Quorum sensing and a global regulator TsrA control expression of type VI secretion and virulence in Vibrio cholerae

Abstract

Vibrio cholerae is a human pathogen that causes the life-threatening diarrheal disease cholera. A type VI secretion system (T6SS) was recently shown to be required for full virulence in the O37 serogroup strain V52, which causes only sporadic human disease, but T6SS is not expressed in seventh pandemic O1 El Tor strains under standard laboratory conditions. In this study, we show that in the O1 El Tor strain C6706, T6SS is repressed by both quorum sensing and the uncharacterized protein VC0070 (TsrA). Disruption of TsrA and the quorum sensing regulator LuxO induces expression and secretion of the T6SS substrate Hcp, and this is dependent on the downstream regulator HapR, which directly binds to the promoter region of the T6SS genes hcp1 and hcp2 to induce expression. The activated T6SS in C6706 is functional and can translocate the effector protein VgrG-1 into macrophage cells, and T6SS activation leads to fecal diarrhea and intestinal inflammation in infant rabbits. Using an infant mouse infection model, we show that deletion of tsrA results in a 9.3-fold increase in intestinal colonization compared with wild type. TsrA functions as a global regulator to activate expression of hemagglutinin protease and repress cholera toxin and toxin coregulated pilus. Our findings provide significant insight into the molecular mechanism of T6SS and ToxT regulon gene regulation by quorum sensing and TsrA.

Fig. 1.

Quorum sensing and TsrA control T6SS. (A) Western blot analysis of Hcp in bacterial pellet (P) and culture supernatant (S) from various C6706 strains. The Western blot was probed with anti-Hcp serum. (B) Effect of mutations in various regulators on the expression of T6SS substrate hcp1 and the T6SS structural element VCA0107 using chromosomal transcriptional lacZ fusions. Bacteria were cultured in LB medium. Values given are means ± SD. (C) Deletion of hapR in ΔluxO and ΔluxO tsrA (C6706) decreased the expression of Hcp. Proteins from bacterial pellet were analyzed by Western blot with anti-Hcp serum.

Fig. 2.

The secretion of Hcp and translocation of VgrG-1 by C6706 after T6SS activation. (A) Western blot analysis of Hcp in bacterial pellet (P) and culture supernatant (S) from various C6706 strains. (B) Actin cross-linking in J744 macrophage cells infected with various C6706 strains. The Western blot was probed with anti–G-actin.

Fig. 3.

T6SS activation by tsrA deletion leads to fecal diarrhea in infant rabbits. Infant rabbits were orogastrically infected with sodium bicarbonate buffer, containing the indicated V. cholerae strains and observed for 3 d. (A) The frequency of infected rabbits that exhibited diarrhea. (B) H&E staining of the large intestine of infected infant rabbits. The arrows indicate signs of edema, congestion, and hemorrhage. (C) The expression of IL-8 in infected infant rabbits was examined with qRT-PCR. Each data point represents an individual animal infected with V. cholerae, and the horizontal bars indicate the mean.

Fig. 4.

Competitive indices of various regulatory deletion mutants (ΔtsrA, ΔtsrA + tsrA, ΔluxO, and ΔluxO tsrA) (LacZ⁻) compared with wild-type C6706 (LacZ⁺) (A) and T6SS mutant ΔtsrA vasK (LacZ⁺) compared with ΔtsrA (LacZ⁻) (B) in the infant mouse model. Each data point represents an individual animal infected with V. cholerae, and the horizontal bars indicate the mean.

Fig. 5.

TsrA and quorum sensing regulate the expression of virulence factors. Effect of deletion mutants in various regulators on expression of the virulence factors TCP and CT (A) and their regulators TcpP, ToxR, and ToxT (B) using chromosomal transcriptional lacZ fusion. Bacteria were cultured in LB medium. Values given are means ± SD. (C) The regulation of TsrA on HA protease production determined by azocasein activity.

Fig. 6.

A model for quorum sensing and TsrA regulation on V. cholerae T6SS and virulent factors. Solid line arrows or T bars denote the characterized positive or negative regulation. Dot line arrows or T bars denote the newly defined positive or negative regulations.