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-cytometri 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 flo 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.Streptococcus pneumoniae remains the principal causative agent of otitis media, pneumonia, and meningitis in both children and adults [1]. The pneumococcus is an extracellular pathogen that uses virulence factors, such as its polysaccharide capsule, to inhibit ingestion and subsequent killing by phagocytes [1,2]. The expression of several virulence-associated proteins-including the choline-binding pneumococcal surface proteins A [3,4] and C (PspC; also referred to as "CbpA" and "SpsA") [5][6][7], as well as pneumolysin [8]-also contribute to this organism's capacity to hinder im-

The pneumococcal virulence factors include capsule, PspA, PspC, and Ply. Cytometric analysis demonstrated that the greatest levels of C3 deposition were on a ⌬ply 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 ⌬ply PspA ؊ PspC ؊ pneumococci compared to the clearance of nonencapsulated pneumococci.Streptococcus pneumoniae possesses virulence factors that function in evasion of complement (3,(27)(28)(29). The capsular polysaccharide (CPS) is considered a key factor in complement resistance (13,21), and the interaction of pneumococci with complement varies according to the CPS type (1,15,19). Ply can activate the classical pathway, diverting complement activation (26,29). Pneumococcal surface protein A (PspA) can also interfere with complement deposition, blocking recruitment of alternative pathway (AP) proteins (4, 24). Pneumococcal surface protein C (PspC; also called CbpA and SpsA) interacts with factor H (7,22). Factor H regulates the AP by serving as a cofactor during factor I-mediated cleavage of C3b to iC3b (10,11,16,25). Studies have demonstrated that more C3b is deposited on nonencapsulated pneumococci (30) and on PspA Ϫ or Ply Ϫ strains (24, 31). PspC mutants are less able to inhibit AP activation (17) and have reduced virulence (9). We investigated C3 deposition, complement inactivation, and blood clearance of pneumococci in the absence of Ply, PspA, and PspC.Pneumococcal strains, growth conditions, and CPS determination. The pneumococci used are listed in Table 1 and include R36A, D39, and isogenic mutants of D39. LM91, TRE108, and TRE121 are insertion-duplication mutants, and ⌬PLY2 and ⌬PAC (generated for this study by deleting ply of TRE121) were generated by allelic replacement (29). Bacteria were grown to mid-log phase as described previously (22). When necessary, erythromycin (0.5 g/ml), tetracycline (15 g/ml), and trimethoprim (50 g/ml) were added to media.To investigate the combined role of Ply, PspA, and PspC in C3 deposition, we generated ⌬PAC. Experiments with ⌬PAC were repeated using independent clones from different transformations to ensure that the results were not due to inadvertent mutations. Growth curves demonstrated that growth of D39 and growth of ⌬PAC were similar (not shown). The amount of CPS was determined by an enzyme-linked immunosorbent assay as previously described (8), using an anti-CPS type 2 monoclonal antibody, monoclonal antibody 2G1 (provided by M. H. Nahm). D39 and mutants of this strain had similar levels of CPS. The endpoint titers for D39, ⌬PAC, and TRE121 were 1,160 Ϯ 655, 1,350 Ϯ 540, and 1,340 Ϯ 530 (means Ϯ standard errors of the means), respectively; these values were significantly different (P ϭ 0.03) from the value for R36A (Յ10). CPS of pneumococci was also detected by flow cytometry (fluorescence-activated cell sorting; FACScan cytometer; Becton Dickinson) as described previously (22) using monoclonal antibody 2G1, w...

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.

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.

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.