Construction and Phenotypic Evaluation of aVibrio vulnificus vvpEMutant for Elastolytic Protease

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Abstract
Vibrio vulnificusis 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 ofV. vulnificusinfections. Among the putative virulence factors is an elastolytic metalloprotease. We cloned and sequenced thevvpEgene encoding an elastase ofV. vulnificusATCC 29307. The functions of the elastase were assessed by constructingvvpEinsertional knockout mutants and evaluating phenotypic changes in vitro and in mice. Although other types of protease activity were still observed invvpEmutants, elastase activity was completely absent in the mutants and was restored by reintroducing the recombinantvvpEgene. In contrast to previous characterization of elastase as a potential virulence factor, which was demonstrated by injecting the purified protein into animals, inactivation of theV. vulnificus vvpEgene 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, avvpEmutant 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 ofV. vulnificusthan would have been predicted by examining the effects of administering purified proteins to animals. However,V. vulnificusutilizes a variety of virulence factors; hence, the effects of inactivation of elastase alone could be masked by other compensatory virulence factors.