Characterization of a novel chromosomal virulence locus involved in expression of a major surface flagellar sheath antigen of the fish pathogen Vibrio anguillarum

Abstract

The fish pathogenic bacterium Vibrio anguilarum 775.17B was mutated by the use of transposon Tn5-132. Two hundred independent exconjugants were isolated and screened for a reduction ofvirulence in experimental infections of rainbow trout (Onchorhynchus mykiss). Two of these exconjugants, VAN20 and VAN70, showed a significant reduction in virulence after both intraperitoneal and immersion infections. The avirulent mutants showed no loss of any previously suggested virulence determinants of V. anguillarum. One of the mutants (VAN70) was further characterized. DNA sequence analysis revealed two open reading frames, the gene into which TnS-132 had been inserted (virA) and a closely linked upstream gene (virB). A virB mutant of 775.17B, NQ706, was isolated and also shown to be avirulent. The deduced amino acid sequences of virA and virB correspond to proteins with molecular weights of 36,000 and 42,000, respectively. Insertional mutagenesis of the corresponding virA and virB genes of a clinical isolate of V. anguiUarum, serotype 01, also resulted in avirulence. In immunoblot experiments, the total cell lysates ofVAN70 (virA) and NQ706 (virB) did not respond to a rabbit polyclonal antiserum directed against whole cells of 775.17B (wild type). This suggests that virA and virB are involved in the biosynthesis of a major surface antigen important for the virulence of V. anguillarum. Immunogold electron microscopy showed that a constituent of the flagellar sheath was expressed by 775.17B

Vibrio anguillarum colonization of rainbow trout integument requires a DNA locus involved in exopolysaccharide transport and biosynthesis

“…2B). For the wild type and the EPS mutants, a clearing around the bacterial cell that also continued around the polar flagellum similar to a sheath seen in V. anguillarum and other Vibrio strains was seen (Allen and Baumann, 1971;Norqvist and Wolf-Watz, 1993). No obvious structural changes were seen with the EPS mutant strains or with strains overexpressing the putative orf1-wbfDCB operon suggesting that these EPS biosynthesis genes do not affect this capsular structure.…”

Real-time PCR detection and quantification of fish probioticPhaeobacterstrain 27-4 and fish pathogenicVibrioin microalgae, rotifer,Artemiaand first feeding turbot (Psetta maxima) larvae

“…In V. anguillarum several genes are responsible of haemolytic activity (Rock and Nelson 2006), and we therefore tried to design primers in other genes to improve primers specificity. Specific primers for V. anguillarum 90-11-287 were successfully designed within the gene virA ⁄ virB, which encodes for a major surface antigen that is essential for the virulence of serotype O1 (Norqvist and Wolf-Watz 1993).…”

A comprehensive review of Vibrio (Listonella) anguillarum: ecology, pathology and prevention

“…The bacterium's only flagellum is comprised of five flagellin proteins which are encoded by genes flaABCDE and the proteins FlaA, FlaD and FlaE serve as direct contributors to V. anguillarum virulence (McGee et al 1996;Milton et al 1996). The flagella contains lipopolysaccharide surface antigens in the outermost flagellar sheath which are encoded by genes virA and virB, resistant to proteinase K, sensitive to periodate anion and serve as essential requirements for virulence in pathogenic V. anguillarum strains (Norqvist & Wolf-Watz 1993). Vibrio anguillarum maintains ordinal migration preference towards intestinal, fish skin and gill mucus, respectively, by means of cheR encoded chemotactic motility due to the presence of amino acids, bile acids and carbohydrates therein which bait the pathogen towards intestinal colonization following host adhesion (O'Toole et al 1999) and the process is essential for virulence among a host organism, although avirulent strains also exhibit chemotaxis in motility .…”