Regulatory Characteristics of Vibrio vulnificus gbpA Gene Encoding a Mucin-binding Protein Essential for Pathogenesis

Abstract

Binding to mucin is the initial step for enteropathogens to establish pathogenesis. An open reading frame, gbpA, of Vibrio vulnificus was identified and characterized in this study. Compared with wild type, the gbpA mutant was impaired in binding to mucin-agar and the mucin-secreting HT29-methotrexate cells, and the impaired mucin binding was restored by the purified GbpA provided exogenously. The gbpA mutant had attenuated virulence and ability of intestinal colonization in a mouse model, indicating that GbpA is a mucin-binding protein and essential for pathogenesis of V. vulnificus. The gbpA transcription was growth phase-dependent, reaching a maximum during the exponential phase. The Fe-S cluster regulator (IscR) and the cyclic AMP receptor protein (CRP) coactivated, whereas SmcR, a LuxR homologue, repressed gbpA. The cellular levels of IscR, CRP, and SmcR were not significantly affected by one another, indicating that the regulator proteins function cooperatively to regulate gbpA rather than sequentially in a regulatory cascade. The regulatory proteins directly bind upstream of the gbpA promoter P gbpA . DNase I protection assays, together with the deletion analyses of P gbpA , demonstrated that IscR binds to two specific sequences centered at ؊164.5 and ؊106, and CRP and SmcR bind specifically to the sequences centered at ؊68 and ؊45, respectively. Furthermore, gbpA was induced by exposure to H 2 O 2 , and the induction appeared to be mediated by elevated intracellular levels of IscR. Consequently, the combined results indicated that IscR, CRP, and SmcR cooperate for precise regulation of gbpA during the V. vulnificus pathogenesis.

Vibrio vulnificus: From Oyster Colonist to Human Pathogen

“…vulnificus strains integrate into marine aggregates (chitin) more efficiently than C-type strains. This study also found that expression of gbpA was significantly higher in C-type strains [20]. While more research is needed, it is clear that environmental context could be an important link to understanding V .…”

“…In fact, V . vulnificus GbpA is also a mucin-binding protein and is essential for pathogenesis in a mouse model [20]. Interestingly, a recent study found E-type V .…”

Identification and characterization of Vibrio vulnificus plpA encoding a phospholipase A2 essential for pathogenesis

“…Furthermore, no genes encoding the PLAs have been identified from V. vulnificus, and thus the molecular mechanisms by which the bacterium modulates the expression of the genes have not yet been characterized. In the present study, we conducted a transcriptome analysis and identified the plpA gene, which is preferentially expressed in V. vulnificus cells exposed to human intestinal HT29-methotrexate (MTX) cells (10,11). The characteristics of V. vulnificus PlpA (VvPlpA), the product of plpA, were verified experimentally.…”

“…Although a few examples of transcriptional coactivation of bacterial virulence genes by multiple activators have been reported (11,48), coactivation by HlyU and CRP has not yet been reported. Each HlyU and CRP binds to the upstream sequence of P plpA and activates the promoter as a solitary regulator, albeit not to levels comparable with that of the wild type (Figs.…”

“…For this purpose, the V. vulnificus strains grown to an A 600 of 0.5 were harvested and suspended in PBS to 10 8 or 10 6 cfu/100 l for intrasgastric or intraperitoneal infection, respectively. Mouse lethality of the V. vulnificus strains was compared as described previously (n ϭ 15 per group) (11). The mice were intraperitoneally injected with 30 g of iron-dextran for each gram of body weight, immediately after which they were administered with 100 l of the inoculum intragastrically.…”