SPECIFIC ROLES OF ALPHA-TOXIN AND BETA-TOXIN DURING STAPHYLOCOCCUS-AUREUS CORNEAL INFECTION

Staphylococcus aureus corneal infection results in extensive inflammat ion and tissue damage. Our previous studies of bacterial mutants have demonstrated a role for alpha-toxin in corneal virulence, This study a nalyzes, by genetic rescue experiments, the virulence of mutants affec ting alpha-toxin and beta-toxin activity and demonstrates the ocular t oxicity of these purified staphylococcal proteins. Three types of isog enic mutants were analyzed: (i) mutants specifically deficient in alph a-toxin (Hla) or beta-toxin (Hlb), (ii) a mutant deficient in both ala and HIb, and (iii) a regulatory mutant, deficient in the accessory ge ne regulator (agr), that produces reduced quantities of multiple exopr oteins, including alpha- and beta-toxins, Plasmids coding for Hla and Hlb (pDU1212 and pCU1hlb, respectively) were used to restore toxin act ivity to mutants specifically deficient in each of these toxins, Eithe r corneas were injected intrastromally with logarithmic-phase S. aureu s or purified alpha- or beta-toxins were administered to normal eyes, Ocular pathology was evaluated by slit lamp examination and myeloperox idase activity of infiltrating polymorphonuclear leukocytes, Corneal h omogenates were cultured to determine the CFU per cornea, Eyes infecte d with the wild-type strain developed significantly greater corneal da mage than eyes infected with Agr(-), Hlb(-), or Hla(-) strains, Epithe lial erosions produced by parent strains were not produced by Agr(-) o r Hla(-) strains, Hlb+ strains, unlike Hlb(-) strains, caused scleral edema. Plasmid pDU1212 restored corneal virulence to strain DU1090 (HI a(-)), and plasmid pCU1hlb restored corneal virulence to strain DU5719 (Hlb(-)). Application of purified alpha-toxin produced corneal epithe lial erosions and iritis, while application of beta-toxin caused scler al inflammation. These studies confirm the role of alpha-toxin as a ma jor virulence factor during S. aureus keratitis and implicate beta-tox in, a mediator of edema, as a lesser contributor to ocular damage.