R-041. Excision and Integration Dynamics of Vibrio Pathogenicity Island-2 (VPI-2) from Vibrio cholerae

S. Almagro-Moreno, E. F. Boyd;
Univ. of Delaware, Newark, DE.

Cholera is a deadly diarrheal disease caused by Vibrio cholerae and is endemic in much of Asia, Africa and South America. We identified a pathogenicity island, which we named Vibrio Pathogenicity Island-2 (VPI-2), that is present among O1 serogroup isolates, and is also present in O139 serogroup isolates recovered in 1992, but is deleted from all O139 isolates recovered since then. VPI-2 encodes a neuraminidase, which removes sialic acid residues from the sialogangliosides converting them into GM1, the receptors of cholera toxin. VPI-2 also encodes, among others, genes putatively involved in sialic acid metabolism as well as an integrase. In order to understand how pathogenic and new variant isolates emerge, we must first determine the mechanisms of acquisition and incorporation into the host genome as well as how the region excises. VPI-2 can excise from V. cholerae genome forming a circular intermediate (CI) after precise excision from its tRNA-serine insertion site, probably a first step in its horizontal transfer. The conditions that induce excision of pathogenicity islands in V. cholerae are not known nor the factors involved. In this study, we quantify by the use of QPCR the rate of excision of VPI-2 under a range of different conditions such as growth stages, nutrient limitation, temperature, NaCl, UV and mitomycin C stress, finding different rates of excision of VPI-2, particularly under UV light treatment. Integrase is directly involved in the excision of VPI-2, we found that the expression of the int gene (VC1758) is increased after UV light treatment. Integrase function in recombination is highly directional and the excision function of an integrase is increased by the presence of a Recombination Directional Factor (RDF). We identified a novel RDF (VC1809) encoded on VPI-2, named vrdF, whose expression was also found to be increased after UV light treatment, then we constructed a deletion mutation to determine the effects on excision. Host chromosome-encoded factors are often required for efficient site-specific recombination such as the integration host factor encoded by himA and himD and the Fis protein. We examined VPI-2 stability in strain SM463, a ΔhimA mutant and strain SM465, a Δfis mutant.