Pneumolysin, PspA, and PspC Contribute to Pneumococcal Evasion of Early Innate Immune Responses during Bacteremia in Mice

Abstract

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...

Fhb, a Novel Factor H-Binding Surface Protein, Contributes to the Antiphagocytic Ability and Virulence of Streptococcus suis

“…As a cofactor of factor I, hFH can also cleave C3b to iC3b (11). In S. pneumoniae, loss of PspC (an hFH-binding surface protein) can cause reduced iC3b and increased C3b/iC3b (compatible with reduced C3 convertase activity) deposition on strains (37,45), which is similar to our data that loss of Fhb could increase C3b/iC3b and reduce iC3b deposition on the mutant. We hypothesized that Fhb could interact with hFH to inhibit activation of the alternative complement pathway by S. suis.…”

Pneumococcal Virulence Gene Expression and Host Cytokine Profiles during Pathogenesis of Invasive Disease

“…Moreover, other proinflammatory factors, such as lipoteichoic acid and Ply, are likely to contribute. Indeed, it was shown that Ply, PspA, and CbpA (PspC) all contribute to pneumococcal evasion of early innate immune responses during bacteremia in mice (62,80). The involvement of proinflammatory cytokines, CXC chemokines, and cell adhesion molecules in pneumococcal meningitis is well established (31,60,74,82).…”

Role of Pore-Forming Toxins in Bacterial Infectious Diseases

“…PLY's complement-activating property functions to reduce the accumulation of T cells, whereas its cytotoxicity increases neutrophil recruitment and contributes to T-cell suppression; neither has a major effect on the accumulation of B cells or macrophages (367). Simultaneously, PLY, aided by pneumococcal surface protein A (PspA), appears to be able to impair the complement system by inhibiting C3 deposition (368,369).…”