The Vibrio cholerae trh gene is coordinately regulated in vitro with type III secretion system genes by VttR(A)/VttR(B) but does not contribute to Caco2-BBE cell cytotoxicity

Abstract

Numerous virulence factors have been associated with pathogenic non-O1/non-O139 serogroup strains of Vibrio cholerae. Among them are the thermostable direct hemolysin (TDH) and the TDH-related hemolysin (TRH), which share amino acid similarities to the TDH and TRH proteins of Vibrio parahaemolyticus, where they have been shown to contribute to pathogenesis. Although TDH and TRH homologs can be encoded on extrachromosomal elements in V. cholerae, type III secretion system (T3SS)-positive strains, such as AM-19226, carry a copy of trh within the T3SS genomic island. Transcriptional fusion analysis showed that in strain AM-19226, trh expression is regulated in a bile-dependent manner by a family of transmembrane transcriptional regulators that includes VttR(A), VttR(B), and ToxR. Genes encoding T3SS structural components are expressed under similar conditions, suggesting that within the T3SS genomic island, genes encoding proteins unrelated to the T3SS and loci involved in T3SS synthesis are coregulated. Despite similar in vitro expression patterns, however, TRH is not required for AM-19226 to colonize the infant mouse intestine, nor does it contribute to bile-mediated cytotoxicity when strain AM-19226 is cocultured with the mammalian cell line Caco2-BBE. Instead, we found that a functional T3SS is essential for AM-19226 to induce bile-mediated cytotoxicity in vitro. Collectively, the results are consistent with a more minor role for the V. cholerae TRH in T3SS-positive strains compared to the functions attributed to the V. parahaemolyticus TDH and TRH proteins.

Fig 1

Bioinformatic analysis of AM-19226 TRH: ClustalW alignment of V. parahaemolyticus RIMD2210633 TDHA (VpTDH), V. parahaemolyticus AQ4037 TRH1 (VpTRH), and V. cholerae AM-19226 TRH (VcTRH). The asterisk denotes identical amino acids. The period denotes conserved amino acids. The predicted N-terminal general signal sequence is indicated by underlined amino acids. Gray shading indicates residues important for hemolytic activity in V. parahaemolyticus TDH.

Fig 2

Bile-mediated trh-lacZ expression requires the transcriptional regulators VttR_A, VttR_B, and ToxR. (A) Wild-type AM-19226 strains carrying a promoterless-lacZ or trh-lacZ transcriptional fusion were grown overnight at 37°C in LB medium alone or LB containing 0.4% bile and then assayed for β-galactosidase activity. (B) The following strains, possessing either the promoterless-lacZ or trh-lacZ transcriptional fusion, were grown overnight at 37°C in LB medium containing 0.4% bile and assayed for β-galactosidase activity: AM-19226 WT (black bars), ΔvttR_A (cross-hatched bars), ΔvttR_B (light gray bars), ΔtoxR (diagonally lined bars), ΔvttR_A ΔvttR_B (checkered bars), ΔvttR_A ΔvttR_B ΔtoxR (lined bars). The data represent a single experiment using three colonies of each strain. The experiment was repeated with similar results.

Fig 3

ΔVttR_A and ΔVttR_B complementation of vcsRTCNS2-lacZ expression. AM-19226 strains carrying a vcsRTCNS2-lacZ transcriptional fusion and pBAD18, pBAD18-vttR_A, or pBAD18-vttR_B were grown overnight at 37°C in LB medium containing 0.4% bile with or without arabinose as indicated and assayed for β-galactosidase activity. Gene expression was examined in the following AM-19226 backgrounds: WT carrying pBAD18 (black bars; A and B), ΔvttR_A carrying pBAD18 (light gray bars; A), ΔvttR_A carrying pBAD18-vttR_A (cross-hatched bars; A), ΔvttR_B carrying pBAD18 (dark gray bars; B), or ΔvttR_B carrying pBAD18-vttR_B (lined bars; B). In panel A, asterisks denote P < 0.05 between the indicated group and AM-19226 ΔvttR_A (pBAD18-vttR_A) grown in 0.005% arabinose (cross-hatched bar). In panel B, asterisks denote P < 0.05 between the indicated group and AM-19226 ΔvttR_B (pBAD18-vttR_B) grown in 0.005% arabinose (lined bar). Data are shown for one experiment using three individual colonies per strain. The experiment was repeated with similar results.

