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.

Endophthalmitis caused by the encapsulated strain is more damaging to retinal function and structural integrity. These findings indicate that capsule is an important virulence factor of S. pneumoniae endophthalmitis, in contrast to keratitis, suggesting that the anatomic host site in pneumococcal ocular infections is important.

Immunization with PLY helps protect the eye from damage caused by pneumococcal endophthalmitis.

Besifloxacin had an 8-fold lower MIC for MRSA than gatifloxacin and moxifloxacin and was significantly more effective than gatifloxacin and moxifloxacin in reducing the number of MRSA in the rabbit cornea 16 hours after infection.

HI-PLY and psiPLY both elicit antibodies that provide passive protection against S. pneumoniae keratitis.

The purpose of this study was to determine whether the in vitro activity and concentration of Streptococcus pneumoniae pneumolysin correlated to the pathogenesis of S. pneumoniae endophthalmitis. Five S. pneumoniae clinical endophthalmitis strains were grown in media to similar optical densities (OD), and extracellular milieu was tested for pneumolysin activity by hemolysis of rabbit red blood cells. Pneumolysin concentration was determined using a sandwich ELISA. Rabbit vitreous was injected with 102 colony-forming units (CFU) of 1 of 2 different strains with low hemolytic activity (n = 10 and 12 for strains 4 and 5, respectively) or 1 of 3 different strains with high hemolytic activity (n = 12 per strain). Pathogenesis of endophthalmitis infection was graded by slit lamp examination (SLE) at 24 hours post-infection. Bacteria were recovered from infected vitreous and quantitated. The SLE scores of eyes infected with strains having high hemolytic activity were significantly higher than the scores of those infected with strains having low hemolytic activity (P < 0.05). Pneumolysin concentration in vitro, however, did not correlate with hemolysis or severity of endophthalmitis. Bacterial concentrations from the vitreous infected with 4 of the strains were not significantly different (P > 0.05). These data suggest that pneumolysin hemolytic activity in vitro directly correlates to the pathogenesis of S. pneumoniae endophthalmitis. The protein concentration of pneumolysin, however, is not a reliable indicator of pneumolysin activity.

Active immunization with pneumococcal capsular polysaccharide and Freund's adjuvant fails to produce opsonizing antibodies, and passive administration of serotype specific opsonizing antibodies offers no protection against pneumococcal keratitis in the rabbit, whereas active immunization with the conserved protein virulence factor PLY and Freund's adjuvant is able to reduce corneal inflammation associated with pneumococcal keratitis, but has variable effects on bacterial loads in the cornea.