The Importance of Vibrio vulnificus rtxE for Virulence and Its Expression Induced by Exposure to Host Cell

School of Life Sciences and Biotechnology, Korea University, Seoul 136-701; Department of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University, Seoul 151-742; Department of Life Science and Interdisciplinary Program of Integrated Biotchnology, Sogang University, Seoul 121-742, South Korea

^* To whom correspondence should be addressed. Email: choish{at}snu.ac.kr.

	Abstract

Numerous secreted virulence factors have been proposed to account for the fulminating and destructive nature of Vibrio vulnificus infections. A mutant of V. vulnificus that exhibited less cytotoxicity toward INT-407 human intestinal epithelial cells was screened from a library of mutants constructed by random transposon mutagenesis. Using a transposon-tagging method, an open reading frame encoding an RTX toxin secretion ATP binding protein, RtxE, was identified and cloned from V. vulnificus. The deduced amino acid sequence of RtxE from V. vulnificus was 91% identical to that reported from V. cholerae. Functions of the rtxE gene in virulence were assessed by the construction of an isogenic mutant whose rtxE gene was inactivated by allelic exchanges, and by evaluating its phenotype changes in virulence in vitro and in mice. The disruption of rtxE blocked secretion of RtxA to the cell exterior and resulted in a significant reduction in cytotoxic activity toward epithelial cells in vitro. Also, the intraperitoneal LD₅₀ of the rtxE mutant was 10⁴-10⁵ times higher than that of the parental wild type, indicating that the RtxE is essential for the virulence of V. vulnificus. Furthermore, the present study demonstrated that the rtxBDE genes are transcribed as one transcriptional unit under the control of a single promoter P_rtxBDE. The activity of V. vulnificus P_rtxBDE is induced by exposure to INT-407 cells, and the induction requires direct contact of the bacteria to the host cells.