Screening of genes expressed in vivo after infection by Vibrio anguillarum M3

Abstract

Aims: Genes uniquely expressed in vivo may contribute to the overall pathogenicity of an organism and are likely to serve as potential targets for the development of new vaccine. This study aims to screen the genes expressed in vivo after Vibrio anguillarum infection by in vivo‐induced antigen technology (IVIAT). Methods and Results: The convalescent‐phase sera were obtained from turbot (Scophthalmus maximus) survived after infection by the virulent V. anguillarum M3. The pooled sera were thoroughly adsorbed with M3 cells and Escherichia coli BL21 (DE3) cells. A genomic expression library of M3 was constructed and screened for the identification of immunogenic proteins by colony immunoblot analysis with the adsorbed sera. After three rounds of screening, 19 putative in vivo‐induced (ivi) genes were obtained. These ivi genes were catalogued into four functional groups: regulator/signalling, metabolism, biological process and hypothetical proteins. Three ivi genes were insertion‐mutated, and the growth and 50% lethal dose (LD50) of these mutants were evaluated. Conclusions: The identification of ivi genes in V. anguillarum M3 sheds light on understanding the bacterial pathogenesis and provides novel targets for the development of new vaccines and diagnostic reagents. Significance and Impact of the Study: To the best of our knowledge, this is the first report describing in vivo‐expressed genes of V. anguillarum using IVIAT. The screened ivi genes in this study could be new virulent factors and targets for the development of vaccine, which may have implications for the development of diagnostic regents.

Vibrio anguillarum as a fish pathogen: virulence factors, diagnosis and prevention

“…Recently, random genome sequencing, suppression subtractive hybridization (SSH) and in vivo ‐induced antigen technology (IVIAT) have been used to identify additional virulence‐related genes of V. anguillarum (Rodkhum et al. 2006b; Wang, Xu, Jia, Chen, Mo & Zhang 2009; Zou, Mo, Hao, Ye, Guo & Zhang 2010). Although these techniques have produced interesting results, the complete mechanism of pathogenesis is still to be unravelled.…”

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

“…The pathogen also contains a 1755-bp chitinase gene vac which is believed to contribute to virulence in crustaceans, but the notion remains unverified (Hirono et al 1998). Genes in the ivi region are believed to play a role in virulence mechanisms, phospholipase signalling, genetic regulation, metabolism and outermembrane assembly, but such functions are left only to speculation at the present time and more research in this area is likely to emerge in future studies (Zou et al 2010). Furthermore, the gene recA is expressed to repair damaged DNA and the gene is required for recombination but not survival (Singer 1989;Singer et al 1996).…”

Identification and real-time expression analysis of selected Toxoplasma gondii in-vivoinduced antigens recognized by IgG and IgM in sera of acute toxoplasmosis patients

“…IVIAT has been applied in the study of several pathogenic microorganisms based on the assumption that conventional genetic and biochemical in-vitro approaches cannot mimic the real complex and dynamic in-vivo situation of human infection [19-28]. In our recent published report, T.gondii genes preferentially expressed in-vivo were identified via immunoscreening of T.gondii expression library with sera from chronically-infected individuals followed by real time PCR to confirm the up-regulation of the identified genes.…”