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

Department of Food Science and Technology, Institute of Biotechnology, Chonnam National University, Kwang-Ju, 500-757, South Korea.
Infection and Immunity (Impact Factor: 3.73). 10/2000; 68(9). DOI: 10.1128/IAI.68.9.5096-5106.2000
Source: PubMed Central


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.

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    • "However, the vvhA mutant did not show a decrease in virulence by intraperitoneal infection using either an iron-normal or iron-overloaded mouse model (Wright and Morris 1991; Fan et al. 2001). In addition, the inactivation of the vvpE gene did not affect the ability of bacteria to infect mice and cause organ damage which indicates that HlyU must regulate the expression of other virulence-related gene(s) (Jeong et al. 2000; Shao and Hor 2000; Fan et al. 2001). We compared the transcriptome profiles of the hlyU mutant and the wild type V. vulnificus strain. "
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    ABSTRACT: HlyU is a master regulator that plays an essential role in the virulence of the human pathogen Vibrio vulnificus. One of the most noteworthy characteristics of HlyU regulation in this organism is its positive control of the expression of the repeat-in-toxin (RtxA1) gene, one of the most important virulence factors accounting for the fulminating and damaging nature of V. vulnificus infections. In this work, we reviewed the latest studies of RtxA1 in this bacterium and highlight the mechanism of gene regulation of rtxA1 expression by HlyU under a broader gene regulatory network.
    MicrobiologyOpen 12/2012; 1(4):502-13. DOI:10.1002/mbo3.48 · 2.21 Impact Factor
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    • "Cytotoxicity to INT-407 cell monolayers. RtxA1 activity was assessed by detachment of INT-407 cell monolayers, as previously described (Jeong et al., 2000). Briefly, INT-407 cells were seeded in a 24-well tissue culture plate for 2 days until monolayers reached 80– 90 % confluency. "
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    ABSTRACT: The GacS/GacA two-component signal transduction system regulates virulence, biofilm formation and symbiosis in Vibrio species. The present study investigated this regulatory pathway in Vibrio vulnificus, a human pathogen that causes life-threatening disease associated with the consumption of raw oysters and wound infections. Small non-coding RNAs (csrB1, csrB2, csrB3 and csrC) commonly regulated by the GacS/GacA pathway were decreased (P<0.0003) in a V. vulnificus CMCP6 ΔgacA : : aph mutant compared with the wild-type parent, and expression was restored by complementation of the gacA deletion mutation in trans. Of the 20 genes examined by RT-PCR, significant reductions in the transcript levels of the mutant in comparison with the wild-type strain were observed only for genes related to motility (flaA), stationary phase (rpoS) and protease (vvpE) (P=0.04, 0.01 and 0.002, respectively). Swimming motility, flagellation and opaque colony morphology indicative of capsular polysaccharide (CPS) were unchanged in the mutant, while cytotoxicity, protease activity, CPS phase variation and the ability to acquire iron were decreased compared with the wild-type (P<0.01). The role of gacA in virulence of V. vulnificus was also demonstrated by significant impairment in the ability of the mutant strain to cause either skin (P<0.0005) or systemic infections (P<0.02) in subcutaneously inoculated, non-iron-treated mice. However, the virulence of the mutant was equivalent to that of the wild-type in iron-treated mice, demonstrating that the GacA pathway in V. vulnificus regulates the virulence of this organism in an iron-dependent manner.
    Microbiology 12/2010; 156(Pt 12):3722-33. DOI:10.1099/mic.0.043422-0 · 2.56 Impact Factor
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    • "To generate the ΔrtxA::nptI mutant, MW064, by homologous recombination, E. coli SM10 λpir, tra (containing pMW0613) (Miller and Mekalanos, 1988) was used as a conjugal donor to V. vulnificus M06-24/O. The conjugation and isolation of the transconjugants were achieved using the methods, in previous studies (Jeong et al., 2000; Rhee et al., 2004; Lee et al., 2006; Lee and Choi, 2006) and a double crossover, in which wild-type rtxA gene being replaced with the ΔrtxA::nptI allele, was confirmed by PCR as shown in Fig. 2B. PCR analysis of genomic DNA from the wild type with primers RTXA-CF1 and RTXA-CF2 (Table 2) produced a 924 bp fragment (Fig. 2B). "
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    ABSTRACT: A mutant exhibiting decreased cytotoxic activity toward INT-407 intestinal epithelial cells and carrying a mutation in the rtx gene cluster that consists of rtxCA and rtxBDE operons was screened from a library of V. vulnificus mutants. The functions of the rtxA gene, assessed by constructing an isogenic mutant and evaluating its phenotypic changes, demonstrated that RtxA is essential for the virulence of V. vulnificus in mice as well as in tissue cultures.
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