Cloning, characterization and molecular analysis of a metalloprotease from Proteus mirabilis

Proteus mirabilis is an important pathogen that is usually found in complicated urinary tracts infection. It possesses a metalloprotease, ZapA, that acts as a virulence factor. The gene encoding ZapA was cloned from P. mirabilis Pm7-a strain isolated from marine environments-and conditionally expressed in Escherichia coli. Zn(2+) and Co(2+) exhibited an apparently positive effect on the enzyme activity of the 54-kDa protease. Ag(+), Cd(2+), Cu(2+), Hg(2+), Pb(2+), EDTA and sulfhydryl reagents including beta-mercaptoethanol and dithiothreitol exhibited an apparently negative effect on enzyme activity. Enzyme activity analysis revealed that the optimum temperature and pH for purified recombinant ZapA were approximately 40 degrees C and 8.0, respectively. Enzyme activity and western immuno-blotting analysis were used for the determination of the extracellular location of ZapA. The simultaneously depressed expression of zapA and swarming motility of Pm7 in the presence of glucose were determined by real-time PCR and swarming motility measurements, respectively. Furthermore, the outer membrane proteins of two bacteria (Enterobacter sp. T41 and Edwardsiella tarda strain TX1-a fish pathogen) were found to be substrates of ZapA proteolysis.Proteus mirabilis is an important pathogen that is usually found in complicated urinary tracts infection. It possesses a metalloprotease, ZapA, that acts as a virulence factor. The gene encoding ZapA was cloned from P. mirabilis Pm7-a strain isolated from marine environments-and conditionally expressed in Escherichia coli. Zn(2+) and Co(2+) exhibited an apparently positive effect on the enzyme activity of the 54-kDa protease. Ag(+), Cd(2+), Cu(2+), Hg(2+), Pb(2+), EDTA and sulfhydryl reagents including beta-mercaptoethanol and dithiothreitol exhibited an apparently negative effect on enzyme activity. Enzyme activity analysis revealed that the optimum temperature and pH for purified recombinant ZapA were approximately 40 degrees C and 8.0, respectively. Enzyme activity and western immuno-blotting analysis were used for the determination of the extracellular location of ZapA. The simultaneously depressed expression of zapA and swarming motility of Pm7 in the presence of glucose were determined by real-time PCR and swarming motility measurements, respectively. Furthermore, the outer membrane proteins of two bacteria (Enterobacter sp. T41 and Edwardsiella tarda strain TX1-a fish pathogen) were found to be substrates of ZapA proteolysis