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Richard J. O'Callaghan, Aihua Tang, Mary Marquart, Armando Caballero; Pseudomonas aeruginosa Small Protease (PASP), a Keratitis Virulence Factor. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6128.
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Secreted bacterial proteases have been correlated with the destruction of the cornea during Pseudomonas keratitis and one protease, protease IV, has been shown to be a key virulence factor. This study was performed to determine if PASP is also a corneal virulence factor.
The whole Pasp gene of P. aeruginosa strain PA103-29 was replaced with the tetracycline resistance gene (Tet) via allelic exchange, which was confirmed by sequencing. A plasmid expressing the Pasp gene (pUCP20-Pasp) was introduced into the PASP-deficient mutant to generate a rescue strain. The presence of the PASP protein and its protease activity in the culture supernatants of the parent, PASP-deficient mutant, and rescue strain was determined by Western blot analysis and gelatin zymography, respectively. Corneal virulence was evaluated in both a rabbit intrastromal model and a mouse topical model of keratitis by slit lamp examination (SLE), bacteria enumeration, and/or histopathological analysis.
In the rabbit keratitis model, the PASP-deficient mutant produced a significantly lower SLE score when compared to the parent or the rescue strain (P ≤ 0.05) at 29 hours postinfection (PI). All the strains grew equally in the rabbit cornea (P = 0.971). Corneas infected with the PASP-deficient mutant showed moderate histopathology compared to the severe pathology including epithelial erosions, corneal edema and PMN infiltration present in the corneas infected with the parent or rescue strain. In the mouse model, eyes inoculated with the PASP-deficient mutant had a significantly lower SLE score than the eyes inoculated with the parent or rescue strain at 24 hours PI (P ≤ 0.05). The bacterial growth in the mouse corneas was equivalent (P = 0.990).
PASP is a secreted protease of all strains tested and it is able to contribute significantly to the tissue damage during experimental Pseudomonas keratitis.
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