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Purpose: Wall teichoic acids (WTAs) are major polyanionic polymer components of the cell wall of Staphylococcus aureus. However, little is known about their role at the host-pathogen interface, especially in endophthalmitis. This study was designed to investigate the extent to which WTAs contribute to the pathogenicity of S. aureus in models of endophthalmitis and to determine whether there would be value in targeting their biosynthesis as a new therapeutic approach., Methods: S. aureus RN6390 and its isogenic WTA-null mutant (RN6390ΔtarO) were used to evaluate the role of WTAs in endophthalmitis. RN6390 and RN6390ΔtarO were cultured in bovine vitreous humor (VH) in vitro or inoculated into the vitreous chamber of C57B6 mice. Changes in the number of bacteria, organ function as determined by electroretinography (ERG), and histopathologic changes were assessed throughout the course of infection. In addition, the efficacy of WTA biosynthesis inhibitors in VH in vitro was examined., Results: It was observed that a component of VH synergized with WTA biosynthesis inhibitors in vitro and killed the S. aureus. This effect was also seen when mutants incapable of expressing WTA were exposed to VH. The killing activity of VH was lost on treatment with a protease inhibitor. RN6390ΔtarO could not survive in mouse eyes and did not affect organ function, nor was it able to establish endophthalmitis., Conclusions: WTAs are essential cellular constituents for the manifestation of virulence by S. aureus in endophthalmitis, and appears to be a viable target for treating the endophthalmitis caused by S. aureus strains.

Purpose: Bacillus endophthalmitis is a highly explosive infection of the eye that commonly results in rapid inflammation and vision loss, if not loss of the eye itself, within a few days. Quorum-sensing-controlled toxins are essential to virulence during infection. Another unique characteristic of this disease is the ability of Bacillus to replicate rapidly and migrate to all parts of the eye. This study was conducted to determine the combined roles of toxins and motility during Bacillus endophthalmitis., Methods: Rabbit eyes were injected intravitreally with approximately 100 cfu of wild type, nonmotile, or nonmotile/quorum-sensing-deficient Bacillus thuringiensis. Infection courses were analyzed by biomicroscopy, histology, electroretinography, and quantitation of bacteria and inflammatory cells., Results: Infection with wild type B. thuringiensis resulted in complete retinal function loss by 18 hours after infection, whereas nonmotile B. thuringiensis infections required 30 hours to achieve a reduction of >90% in retinal function. Further attenuation of infection resulted from infection with the nonmotile/quorum-sensing-deficient B. thuringiensis strain, with approximately 90% retinal function loss occurring at 36 hours. Overall, the nonmotile and nonmotile/quorum-sensing-deficient mutants were significantly less virulent than wild-type B. thuringiensis., Conclusions: The results demonstrate that, in addition to quorum-sensing-controlled toxin production, bacterial migration within the eye contributed to the rapidly fulminant and destructive course of Bacillus endophthalmitis. Motility and quorum-sensing may therefore represent possible targets for the development of therapies designed to attenuate the devastating effects of Bacillus in the eye during endophthalmitis.

Purpose: To determine the role of complement and Fas Ligand (FasL) in the host defense against Staphylococcus aureus endophthalmitis., Methods: C3-/-, FasL defective gld, and C57/BL6 (wild-type [WT]) mice were infected intravitreally with 500 and 5000 CFU S. aureus, and the course of infection was followed by determining the intraocular bacteria counts, retinal function by ERG, and morphologic damage and inflammation by histopathology and flow cytometry., Results: In WT eyes injected with 500 CFU, S. aureus grew to 1 x 10(7) CFU/mL by 24 hours, but was cleared by 96 hours. In the WT eyes injected with 5000 CFU, S. aureus grew to 2 x 10(9) CFU/mL by 72 hours, resulting in corneal perforation. C3-/- eyes injected with 500 CFU reached transiently higher levels than their WT counterparts (P < 0.001), but eventually followed a similar course. Bacterial counts in gld eyes infected with 500 CFU were similar to those in WT eyes infected with 5000 CFU. In WT and C3-/- eyes injected with 500 CFU, retinal function decreased only transiently and recovered to 66% in 72 hours. In WT eyes injected with 5000 CFU and gld eyes infected with 500 CFU, retinal function was completely lost by 24 hours. By 24 hours, WT and C3-/- eyes injected with 500 CFU were infiltrated with a similar number of granulocytes, but recruitment was significantly impaired in gld eyes (P < 0.005). Cell counts in WT and C3-/- eyes decreased thereafter without overt retinal disease. In eyes injected with 5000 CFU and gld eyes infected with 500 CFU, inflammatory cells completely filled the intraocular space by 48 hours. Retinal and uveal tissue was destroyed by that time., Conclusions: The tipping point for a good versus a bad outcome in this murine model of endophthalmitis lies between 500 and 5000 CFU S. aureus. This point is identical in animals deficient in complement activation, suggesting that complement does not play a significant role in the ocular defense against intraocular bacteria. In contrast, FasL was found to be critical for clearance, since animals deficient in FasL signaling were unable to control infection with 500 CFU.

Purpose: CAP37 is a polymorphonuclear neutrophil (PMN)-derived inflammatory protein with potent antibiotic and chemotactic activity. To further investigate the biological significance of CAP37 in infection and inflammation, a well-characterized in vivo rabbit model of bacterial keratitis was selected to study its contribution to host defenses., Methods: One hundred colony-forming units of log phase Staphylococcus aureus was injected intrastromally. Eyes were enucleated at 5 to 25 hours after infection and CAP37 detected by immunohistochemistry. To identify the mechanism of CAP37 upregulation in corneal epithelium, in vitro studies using immortalized human corneal epithelial cells (HCECs) were undertaken to determine whether proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), induce CAP37. Because adhesion of leukocytes is important in leukocyte-epithelium interactions, the effect of CAP37 on expression of intercellular adhesion molecule (ICAM)-1 on HCECs was determined by flow cytometry., Results: Strong staining for CAP37 was demonstrated in the corneal epithelium, stromal fibroblasts, ciliary epithelium, related limbus, ciliary vascular endothelium, and bulbar conjunctiva in rabbits injected with S. aureus. The most dramatic expression of CAP37 aside from that in the PMNs occurred in the corneal epithelium. The in vitro studies suggest that CAP37 induction is regulated by TNF-alpha and IL-1beta. In addition, ICAM-1 expression on HCECs was increased in response to CAP37. Molecular cloning of corneal epithelial CAP37 indicated strong sequence identity with an extensive region of PMN-CAP37., Conclusions: The findings in this study describe the extraneutrophilic expression of CAP37 in response to infection and suggest a role for CAP37 in host defense against infection in the eye.