Identification and characterization of Vibrio vulnificus plpA encoding a phospholipase A(2) essential for pathogenesis

Abstract

The marine bacterium Vibrio vulnificus causes food-borne diseases, which may lead to life-threatening septicemia in some individuals. Therefore, identifying virulence factors in V. vulnificus is of high priority. We performed a transcriptome analysis on V. vulnificus after infection of human intestinal HT29-methotrexate cells and found induction of plpA, encoding a putative phospholipase, VvPlpA. Bioinformatics, biochemical, and genetic analyses demonstrated that VvPlpA is a phospholipase A(2) secreted in a type II secretion system-dependent manner. Compared with the wild type, the plpA mutant exhibited reduced mortality, systemic infection, and inflammation in mice as well as low cytotoxicity toward the human epithelial INT-407 cells. Moreover, plpA mutation attenuated the release of actin and cytosolic cyclophilin A from INT-407 cells, indicating that VvPlpA is a virulence factor essential for causing lysis and necrotic death of the epithelial cells. plpA transcription was growth phase-dependent, reaching maximum levels during the early stationary phase. Also, transcription factor HlyU and cAMP receptor protein (CRP) mediate additive activation and host-dependent induction of plpA. Molecular biological analyses revealed that plpA expression is controlled via the promoter, P-plpA, and that HlyU and CRP directly bind to P-plpA upstream sequences. Taken together, this study demonstrated that VvPlpA is a type II secretion system-dependent secretory phospholipase A(2) regulated by HlyU and CRP and is essential for the pathogenicity of V. vulnificus.