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R. J. O'Callaghan, IV, C. C. McCormick, A. R. Caballero; Chemical Inhibition of Alpha-Toxin, a KeyCorneal Virulence Factor of Staphylococcus aureus. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4485. doi: https://doi.org/.
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Staphylococcus aureus is a leading cause of bacterial keratitis and studies of experimental infections have shown that alpha-toxin production accounts for much of the corneal pathology. Mutants lacking this gene have significantly reduced virulence; restoration of the gene restores the virulence. Injection of purified toxin in nanogram amounts causes corneal epithelial erosion and inflammation. Antibody to the toxoid protects the cornea during experimental infection. The toxin has a lytic action that involves an interaction with cholesterol containing lipid rafts. This study describes the first means to chemically inhibit this toxin in the cornea, a finding of potential value for human infections.
Alpha-toxin activity was determined by a hemolytic assay using rabbit erythrocytes (107 per well). Inhibition of the toxin in vitro using a water soluble cholesterol (0.1 %) inhibitor was assayed by hemolytic inhibition. Inhibition of alpha-toxin activity in vivo by prophylactic administration of the inhibitor (1.0 %) was analyzed by measuring epithelial erosions at 1, 2, and 3 hours following intrastromal corneal injection of the toxin (12 hemolytic units). Inhibitor solution was injected intrastromally (10 µl) 1 hour prior to toxin injection and a single drop topically applied at 60, 40, and 20 minutes prior to toxin injection.
Purified alpha-toxin assayed in the presence of a 0.1 % inhibitor solution demonstrated a ~8,000-fold reduction in hemolytic titer. Injection of 12 hemolytic units of alpha-toxin into normal rabbit corneas (n = 8) caused epithelial erosions beginning 1-2 hours after toxin injection and reaching a maximum diameter of 6 mm by 3 hours after injection. In contrast, corneas (n = 8) treated with inhibitor solution prior to toxin injection demonstrated minimal epithelial erosions (≤ 2 mm) through 3 hours post-injection. At 2 hours after toxin injection, the area of the cornea eroded was 18.65 mm2 in the untreated eyes and 4.91 mm2 in the inhibitor-treated eyes, a 74 % reduction. At 3 hours, the inhibitor treatment caused a 58 % reduction (17.08 mm2 versus 7.11 mm2) in the area of cornea eroded by alpha-toxin.
The cholesterol-containing inhibitor solution inhibits alpha-toxin in vitro and in the rabbit cornea, evidencing its potential ability to limit the corneal damage mediated by Staphylococcus aureus keratitis.
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