Fig 4

VttR_B alone can promote trh expression. Strains are represented as follows: AM-19226 WT, black bars; ΔvttR_A, cross-hatched bars; ΔvttR_B, light gray bars; ΔtoxR, diagonally lined bars; ΔvttR_A ΔvttR_B, checkered bars; ΔvttR_A ΔvttR_B ΔtoxR, lined bars. Strains possessing either the promoterless-lacZ or trh-lacZ transcriptional reporter fusion were assayed for β-galactosidase activity. (A) Results of assays using strains that carried the pBAD18-vttR_A-complementing plasmid, grown overnight at 37°C in LB medium with 0.4% bile and 0.01% arabinose. (B) Results of assays using strains that carry the pBAD18-vttR_B-complementing plasmid, grown overnight at 37°C in LB medium with 0.4% bile and 0.0025% arabinose. In all cases, three colonies from each strain were assayed, and results were compared to strains grown in the absence of arabinose. Promoterless-lacZ reporter fusions exhibited <20 units of activity in all strain backgrounds (data not shown). *, P < 0.05 between the no arabinose and 0.01% arabinose (A) or 0.0025% arabinose (B) conditions for each AM-19226 strain background. Each experiment was repeated with similar results.

Fig 5

ToxR may positively and negatively regulate trh transcription. AM-19226 WT (black bars), ΔvttR_A (cross-hatched bars), ΔvttR_B (light gray bars), ΔtoxR (diagonally lined bars), ΔvttR_A ΔvttR_B (checkered bars), and ΔvttR_A ΔvttR_B ΔtoxR (lined bars) backgrounds possessing either the promoterless-lacZ or trh-lacZ transcriptional fusion were examined for β-galactosidase activity. Strains carried either the pBR322 empty vector or pVM7 to express toxR from the constitutive tetracycline promoter. Three colonies from each strain were grown overnight at 37°C in LB medium with 0.4% bile. Promoterless-lacZ fusion strains exhibited less than 5 units activity and are not shown. Asterisks indicate significant differences (P < 0.05) between an AM-19226 strain carrying pBR322 and the isogenic strain carrying pVM7. The experiment was repeated with similar results.

Fig 6

TRH is not required for infant mouse colonization. Infant mouse competition assays were performed using an inoculum containing a 1:1 ratio of a lacZ mutant derivative of AM-19226 and a lacZ ⁺ AM-19226 trh deletion strain. The results shown are from a single experiment; each triangle represents the CI for one mouse (n = 10), and the horizontal bar represents the mean. The experiment was repeated with similar results.

Fig 7

trh and vcsRTCNS2 expression during mammalian cell coculture. trh-lacZ, vcsRTCNS2-lacZ, or promoterless-lacZ activity was examined in AM-19226 Δ3 strains after 2 h of coculture with Caco2-BBE cells. AM-19226 strains were grown overnight at 37°C in LB medium. Caco2-BBE cells containing fresh culture medium with or without 0.2% bile were infected with AM-19226 at a multiplicity of infection of ∼10 and cocultured for 2 h at 37°C, 5% CO₂. Culture medium without mammalian cells was also inoculated with AM-19226 and cultured for 2 h. Bacteria were then harvested, resuspended in Z-buffer, and added back to the mammalian cells for processing. β-Galactosidase activity was measured by a standard kinetic assay. Data shown are from one experiment using three colonies per strain. Asterisks denote significant differences (P < 0.05) between the indicated group and AM-19226 trh-lacZ cultured with Caco2-BBE cells in medium containing 0.2% bile. The experiment was repeated with similar results.

Fig 8

The T3SS and not TRH is required for bile-induced Caco2-BBE cytotoxicity. Three colonies of each AM-19226 strain were grown overnight at 37°C in LB medium and used to infect Caco2-BBE cells with fresh medium with or without 0.2% bile. (A) After 3 h of coculture, representative images were taken for each coculture condition. (B) Immediately following imaging, LDH release was measured from each sample after 3 h of coculture to quantify cytotoxicity. Data are shown for mammalian cells cocultured with various AM-19226 strains in the presence of 0.2% bile. All strains were T3SS competent except those containing a deletion in vcsN2. The experiment was repeated with similar results.