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AR Caballero, B Thibodeaux, M Marquart, MR Traidej, R O'Callaghan; Pseudomonas Protease And Corneal Virulence . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1596.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:To analyze the correlation between corneal virulence and protease IV production by P. aeruginosa strain PA103-29, an organism deficient in elastase and alkaline protease. Methods:An allele replacement mutant of the protease IV gene was prepared by inserting a tetracycline resistant marker into the cloned protease IV gene and allowing this construct to recombine with the PA103-29 chromosome by tri-parental mating. This protease IV knockout strain was confirmed by PCR and Southern blotting. Protease IV production (or lack of) was assayed by casein degradation, zymography, colorimetric assays using a chromogenic peptide, and Western blot. Tissue damage caused by the knockout mutant relative to the wild type strain was assayed by analysis of 7 different parameters during slit lamp examination in a rabbit intrastromal model of keratitis. Results:The parental strain PA103-29 was positive for protease IV production as evidenced by casein degradation, the presence of a 200 kda band on zymograms, cleavage of the chromogenic peptide, and reaction with specific antisera resulting in a 30 kda band on Western blots. The protease IV knockout strain was negative for protease IV production by zymography, colorimetric assay and Western blotting relative to the parental strain. The knockout strain, however, was still able to degrade casein. The protease IV knockout strain produced an average SLE score of 9.88 relative to the parental strain score of 12.38 (p= 0.035). Conclusion:Elimination of the protease IV gene of PA103-29 significantly reduced but did not abolish corneal virulence. The in vitro analysis suggests that there could be an uncharacterized protease responsible for this mutant’s corneal virulence.
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