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A. Tang, M. E. Marquart, C. C. McCormick, A. R. Caballero, R. J. O'Callaghan; Distribution and Further Characterization of Pseudomonas aeruginosa Small Protease. Invest. Ophthalmol. Vis. Sci. 2007;48(13):725.
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© ARVO (1962-2015); The Authors (2016-present)
To study the distribution and expression of the Pseudomonas aeruginosa small protease (PASP) gene, a recently discovered ocular virulence factor, by strains of P. aeruginosa and non-aeruginosa Pseudomonas species, and to further characterize its proteolytic activity.
Twenty-five strains of P. aeruginosa and a strain each of Pseudomonas putida, stutzeri, alcaligenes, and otiditis were analyzed by PCR using two sets of primers specific for the PASP gene of Pseudomonas aeruginosa. Concentrated culture supernatants from all strains were analyzed for the presence of the PASP protein by Western blot, using antibody against recombinant PASP made in rabbits (1:1000), and for proteolytic activity by gelatin zymography. Furthermore, the proteolytic activity of purified recombinant PASP against immunologically important molecules active in ocular defense (fibrinogen, IgA, IgM, IgG, C1q and C3) was examined in vitro by incubation at 37°C for 1 hour followed by SDS-PAGE for gel shift assays.
All 25 strains of P. aeruginosa were positive for the presence of the PASP gene by PCR. P. stutzeri and P. otitidis were positive for the PASP gene whereas P. putida, P. alcaligenes were negative. Western blot analysis showed the presence of the PASP protein in the concentrated culture supernatants of all P. aeruginosa strains, but not in the supernatants from any non-aeruginosa species. Zymography demonstrated PASP activity in supernatants of P. aeruginosa strains PAO-1, PA103, 23, 70, 13014, 30099, 30132 and 30244. None of the supernatants from non-aeruginosa species exhibited PASP or any other proteolytic activity. PASP was shown in vitro to degrade fibrinogen, C1q, and C3, but not IgA, IgM, nor IgG.
The PASP gene, its protein product, and enzymatic activity were present in P. aeruginosa strains; however, only the PASP gene was detected in P. stutzeri and P. otiditis. PASP was shown to degrade several proteins including two complement components active in the host response to bacterial infection.
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