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A. R. Caballero, C. C. McCormick, A. Tang, R. J. O'Callaghan; Isolation and Characterization of a New Staphylococcus aureus Protease. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5514.
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The corneal virulence of Staphylococcus aureus has been established to be mediated by alpha-toxin and, to a lesser extent, gamma-toxin; toxins produced by about 70% and >90% of the isolates respectively. Strain Newman is a prototype strain that produces little alpha-toxin yet is fully virulent in the cornea. A mutant of Newman unable to produce alpha- or gamma-toxins retained extensive corneal virulence. This study attempted to define the virulence mechanism that could be important for corneal virulence in ~30% of the isolates.
Mutant of strain Newman deficient in alpha- and gamma-toxin was grown in M9 chemically defined medium. Culture supernatants were concentrated and fractionated by ultra-filtration and molecular sieve chromatography. Fractions were tested for corneal toxicity by intrastromal injection of aliquots into rabbit corneas. An active fraction was analyzed for proteolytic activity by digestion of fluorescent casein. The molecular size of the protease was measured by zymography and on SDS-PAGE gels. N-terminal analysis was performed, the gene coding for the protease identified, and PCR reaction for the gene developed. The PCR product was cloned and expressed as a 6xHis-tagged recombinant protein.
The active fraction of the culture supernatant caused extensive pathology in rabbit corneas. The fraction contained a protease that digested casein and migrated on zymograms as a major band of ~37 kDa, and on SDS-PAGE at ~ 22 kDa. The N-terminal sequence of the isolated protein was identified and a blast search indicated that it was the gene product of the chromosomal gene NC_009641, a site identified as a potential toxin with possible super-antigen activity. The recombinant protein expressed in E. coli was inactive, but re-naturation allowed restoration of limited proteolytic activity. The protease was found to cleave fibrinogen, but not IgA, IgM, or IgG .
The isolation and initial characterization of a potentially important virulence factor indicates that the molecule, suspected from its structure to be a super-antigen toxin, is a protease that has not been previously characterized or identified as a protease.
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