Abstract
Purpose: :
To characterize further the virulence mechanisms of S. aureus operative in experimental keratitis.
Methods: :
S. aureus strain Newman deficient in alpha– and gamma–toxins (Δhlg hla) was grown in M9 medium for 24 hrs. Culture supernatants were filtered and concentrated for analysis and for fractionation by molecular sieve chromatography. Aliquots (20 µl) were analyzed by SDS–PAGE and for toxicity in both the native and heat–denatured forms by intrastromal injection into rabbit corneas. Slit lamp examination scoring was performed at 2, 4 and 24 hrs after protein injection. Protein of interest was analyzed by N–terminal amino acid sequencing.
Results: :
Strain Newman (Δhlg hla) was fully virulent in the rabbit eye and intrastromal injection of its culture supernatant produced severe inflammation. Fractions obtained by molecular sieve chromatography that had corneal toxicity contained two major protein bands on SDS–PAGE; this toxicity was sensitive to heat inactivation, suggesting that this was not an immune response to the protein. The toxicity included chemosis, injection, severe corneal infiltration, and iritis that appeared by 4 hours post–injection and reached a maximum by 24 hours post–injection. The protein band that was most dominant in the toxic fractions and that was common to all fractions with corneal toxicity had a molecular weight of 12–14 kDa. This protein had an N–terminal sequence of 20 amino acids that precisely matched an open reading frame of the genome of S. aureus strain 8325–4. The genomic site identified was not previously associated with toxicity, suggesting that this is evidence of a new staphylococcal toxin.
Conclusions: :
These studies have implicated a protein–based toxicity that mediates considerable corneal damage during experimental S. aureus keratitis.
Keywords: Staphylococcus • keratitis • protein purification and characterization