Construction and Phenotypic Evaluation of a Vibrio vulnificus vvpE Mutant for Elastolytic Protease

Abstract

Vibrio vulnificus is an opportunistic gram-negative pathogen that commonly contaminates oysters. Predisposed individuals who consume raw oysters can die within days from sepsis, and even otherwise healthy people are susceptible to serious wound infection after contact with contaminated seafood or seawater. Numerous secreted and cell-associated virulence factors have been proposed to account for the fulminating and destructive nature of V. vulnificus infections. Among the putative virulence factors is an elastolytic metalloprotease. We cloned and sequenced the vvpE gene encoding an elastase of V. vulnificus ATCC 29307. The functions of the elastase were assessed by constructing vvpE insertional knockout mutants and evaluating phenotypic changes in vitro and in mice. Although other types of protease activity were still observed in vvpE mutants, elastase activity was completely absent in the mutants and was restored by reintroducing the recombinant vvpE gene. In contrast to previous characterization of elastase as a potential virulence factor, which was demonstrated by injecting the purified protein into animals, inactivation of the V. vulnificus vvpE gene did not affect the ability of the bacteria to infect mice and cause damage, either locally in subcutaneous tissues or systemically in the liver, in both iron-treated and normal mice. Furthermore, a vvpE mutant was not affected with regard to cytolytic activity toward INT407 epithelial cells or detachment of INT407 cells from culture dishes in vitro. Therefore, it appears that elastase is less important in the pathogenesis of V. vulnificus than would have been predicted by examining the effects of administering purified proteins to animals. However, V. vulnificus utilizes a variety of virulence factors; hence, the effects of inactivation of elastase alone could be masked by other compensatory virulence factors.