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M.E. Marquart, A.R. Caballero, R.J. O'Callaghan; Pseudomonas Aeruginosa Small Protease (PASP) Causes Corneal Epithelial Erosions . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5076.
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Purpose: One or more known Pseudomonas proteases are involved in corneal damage. The purpose of this study was to further analyze the properties of the newly discovered Pseudomonas aeruginosa small protease (PASP), including its ability to be expressed as an enzymatic recombinant molecule from a cloned gene in Escherichia coli and the ability of the native enzyme to cause corneal damage. Methods: PASP was purified by ion–exchange chromatography from stationary phase culture supernatant of P. aeruginosa strain PA103. The N–terminal sequence of PASP was used to locate the corresponding P. aeruginosa PAO1 sequence, and the gene encoding PASP (strain PA103) was cloned and expressed in E. coli, then purified from inclusion bodies using guanidine as a denaturant. Recombinant PASP was refolded by gradual removal of the guanidine, and protease activity was verified by gelatin zymography. The gene of PA103 pasp was sequenced and compared to the PAO1 sequence for pasp. The left corneas of New Zealand white rabbits (n = 3) were injected with 7 µg of purified PASP, and the right corneas (n = 3) were injected with 7 µg of heat–inactivated PASP. Corneas were monitored periodically for 48 hours. Erosions were stained with fluoroscein and measured. Microplate assays of PASP activity using fluorescence–conjugated bovine collagen types I and IV as substrates were performed. Results: Plasmid–encoded PASP, expressed in E. coli, produced a recombinant enzyme in denatured form. The enzymatic activity of this recombinant protease was restored with gradual removal of guanidine from the protease suspension. The PA103 pasp gene was determined to be 99.5% identical to the PAO1 pasp gene (PA0423). Intrastromal injection of active PASP produced corneal epithelial erosions in all eyes with diameters ranging from 1.0 to 2.5 mm at 5 hours and 0.5 to 6.0 mm by 10 hours post–injection (n = 3). All of the erosions healed by 48 hours. In contrast, intrastromal injection of heat–inactivated PASP failed to produce any epithelial erosions or defects (n = 3). Because PASP damaged corneas in vivo, the ability of this enzyme to digest collagen, the primary component of the corneal stroma, was tested in vitro. PASP cleaved both types of collagen tested, especially collagen type I. Conclusions: Production of recombinant PASP will increase yields of this enzyme for further characterization. PASP damages the corneal epithelium in vivo and degrades collagen in vitro, and therefore could be a virulence factor involved in Pseudomonas keratitis.
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