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

摘要

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 and 40 degrees C. It showed stability at pH 6-9 for 1 h and up to 60 degrees C for 30 min with midpoint temperature (T-m) at 61.3 degrees C and isoelectric value (pI) at 5.6 +/- 0.2. Its K-m and catalytic efficiency (K-cat/K-m) for the substrate azocasein were 1.3 mg/mL and 4.62x10(7) M(-1)s(-1), respectively. On contrary to most P. aeruginosa proteases, Zn2+, EDTA, 2,2-bipyridine and o-phenanthroline showed slight inhibition upon its activity, while, the elastase inhibitors (elastatinal and elastase inhibitor II) and the serine protease inhibitors (TLCK, PMSF, SBTI, and aprotinin) markedly decreased the enzymatic activity. Taken together, we suggest that ZuhP13 is a serine elastase-type. Interestingly, the tested enzyme showed both hemolytic and hemorrhagic activities in vivo. Furthermore, it induced nuclear lysis yielding hyperchromatism within leaky and malformed hepatocytes, suggesting ZuhP13 elastase as a high molecular weight potential pathological agent.