The Vibrio cholerae type VI secretion system displays antimicrobial properties

Abstract

The acute diarrheal disease cholera is caused by the marine bacterium Vibrio cholerae. A type VI secretion system (T6SS), which is structurally similar to the bacteriophage cell-puncturing device, has been recently identified in V. cholerae and is used by this organism to confer virulence toward phagocytic eukaryotes, such as J774 murine macrophages and Dictyostelium discoideum. We tested the interbacterial virulence of V. cholerae strain V52, an O37 serogroup with a constitutively active T6SS. V52 was found to be highly virulent toward multiple Gram-negative bacteria, including Escherichia coli and Salmonella Typhimurium, and caused up to a 100,000-fold reduction in E. coli survival. Because the T6SS-deficient mutants V52ΔvasK and V52ΔvasH showed toxicity defects that could be complemented, virulence displayed by V. cholerae depends on a functional T6SS. V. cholerae V52 and strains of the O1 serogroup were resistant to V52, suggesting that V. cholerae has acquired immunity independently of its serogroup. We hypothesize that the T6SS, in addition to targeting eukaryotic host cells, confers toxicity toward other bacteria, providing a means of interspecies competition to enhance environmental survival. Thus, the V. cholerae T6SS may enhance the survival of V. cholerae in its aquatic ecosystem during the transmission of cholera and between epidemics.

Fig. 1.

V. cholerae uses its T6SS to target bacteria with specificity toward Gram-negative species. Survival of rifampicin-resistant Gram-negative bacterial prey is shown: (A) E. coli K-12 MG1655 (red), S. Typhimurium (green), C. rodentium (blue), and P. aeruginosa (orange) and (B) E. coli K-12 MG1655 (red), EHEC (purple), and EPEC (orange) were determined by measuring cfu following exposure to the indicated rifampicin-sensitive predator listed on the x axis with each prey exposed to predatory self, V52, and V52ΔvasK. The data represent three independent experiments.

Fig. 2.

V. cholerae is highly virulent toward E. coli in a T6SS-dependent manner. (A) Killing assay: Survival of rifampicin-resistant E. coli was determined by measuring cfu following exposure to the indicated rifampicin-sensitive predator listed on the x axis. The data represent three independent experiments. (B) Survival curve: Survival of E. coli prey was measured following varying lengths of exposure to V52. Predator and prey were mixed at an MOI of 1 (■) or 10 (◆). The results shown are the mean (±SD) of experimental duplicates.

Fig. 3.

E. coli cells are undetectable following exposure to V. cholerae. Detection of V. cholerae (green) and E. coli (red) by fluorescence microscopy before and after coincubation with V52 is shown. MG1655R/pTV-mCherry was mixed with V. cholerae strains V52/pBSW208lacP::GFP (A) and V52ΔvasK/pBSW208lacP::GFP (B) at an MOI of 0.1 and spotted onto LB for 4 h at 37 °C. Cells were visualized before (t = 0) and after (t = 4) coincubation by immobilization on glass slides.

Fig. 4.

Killing is contact-dependent. Contact-dependent killing assay: Survival of rifampicin-resistant E. coli was determined by measuring cfu following exposure to the indicated rifampicin-sensitive predator listed on the x axis with (green) or without (red) a 0.22-μm filter separating predator and prey. The data represent three independent experiments.

Fig. 5.

T6SS-mediated killing of E. coli does not involve actin cross-linking. Killing assay: Survival of rifampicin-resistant E. coli was determined by measuring cfu following exposure to the indicated rifampicin-sensitive predator listed on the x axis. The data represent three independent experiments.

Fig. 6.

O1 pandemic strains of V. cholerae are not virulent toward E. coli. Killing assay: Survival of rifampicin-resistant E. coli was determined by measuring cfu following exposure to the indicated rifampicin-sensitive predator listed on the x axis. The data represent three independent experiments.

Fig. 7.

V. cholerae is resistant to T6SS-mediated bacterial virulence. Killing assay: Survival of the indicated rifampicin-resistant E. coli and V. cholerae prey as listed on the x axis was determined by measuring cfu following exposure to rifampicin-sensitive predator V52 or V52ΔvasK. Bars represent the mean cfu per milliliter of surviving E. coli and V. cholerae prey from three independent experiments (±SD).