Regulation by ToxR-Like Proteins Converges on vttR _B Expression To Control Type 3 Secretion System-Dependent Caco2-BBE Cytotoxicity in Vibrio cholerae

Abstract

Genes carried on the type 3 secretion system (T3SS) pathogenicity island of Vibrio cholerae non-O1/non-O139 serogroup strain AM-19226 must be precisely regulated in order for bacteria to cause disease. Previously reported results showed that both T3SS function and the presence of bile are required to cause Caco2-BBE cell cytotoxicity during coculture with strain AM-19226. We therefore investigated additional parameters affecting in vitro cell death, including bacterial load and the role of three transmembrane transcriptional regulatory proteins, VttR A , VttR B , and ToxR. VttR A and VttR B are encoded on the horizontally acquired T3SS genomic island, whereas ToxR is encoded on the ancestral chromosome. While strains carrying deletions in any one of the three transcriptional regulatory genes are unable to cause eukaryotic cell death, the results of complementation studies point to a hierarchy of regulatory control that converges on vttR B expression. The data suggest both that ToxR and VttR A act upstream of VttR B and that modifying the level of either vttR A or vttR B expression can strongly influence T3SS gene expression. We therefore propose a model whereby T3SS activity and, hence, in vitro cytotoxicity are ultimately regulated by vttR B expression. IMPORTANCEIn contrast to O1 and O139 serogroup V. cholerae strains that cause cholera using two main virulence factors (toxin-coregulated pilus [TCP] and cholera toxin [CT]), O39 serogroup strain AM-19226 uses a type 3 secretion system as its principal virulence mechanism. Although the regulatory network governing TCP and CT expression is well understood, the factors influencing T3SS-associated virulence are not. Using an in vitro mammalian cell model to investigate the role of three ToxR-like transmembrane transcriptional activators in causing T3SS-dependent cytotoxicity, we found that expression levels and a hierarchical organization were important for promoting T3SS gene expression. Furthermore, our results suggest that horizontally acquired, ToxR-like proteins act in concert with the ancestral ToxR protein to orchestrate T3SS-mediated pathogenicity. P athogenic bacteria must effectively control the expression of virulence factors in order to achieve productive infection. As a waterborne pathogen, Vibrio cholerae senses and responds to signals from two distinct environments, the aquatic reservoir and the human host, requiring appropriate temporospatial changes in gene expression. Epidemic-causing O1 and O139 serogroup strains use the ToxR-ToxT regulatory hierarchy to modulate the expression of genes encoding factors responsible for colonization (toxin-coregulated pilus [TCP]) and diarrhea (cholera toxin [CT]) (1, 2). Whereas toxR is an ancestral gene found in all V. cholerae strains, toxT is carried on horizontally acquired Vibrio pathogenicity island 1 (VPI-1), which encodes the TCP. ToxR can directly activate transcription from the toxT promoter (3, 4). VPI-1 also encodes the TcpPH proteins, which can increase toxT transcription and are required for...

Advances on Vibrio parahaemolyticus research in the postgenomic era

“…15,22 In the V. cholerae AM-19226 strain, the homologous proteins of VtrA and VtrB (VttRA and VttRB, respectively) control T3SS genes and are required for T3SS-mediated pathogenicity. 30,31 These findings indicate the evolutionary conservation of the VtrA-VtrB regulatory cascade in both the T3SS2α and T3SS2β lineages and its central role in virulence regulation via T3SS2 gene expression.…”

“…The vtrA and vtrB genes are prevalent in the V. parahaemolyticus TH3996 strain (T3SS2β type) and in the non‐O1/non‐O139 V. cholerae strains AM‐19226 (T3SS2α type) and 1587 (T3SS2β type) . In the V. cholerae AM‐19226 strain, the homologous proteins of VtrA and VtrB (VttRA and VttRB, respectively) control T3SS genes and are required for T3SS‐mediated pathogenicity . These findings indicate the evolutionary conservation of the VtrA–VtrB regulatory cascade in both the T3SS2α and T3SS2β lineages and its central role in virulence regulation via T3SS2 gene expression.…”

Characterization ofV.choleraeT3SS-dependent cytotoxicity in cultured intestinal epithelial cells

“…While we currently do not understand the precise relationship between adherence, MOI, and cytotoxicity, we do know that 20-30% of the bacterial population is adherent at 2 h using an MOI of~10, and that cytotoxicity remains at~80% at 3 h if the MOI drops to~5. The MOI must be decreased 10-fold tõ 1 in order to observe a decrease in cytotoxicity, measured as twofold (to~40% at 3 h) (Miller et al, 2016). If the proportion of adherent bacteria at 2 h, when little replication has occurred, remains similar regardless of MOI, then a 10-fold decrease in adherence is required to elicit the twofold decrease in cytotoxicity observed by decreasing the MOI 10-fold to~1.…”

“…6A). Previous studies determined that a VopX deletion strain had altered T3SS gene expression because of regulatory sequences that overlap the vopX coding region, so we constructed and assayed separately an AM-19226 strain carrying a copy of the vopX gene that produces a truncated protein (vopX ′) (Miller et al, 2016). Co-culture of Caco2-BBE cells with a strain unable to produce VopX resulted in cytotoxicity similar to the parental strain (Fig.…”

“…pBAD18 was converted to a Gateway compatible vector using the Gateway vector conversion system (Invitrogen) to result in pBAD18rfaKM, which was then used in standard Gateway protocols to produce pBAD18-vopH, pBAD18-vopI, and pBAD18-vopW. The vopX ′ truncation was constructed by introducing a single nucleotide substitution (A157T) resulting in amino acid change K54X, which introduced a unique BglII site as described (Miller et al, 2016).…”