vttRA and vttRB Encode ToxR family proteins that mediate bile-induced expression of type three secretion system genes in a non-O1/non-O139 Vibrio cholerae strain

Abstract

Strain AM-19226 is a pathogenic non-O1/non-O139 serogroup Vibrio cholerae strain that does not encode the toxin-coregulated pilus or cholera toxin but instead causes disease using a type three secretion system (T3SS). Two genes within the T3SS pathogenicity island, herein named vttR(A) (locus tag A33_1664) and vttR(B) (locus tag A33_1675), are predicted to encode proteins that show similarity to the transcriptional regulator ToxR, which is found in all strains of V. cholerae. Strains with a deletion of vttR(A) or vttR(B) showed attenuated colonization in vivo, indicating that the T3SS-encoded regulatory proteins play a role in virulence. lacZ transcriptional reporter fusions to intergenic regions upstream of genes encoding the T3SS structural components identified growth in the presence of bile as a condition that modulates gene expression. Under this condition, VttR(A) and VttR(B) were necessary for maximal gene expression. In contrast, growth in bile did not substantially alter the expression of a reporter fusion to the vopF gene, which encodes an effector protein. Increased vttR(B) reporter fusion activity was observed in a DeltavttR(B) strain background, suggesting that VttR(B) may regulate its own expression. The collective results are consistent with the hypothesis that T3SS-encoded regulatory proteins are essential for pathogenesis and control the expression of selected T3SS genes.

FIG. 1.

(A) The flanking genes for the ancestral toxR gene (hatched arrow) and the region of the T3SS pathogenicity island encoding the structural components (dark-gray arrows) and the two putative transcriptional regulators (black and checkered arrows) are shown. White arrows represent genes encoding hypothetical or conserved hypothetical proteins. The dotted arrow represents a gene present in our annotation but not annotated by the J. Craig Venter Institute. Genes encoding known proteins are shown in light gray. The seven small arrows above the genes indicate the locations of predicted promoter sequences. (B) Multiple-sequence alignment (ClustalW2 with default settings) of the ToxR_N16961, ToxR_AM-19226, A33_1664, and A33_1675 protein sequences. Amino acid residues that constitute the predicted transmembrane domains are in bold and underlined. The predicted secondary structures of the ToxR domains comprising the winged HTH motif are indicated above the N16961 ToxR sequence. Residues forming beta sheets are indicated by thin lines, those forming alpha helices are indicated by thick lines, and wing residues are indicated by the letter W. (C) Hydrophilicity plots of ToxR and the ToxR-related proteins using Kyte-Doolittle analysis. Hydrophilic residues have a negative score and hydrophobic residues have a positive score on the plot. The numbers at the bottom of each panel refer to amino acid positions within the four proteins. (D) Domain structure and membrane localization of ToxR paralogs based on TMHMM analysis, hydrophilicity plots, and phoA fusion analysis. OM, outer membrane; IM, inner membrane.

FIG. 2.

ToxR homologs are essential for full colonization in the infant mouse model. Competition assays with CD-1 infant mice were performed using a lacZ mutant derivative of strain AM19226 (MD996) and a strain with the following gene deleted: ΔA33_1664 (strain AAC228, diamonds), ΔA33_1675 (strain AAC40, triangles), or ΔtoxR (strain AD10, circles). The results of a single experiment are shown, where each symbol represents the CI from a single animal (n = 9, n = 8, and n = 8, respectively). The bars indicate the mean CI for each experiment. Experiments were repeated with similar results.

FIG. 3.

The transcriptional organization of the two main operons encoding the VcsVUQ2 and VcsRTCNS2 structural components are depicted in panels A and D. Primer pairs were designed to amplify the regions shown by the bars above the genes. The open bars above the genes in panels A and D indicate that RT-PCR did not produce an amplicon, whereas the solid bars indicate that an amplicon was obtained using RT-PCR. The numbers above the bars correspond to the gel lanes in panels B, C, E, and F, showing the results of RT-PCR analyses using RNA extracted from cells grown in LB broth with 0.04% deoxycholate as the template (B and E) and PCR using the same primer pairs with gDNA as the template (C and F). Lanes marked “No RT” in panels B and E show representative PCRs conducted using RNA as the template, indicating that no product was observed, consistent with a lack of gDNA contamination.

FIG. 4.

The growth phase influences the expression of lacZ transcriptional fusions to T3SS genes. β-Galactosidase activity levels were measured in strains containing single-copy transcriptional lacZ fusions to the structural genes vcsRTCNS2, vspD, vcsJ2, and vcsVUQ2, the vopF gene (encoding an effector protein), and the putative transcriptional regulators A33_1675 and A33_1664. The promoterless lacZ fusion was integrated as a control for the basal level of reporter gene expression. Strains were grown in LB medium at 37°C to exponential phase (dark gray bars) or to stationary phase (white bars). The data shown represent the results of one experiment. Experiments were performed twice using three individual colonies each time and produced similar results. Note that the two graphs have different y-axis scales.

FIG. 5.

Deoxycholate and bile increase the expression of T3SS promoter-lacZ transcriptional fusions. β-Galactosidase activity levels were measured in strains containing single-copy chromosomal transcriptional lacZ fusions to the indicated promoter regions. Strains were grown at 37°C overnight in LB medium alone (white bars), LB broth containing 0.04% Na-deoxycholate (dark gray bars), or LB broth containing 0.4% bile (light gray bars). The data shown represent the results of a single experiment using three individual colonies of each strain. The experiment was repeated twice with similar results. Note that the two graphs have different y-axis scales.

FIG. 6.

T3SS pathogenicity island-encoded transcriptional regulators VttR_A (encoded by A33_1664) and VttR_B (encoded by A33_1675) regulate T3SS structural gene expression when strains are grown in LB broth containing 0.4% bile but not when they are grown in LB broth alone. Single-copy transcriptional lacZ fusions in five different genetic backgrounds were assayed after overnight growth at 37°C in LB medium alone (A) or containing 0.4% bile (B). The β-galactosidase activity for each fusion was measured in each of the following five backgrounds: wild type (light gray bars), ΔA33_1664 (checkered bars), ΔA33_1675 (black bars), ΔA33_1664 ΔA33_1675 (striped bars), and ΔtoxR (hatched bars). The data presented represent the results of three experiments using at least three individual colonies of each strain. All values are background subtracted (using strains expressing the promoterless construct), and the activity for each fusion in the isogenic parent strain was assigned a value of 100%. The percent activity for each reporter fusion in each deletion strain was calculated relative to the expression obtained with the isogenic parent strain, and the standard deviation was calculated based on at least three experiments.