Membrane-bound proteases of ompT+ and ompT- Vibrio cholerae strains]

Detection ofproteases in outer membranes (OM) of ompT+ and ompT- Vibrio cholerae strains of O1 and O139 serogroups.Specific sterile preparations of OM were obtained by lysis of live V. cholerae cells by 4.5 M urea solution with subsequent differential centrifugation and treatment by nucleases. Extraction of OM proteins previously treated by sodium sarcosinate was carried out by Triton X-100 in the presence of EDTA. Protease and polypeptide spectra were studied in substrate and SDS electrophoresis. Sensitivity of proteases to inhibitors was determined in diffusion test in agarose gel containing substrate by using soy trypsin inhibitor (STI) and phenylmethylsulfonyl fluoride (PMSF). The presence of ompT was determined in PCR by using specific primers.According to PCR data 13 Vibrio cholerae O1 strains and 3 V. cholerae O139 strains isolated from clinical material as well as 22 V. cholerae O1 strains isolated from environmental objects contained ompT gene. 2 V. cholerae O1 human isolated strains, 9 V. cholerae O1 strains and 2 V. cholerae O139 strains isolated from the environment did not have ompT gene. By using SDS- and enzyme-electrophoresis in polyacrylamide gel quantitative and qualitative differences in composition of polypeptides and proteases of OM ompT+ and ompT- V. cholerae strains that hydrolyze gelatin, casein and protamine sulfate were detected. Inhibition of OM by STI and PMSF resulted in a decrease of their proteolytic activity.In preparations and extracts of ompT+ and ompT- V. cholerae OM up to 3 proteases some of which may be related to ompT-like were detected.