Biochemical characterization of extracellular proteases from Vibrio cholerae

Abstract

Isoelectric focusing of culture supernatants from Vibrio cholerae El Tor 1621 and high protease-producing mutant strain 1621 hip revealed the presence of three different types of extracellular protease. Type I protease was the major activity in the wild-type strain and was inhibited by phenylmethylsulfonyl fluoride and by the lima bean trypsin inhibitor. Type II protease was present in the wild type and was the major activity in the high protease-producing mutant. It was resistant to inhibitors of metalloproteases and serine proteases. Two peaks of type II protease differed by 1.2 pI units in isoelectric point and by 1,500 in molecular weight. Type II protease had broad specificity, acted as a mucinase, and caused degradation of some other V. cholerae extracellular proteins, including DNase and cholera toxin. Type III protease was EDTA inhibitable and was detected only in the high protease producer. Possible roles of extracellular proteases as virulence factors in cholera pathogenesis are discussed.

Purification and characterization of an extracellular protease of Legionella pneumophila

“…proteins demonstrated proteolytic activity and were antigenically indistinguishable. Possession of multiple enzyme forms, as observed here, is a common feature among zinccontaining proteases from other bacterial species (9,11,27) and other enzymes in general (19). The molecular microheterogeneity observed following electrophoresis could be a consequence of single amino acid residue changes (5), posttranslational covalent modifications, autodigestion (9), or modification occurring as a result of purification (19).…”

Role of rpoS in Stress Survival and Virulence of Vibrio cholerae

“…During the course of these experiments, we observed that SDS-PAGE analysis of V. cholerae outer membrane proteins revealed marked degradation of many proteins in lysates of the wildtype strain but not in lysates prepared with the rpoS::cm mutant. We reasoned that the rpoS mutation may have affected either the secretion or production of one or more of the previously described V. cholerae O1 proteases (55). This possibility was examined by measuring the protease activity secreted into culture supernatants by the wild-type strain, the rpoS::cm mutant, and the mutant with complementing plasmids.…”

The effect on enterotoxicity of protease purified from Vibrio cholerae O1

“…Protease-deficient mutants are known to be less virulent [1]. Young and Broadbent reported the purification of three types of protease from V. cholerae O1: phenylmethylsulfonyl fluoride inhibitable type 1, metalloprotease and serine protease inhibitor resistant type II, and EDTA inhibitable type III [2]. Booth et al [3,4] reported that soluble HA/protease is a metalloenzyme that can be inhibited by a variety of chelating agents and inhibitors of zinc * Corresponding author.…”