B-158. A Phospholipase/Hemolysin of Vibrio vulnificus has no Role in Virulence in a Mouse Model

J. L. Joseph, P. A. Gulig;
Univ. of Florida Coll. of Med., Gainesville, FL.

Vibrio vulnificus causes sepsis and skin infection due to consumption of shellfish and contamination of wounds, respectively. V. vulnificus secretes a wide array of toxins, hemolysins, phospholipases, and lipases. Phospholipase activity has been reported to be important in virulence of V. vulnificus; however, a phospholipase mutant was not examined. The gene VV2_1483 encodes a protein with homology to phospholipases, lecithinases, and hemolysins. This protein has >70% identity to the thermolabile hemolysins of Vibrio parahaemolyticus and Vibrio cholerae O1 El tor. To investigate the role of phospholipase in pathogenesis, we deleted VV2_1483 from V. vulnificus CMCP6. A deletion vector was constructed using USER cloning (New England Biolabs) to create a plasmid with the VV2_1483 upstream and downstream sequences flanking a kanamycin resistance gene. CMCP6 was transformed with sheared plasmid DNA using chitin-induced transformation, originally described for V. cholerae. Recipient V. vulnificus cells were grown in seawater with crab shell to induce natural competence allowing uptake of linear DNA. Transformants were selected for kanamycin resistance, and allelic exchange was verified by PCR. The deletion mutant was hemolytic on 5% sheep blood agar plates. Lecithinase activity, observed on TSA plates containing 5% egg yolk agar, was similar in the mutant and parent strains. CMCP6ΔVV2_1483 was tested for virulence in subcutaneously inoculated, iron dextran-treated mice. CMCP6ΔVV2_1483 was fully virulent at an inoculum of 1000 CFU, approximately 3 times the minimum lethal dose for wild type CMCP6. While the annotation suggests that the VV2_1483 gene product could contribute to hemolysis and cytotoxicity due to phospholipase activity, this gene is dispensable for virulence in a mouse model of infection.