Abstract
Purpose: :
S. aureus is a common cause of ocular infections often characterized by severe tissue damage. The pathogenesis has been related mainly to the secreted cytolysins alpha- and gamma-toxins, as well as a superantigen-like toxin (Ocular Pathology of a Staphylococcus aureus Mutant Lacking a Recently Discovered Virulence Factor ARVO 2010 3891/A27). Other secreted factors, however, could also be important. This study focuses on the corneal pathogenesis of endoproteinase Glu-C (V8 protease), a known virulence factor in other animal models of infection.
Methods: :
Strain Newman deficient in alpha- and gamma-toxins was grown in minimal medium, the supernatant concentrated, and then fractionated by ionic exchange and /or molecular sieve chromatography. Concentrated and dialyzed fractions were injected into rabbit corneas and monitored for 24 hours for pathology. Fractions with maximum corneal pathology were electrophoresed in a 12% SDS-PAGE and prominent proteins were excised and analyzed by Mass Spectrometry followed by BLAST search against sequenced S. aureus genomes. Selected proteins were tested for corneal virulence by intrastromalinjection into rabbit corneas. Slit lamp examination (SLE) was performed at 3 and 6 hours PI.
Results: :
Mass spectrometry of isolated protein bands revealed the presence of two proteins with potential for corneal toxicity; a superantigen-like toxin which we have previously demonstrated to be a virulence factor, and the serine protease V8. A database search shows the gene for V8 protease to be present in most S.aureus genomes sequenced to date. Purified V8 protease obtained from commercial sources was electrophoresed on SDS-PAGE to confirm its purity. Gelatin zymography detected protease activity similar to the activity seen in fractions from S. aureus culture supernatants. Intrastromal injection of heat-inactivated V8 protease (30 minutes / 100°C) did not result in any corneal pathology by 6 hours PI. Injection of 0.8 units of active V8 protease in 10 µl PBS (n ≥ 6) resulted by 3 hours PI in moderate injection, mild chemosis, and iritis. At 6 ours PI, however, large ulcers developed in all injected eyes measuring 4-6 mm in diameter.
Conclusions: :
S. aureus V8 protease can cause severe pathology in the rabbit cornea.
Keywords: Staphylococcus • proteins encoded by disease genes • keratitis