississippi in the prevaccine era (1999-2000). These isolates were compared by capsular typing, pneumococcal surface protein A (PspA) family typing, antibiotic susceptibility, and DNA fingerprinting. Our study shows that there is great genetic variability among pneumococcal clinical isolates of otitis media, except with regard to PspA. Therefore, efforts focused on the development of a PspA-based pneumococcal vaccine would be well placed.

Pneumolysin, PspA, and PspC contribute to pneumococcal evasion of early innate immune responses during bacteremia in mice.

The pneumococcal virulence factors include capsule, PspA, PspC, and Ply. Cytometric analysis demonstrated that the greatest levels of C3 deposition were on a Deltaply PspA(-) PspC(-) mutant. Also, Ply, PspA, and PspC expression resulted in C3 degradation in vitro and in vivo. Finally, blood clearance assays demonstrated that there was enhanced clearance of Deltaply PspA(-) PspC(-) pneumococci compared to the clearance of nonencapsulated pneumococci.

Interaction of clinical isolates of Streptococcus pneumoniae with human complement factor H.

PspC recruits complement factor H (FH) to the pneumococcal surface. While there is differential expression of pspC during infection, detection of PspC on the surface of viable pneumococci is difficult due to variability among PspCs. We analyzed FH binding to detect PspC expression on the surface of pneumococcal isolates from different pathological sources. Using flow cytometry, we investigated FH-binding to 89 low-passage clinical isolates classified by disease manifestation (systemic, mucosal, or carriage). Carriage isolates recruited significantly more FH to their surfaces than either systemic or mucosal isolates, and this binding was independent of capsular serotype.

Cellular location of polyamine transport protein PotD in Streptococcus pneumoniae.

Streptococcus pneumoniae encodes a transporter for polyamines that contributes to virulence in an animal model. The putative polyamine-binding protein, PotD, has an amino-terminal secretory peptide but no other domains known to be involved in anchoring proteins to the surface of Gram-positive bacteria. Cell fractionation and immunoblotting, along with flow cytometry, suggest that PotD is surface-exposed and anchored to the cytoplasmic membrane by a potentially novel mechanism.

Enhanced protective immunity against pneumococcal infection with PspA DNA and protein.

The effect of priming and boosting with pspA/EF5668 and purified recombinant PspA/EF5668 was examined. With this strategy CBA/N mice were protected against fatal challenge with Streptococcus pneumoniae EF5668. Anti-PspA antibody titers were elevated, and Western analysis with the immune serum demonstrated cross-reactivity with PspA from several different pneumococcal isolates, representing different PspA clades. Immune serum localized cross-reactive epitopes to the alpha-helical domain of PspA/Rx1 and PspA/EF5668. We demonstrated that DNA/protein prime-boost immunizations can enhance protective immunity against pneumococcal challenge.

In vivo binding of complement regulator factor H by Streptococcus pneumoniae.

Pneumococcal surface protein C (PspC) binds to the complement regulatory protein factor H (FH), which inhibits alternative pathway activation. In the present study, using a mouse model of systemic infection and flow-cytometric analyses, we demonstrated an in vivo interaction between FH and pneumococci and showed differential FH binding during bacteremia. Flow-cytometric analyses of pneumococci harvested after intraperitoneal (ip) challenge demonstrated increased binding of FH, compared with that after intravenous (iv) challenge. Real-time polymerase chain reaction analyses of PspC mRNA showed that, relative to pneumococci grown in vitro, those recovered from the blood of mice 24 h after iv challenge exhibited 23-fold higher mRNA levels; however, after ip challenge, PspC mRNA induction was increased 870-fold. A subsequent increase in PspC expression was detected by flow cytometry using a monoclonal antibody against PspC. Furthermore, pneumococci with FH bound to complement before exposure had increased proliferation, compared with pneumococci not pretreated with FH. These results suggest that the interaction between PspC and FH contributes to pneumococcal virulence.