Isolation of a putative virulence agent, cytotoxic serine-elastase,from a newly isolated Pseudomonas aeruginosa ZuhP13

ABSTRACT

A 48 kDa ZuhP13 elastase from P. aeruginosa isolated from a urine sample was successfully purified to 8.8-fold and 39%recovery by DEAE-Sepharose CL-6B and Sephadex G-100 chromatography. Its ideal reaction values were pH 7.5 and40C. It showed stability at pH 6–9 for 1 h and up to 60C for 30 min with midpoint temperature (Tm) at 61.3Cand isoelectric value (pI) at 5.6±0.2. Its Km and catalytic efficiency (Kcat/Km) for the substrate azocasein were 1.3 mg/mLand 4.629107 M-1s-1, respectively. On contrary to most P. aeruginosa proteases, Zn2?, EDTA, 2,20-bipyridine ando-phenanthroline showed slight inhibition upon its activity, while, the elastase inhibitors (elastatinal and elastase inhibitorII) and the serine protease inhibitors (TLCK, PMSF, SBTI, and aprotinin) markedly decreased the enzymatic activity. Takentogether, we suggest that ZuhP13 is a serine elastase-type. Interestingly, the tested enzyme showed both hemolytic andhemorrhagic activities in vivo. Furthermore, it induced nuclear lysis yielding hyperchromatism within leaky and malformedhepatocytes, suggesting ZuhP13 elastase as a high molecular weight potential pathological agent.