The present study demonstrates that CD-cholesterol is an effective inhibitor of α-toxin that can block the lytic action on erythrocytes and, more importantly, protect the cornea and surrounding tissues from toxin action during infection or when toxin is injected into the cornea. CD-cholesterol limited toxin action on infected eyes, but did not reduce bacterial growth or kill infecting bacteria. This implies that CD-cholesterol inhibition of α-toxin achieved an effective therapy of the infected eyes despite the continued growth of bacteria and the continuing production of α-toxin. CD-cholesterol inhibition of α-toxin was not strain specific; that is, the inhibitor was active for commercial toxin as well as toxin produced by a variety of strains, including ocular clinical isolates
(Table 1) .
The mechanism by which CD-cholesterol inhibits α-toxin has not yet been demonstrated. The effectiveness of cholesterol as an inhibitor of the toxin is not unexpected, because Raff et al.
31 previously demonstrated that α-toxin activity was weakly inhibited by a high concentration of hydrocortisone or methylprednisolone. It has been determined that caveolin, as found in lipid rafts, is an important glucocorticosteroid receptor that could bind molecules like methylprednisolone, hydrocortisone, or, in the present study, cholesterol.
40 Pany et al.
32 and Vijayvargia et al.
33 35 have demonstrated that α-toxin activity is dependent on caveolin-1. Vijayvargia et al.
35 have also demonstrated that α-toxin activity on cells can be delayed by sequestering cholesterol, or inhibited altogether when cholesterol is depleted from cells. Therefore, a possible mechanism for the effectiveness of CD-cholesterol as an inhibitor of α-toxin could be that CD-cholesterol competes for the same cellular target as α-toxin. This competitive binding to the cellular target molecule would reduce the number of caveolin molecules available to α-toxin resulting in fewer cells being lysed as a consequence of α-toxin pore formation.
There have also been reports of α-toxin inhibition by modified CD.
36 Karginov et al.
36 showed that hepta-6–substituted CD molecules can create a molecule that interferes with the lytic action of the toxin. These modified CD molecules are thought to have affinity for the α-toxin heptamer. Specifically, the modified CD molecules are thought to occlude the central pore of the toxin. Relative to the present study, one could speculate that the cholesterol molecule in CD-cholesterol interacts with the central portion of the α-toxin pore. Thus, the cholesterol in complex with CD is envisioned to occlude the toxin pore in a manner similar to modified-CD inhibitors of α-toxin described by Karginov et al.
36
The CD molecule alone had variable effects on the action of α-toxin. In the in vitro hemolysis assay, CD alone failed to significantly inhibit the hemolytic action of α-toxin. In contrast, when CD was mixed with α-toxin then injected into rabbit corneas, there were some protective effects mediated by CD; that is, at 2 hours postinjection the corneas showed staining with fluorescein, but the epithelium was still intact. The fluorescein had penetrated under the epithelial layer suggesting that the layer had been partially loosened by the toxin. However, at 5 hours postinjection, the eyes developed erosions comparable to eyes injected with toxin mixed with PBS. Furthermore, the application of CD alone to eyes infected with S. aureus 8325-4 reduced the size of corneal erosions, but CD alone did not reduce the overall SLE score. These findings can be understood if one considers that, in the eye, the CD molecule can react with cholesterol or other lipid present in the tear film or surrounding tissues to form a complex that has significant inhibitory action on α-toxin. Because of the greater availability of lipids in the eye (e.g., tear film) than in the erythrocyte lysis assay, such spontaneous formation of an inhibitory complex could be more likely to occur in vivo than in the erythrocyte suspension used for the hemolysis assays in vitro.
α-Toxin, as has been demonstrated by virulence studies of bacterial mutants in rabbits and mice, can cause the majority of the pathologic changes observed during ocular infection.
14 15 16 28 29 Studies with purified α-toxin injected into rabbit corneas show that nanogram quantities of α-toxin are toxic.
16 α-Toxin is produced in readily detectable quantities by most isolates. Strain Newman was reportedly deficient in α-toxin production, but more recent studies showed that the very small amount of α-toxin produced by this strain made a significant contribution to its virulence in the rabbit cornea.
37 38 Additional findings on the importance of α-toxin to corneal virulence have been obtained through the study of α-toxin neutralizing antibody.
39 Neutralizing antibody was capable of inhibiting the damage associated with α-toxin during keratitis infection without affecting the growth of bacteria. Although these studies were performed in a rabbit model of keratitis, the action of α-toxin on human cells is very similar to its action on rabbit cells indicating that the results could be relevant to humans.
Based on the demonstrated importance of α-toxin to keratitis, the treatment of Staphylococcus keratitis with an inhibitor of α-toxin could be a valuable adjunctive therapy that could potentially limit corneal damage while an antibiotic therapy kills the infecting bacteria. The inhibition of α-toxin activity could be especially beneficial in those cases in which the antibiotic therapy is slow or ineffective at killing the infecting bacteria in the cornea.