Quincy C. Moore

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 Δply 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 Δply PspA− PspC− pneumococci compared to the clearance of nonencapsulated pneumococci.

Factor H Binding to PspC of Streptococcus pneumoniae Increases Adherence to Human Cell Lines In Vitro and Enhances Invasion of Mouse Lungs In Vivo

ABSTRACT 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.

The Streptococcus pneumoniae Capsule Is Required for Full Virulence in Pneumococcal Endophthalmitis

PURPOSE To determine whether Streptococcus pneumoniae capsule was necessary for pathogenesis of pneumococcal endophthalmitis. METHODS An isogenic capsule-deficient strain was created using homologous recombination. New Zealand White rabbits were injected intravitreously with 10(2) colony-forming units (CFU) of the parent strain or the capsule mutant. Slit lamp examination (SLE), electroretinography, and myeloperoxidase activity were performed 24 and 48 hours postinfection (PI). Serial dilutions of vitreous were plated to quantitate CFU, eyes were extracted for histology, and host cytokine mRNA expression was determined. RESULTS Eyes infected with the parent strain had significantly higher SLE scores than eyes infected with the capsule-deficient strain 24 and 48 hours PI (P < 0.001). CFU recovered from eyes infected with the capsule mutant were significantly fewer than CFU recovered from eyes infected with the parent strain 24 and 48 hours PI (P < 0.001). The parent strain caused a significantly greater decrease in retinal function and more retinal destruction than the mutant strain 48 hours PI (P = 0.026). Vitreal IL-1β, IL-6, and TNF-α were upregulated by both the parent and mutant strain 12 hours PI. By 48 hours PI, there was significantly more neutrophil infiltration in the vitreous infected with the parent strain. CONCLUSIONS 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.

Efficacy of Besifloxacin in an Early Treatment Model of Methicillin-Resistant Staphylococcus Aureus Keratitis

PURPOSE To determine the effectiveness of topically applied besifloxacin, gatifloxacin, and moxifloxacin for the early treatment of experimental methicillin-resistant Staphylococcus aureus (MRSA) keratitis. METHODS Ten hours post-MRSA infection, rabbit eyes were treated topically with 19 doses of phosphate-buffered saline (PBS), besifloxacin, gatifloxacin, or moxifloxacin. Slit-lamp examinations were performed before and after the inoculation. Corneas were harvested for bacterial quantitation and minimal inhibitory concentrations (MICs) were determined. RESULTS All 3 fluoroquinolones significantly lowered the clinical severity of the infection as compared to treatment with PBS (P < 0.05). However, the mean log(10) colony-forming unit (CFU) recovered from besifloxacin-treated corneas was significantly lower than all other treatment groups (P < 0.01). CFU recovered from corneas treated with moxifloxacin and PBS showed no significant difference (P = 0.12). Corneas treated with gatifloxacin had a significantly lower log(10) CFU recovered as compared to PBS-treated corneas (P < 0.01). The MICs for gatifloxacin and moxifloxacin were 8 microg/mL, whereas the MIC for besifloxacin was 1 microg/mL. CONCLUSIONS All 3 fluoroquinolones significantly lowered the clinical severity of the infection. 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.

Active Immunization with Pneumolysin versus 23-Valent Polysaccharide Vaccine for Streptococcus pneumoniae Keratitis

PURPOSE The purpose of this study was to determine whether active immunization against pneumolysin (PLY), or polysaccharide capsule, protects against the corneal damage associated with Streptococcus pneumoniae keratitis. METHODS New Zealand White rabbits were actively immunized with Freunds adjuvant mixed with pneumolysin toxoid (ψPLY), Pneumovax 23 (PPSV23; Merck, Whitehouse Station, NJ), or phosphate-buffered saline (PBS), before corneal infection with 10⁵ colony-forming units (CFU) of S. pneumoniae. Serotype-specific rabbit polyclonal antisera or mock antisera were passively administered to rabbits before either intravenous infection with 10¹¹ CFU S. pneumoniae or corneal infection with 10⁵ CFU of S. pneumoniae. RESULTS After active immunization, clinical scores of corneas of the rabbits immunized with ψPLY and Freunds adjuvant were significantly lower than scores of the rabbits that were mock immunized with PBS and Freunds adjuvant or with PPSV23 and Freunds adjuvant at 48 hours after infection (P ≤ 0.0010), whereas rabbits immunized with PPSV23 and Freunds adjuvant failed to show differences in clinical scores compared with those in mock-immunized rabbits (P = 1.00) at 24 and 48 hours after infection. Antisera from rabbits actively immunized with PPSV23 and Freunds adjuvant were nonopsonizing. Bacterial loads recovered from infected corneas were higher for the ψPLY- and PPSV23-immunized rabbits after infection with WU2, when compared with the mock-immunized rabbits (P ≤ 0.007). Conversely, after infection with K1443, the ψPLY-immunized rabbits had lower bacterial loads than the control rabbits (P = 0.0008). Quantitation of IgG, IgA, and IgM in the sera of ψPLY-immunized rabbits showed high concentrations of PLY-specific IgG. Furthermore, anti-PLY IgG purified from ψPLY-immunized rabbits neutralized the cytolytic effects of PLY on human corneal epithelial cells. Passive administration of serotype-specific antisera capable of opsonizing and killing S. pneumoniae protected against pneumococcal bacteremia (P ≤ 0.05), but not against keratitis (P ≥ 0.476). CONCLUSIONS Active immunization with pneumococcal capsular polysaccharide and Freunds 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 Freunds adjuvant is able to reduce corneal inflammation associated with pneumococcal keratitis, but has variable effects on bacterial loads in the cornea.

