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

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 ΔvttR_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.