Characterization of Vibrio cholerae protease activities with peptide digest analysis

Abstract

A simple method for the analysis of microbial proteases is described that was used to characterize the proteolytic activities of various Vibrio cholerae isolates. This method utilized the unique peptides generated from the degradation of a standard protein by proteases of various specificities. These peptides were anaâ lyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The unique patterns of peptides seen in gels can be used to type proteases according to their relative specificities. Culture supernatants of V. cholerae isolates from a variety of environmental and human sources were analyzed for the presence of a protease previously isolated and characterized in this laboratory from V. cholerae strain CA401. Supernatants from most isolates showing dimethyl casein proteolytic activity exhibited the presence of enzymes similar to the CA401 protease in their peptide digest patterns against bovine serum albumin and in their immunological reactivities. The probable widespread presence of this virulence-associated protease in V. cholerae isolates is discussed.

Biochemical characterization of extracellular proteases from Vibrio cholerae

“…Our results are in agreement with previous reports on the wide variation in extracellular protease production among different strains of V. cholerae (11,24). Among V. cholerae El Tor isolates, we found a 100-fold variation in protease production, and the two classical strains we tested differed by a factor of 80.…”

“…Serological analysis will be of use in the further investigation of this phenomenon. Schneider et al (24) have also reported the occurrence of two different molecular weight forms of type II protease activity. Serological and biochemical evidence presented by these authors (24) indicates that type II protease is widespread among V. cholerae strains, and our own results have shown that it is the dominant activity in all the high and medium protease producers so far investigated.…”

“…Serological studies by Hsieh and Liu (10) indicated that a single type of protease was shared by several strains of V. cholerae 01 and by a number of non-agglutinable vibrios. Schneider et al (24) have also produced evidence that a single type of protease, in terms of both antigenicity and catalytic specificity, is shared by a wide range of V. cholerae isolates. Earlier studies by Sandvik and Dahle, however, indicated the presence of electrophoretically and antigenically distinct proteases in V. cholerae strains (3,22).…”

Cloning and genetic analysis of the Vibrio cholerae aminopeptidase gene

“…Only one V. cholerae extracellular protease gene, the soluble hemagglutinin/protease (hap) (12), has been cloned and sequenced to date. However, the biochemical characterization of extracellular proteases from V. cholerae suggests the existence of more than one type of protease (24,32). Furthermore, hap-deficient mutants have shown residual proteolytic activity that was not inhibited by anti-hemagglutinin/protease (anti-HA/P)-specific antiserum (12).…”