Protection from Streptococcus pneumoniae Keratitis by Passive Immunization with Pneumolysin Antiserum

PURPOSE To determine whether passive immunization with pneumolysin antiserum can reduce corneal damage associated with pneumococcal keratitis. METHODS New Zealand White rabbits were intrastromally injected with Streptococcus pneumoniae and then passively immunized with control serum, antiserum against heat-inactivated pneumolysin (HI-PLY), or antiserum against cytotoxin-negative pneumolysin (psiPLY). Slit lamp examinations (SLEs) were performed at 24, 36, and 48 hours after infection. An additional four corneas from rabbits passively immunized with antiserum against psiPLY were examined up to 14 days after infection. Colony forming units (CFUs) were quantitated from corneas extracted at 20 and 48 hours after infection. Histopathology of rabbit eyes was performed at 48 hours after infection. RESULTS SLE scores at 36 and 48 hours after infection were significantly lower in rabbits passively immunized with HI-PLY antiserum than in control rabbits (P < or = 0.043). SLE scores at 24, 36, and 48 hours after infection were significantly lower in rabbits passively immunized with psiPLY antiserum than in control rabbits (P < or = 0.010). The corneas of passively immunized rabbits that were examined up to 14 days after infection exhibited a sequential decrease in keratitis, with an SLE score average of 2.000 +/- 1.586 at 14 days. CFUs recovered from infected corneas were not significantly different between each experimental group and the respective control group at 20 or 48 hours after infection (P > or = 0.335). Histologic sections showed more corneal edema and polymorphonuclear leukocyte (PMN) infiltration in control rabbits compared with passively immunized rabbits. CONCLUSIONS HI-PLY and psiPLY both elicit antibodies that provide passive protection against S. pneumoniae keratitis.

Peritoneal Challenge Modulates Expression of Pneumococcal Surface Protein C during Bacteremia in Mice

ABSTRACT 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.

A comparison of pneumolysin activity and concentration in vitro and in vivo in a rabbit endophthalmitis model

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.

Immunization with Pneumolysin Protects Against Both Retinal and Global Damage Caused by Streptococcus pneumoniae Endophthalmitis

PURPOSE To determine whether immunization with pneumolysin (PLY) protects against pneumococcal endophthalmitis. METHODS New Zealand white rabbits were immunized with a mutant form of PLY that retains only 1% of its cytolytic activity until serum IgG titers were ≥51,200. For a negative control, rabbits were immunized with phosphate-buffered saline (mock). Each vitreous was injected with 10(2) colony-forming units of a clinical endophthalmitis isolate of Streptococcus pneumoniae. Severity of endophthalmitis was graded by slit lamp examination at 24 and 48 h postinfection (PI). Serial dilutions of vitreous were plated for bacterial colony-forming units quantitation, eyes were extracted for histology, and a whole blood survival assay was performed. RESULTS Immunized rabbits had a significantly lower mean slit lamp examination score at 24 and 48 h PI when compared to mock immunized rabbits (P ≤ 0.002). There was not a significant difference in bacterial load in the vitreous at 24 or 48 h PI. Histological sections showed that retinas of mock immunized rabbits appeared to be destroyed, whereas those of PLY immunized rabbits remained largely intact. Damage spread to the aqueous humor, stroma, and conjunctiva of mock immunized rabbits by 48 h PI. Minimal damage was observed in the vitreous of PLY immunized rabbits and did not spread to other parts of the eye. Whole blood from immunized rabbits inhibited the growth of bacteria better than whole blood from mock immunized rabbits. CONCLUSION Immunization with PLY helps protect the eye from damage caused by pneumococcal endophthalmitis.

Pathogenesis of A Clinical Ocular Strain of Streptococcus pneumoniae and the Interaction of Pneumolysin with Corneal Cells

Streptococcus pneumoniae is an important cause of bacterial keratitis, an infectious disease of the cornea. This study aimed to determine the importance of pneumolysin (PLY), a pneumococcal virulence factor, in keratitis using a clinical keratitis isolate (K1263) and its isogenic mutant deficient in PLY (K1263ΔPLY) and determine the effect of these strains on primary rabbit corneal epithelial (RCE) cells. Each strain was injected into the corneal stromas of rabbits, clinical examinations were performed, and the recovered bacterial loads were determined. Bacterial extracts were exposed to RCE cells, and morphology and viability were assessed. The mutant strain deficient in PLY, K1263ΔPLY, caused significantly lower ocular disease scores than the parent strain (K1263), although a higher bacterial load was recovered from corneas infected with the mutant strain. Histological examination showed increased inflammatory cells in the anterior chamber and increased edema in eyes infected with the parent strain. RCE cells exposed to the parent strain had significantly decreased cell viability and showed increased evidence of cellular damage. This study confirms that in a strain that can cause clinical keratitis, PLY is a significant cause of the damage associated with pneumococcal keratitis. It also shows for the first time that the results from an in vitro model using RCE cells correlates with in vivo results thereby establishing a less invasive way to study the mechanisms of pneumococcal keratitis.