The phospholipase effector Tle1Vc promotes Vibrio cholerae virulence by killing competitors and impacting gene expression

Abstract

Vibrio cholerae utilizes the Type VI secretion system (T6SS) to gain an advantage in interbacterial competition by delivering anti-prokaryotic effectors in a contact-dependent manner. However, the impact of T6SS and its secreted effectors on physiological behavior remains poorly understood. In this study, we present Tle1^Vc, a phospholipase effector in atypical pathogenic V. cholerae E1 that is secreted by T6SS via its interaction with VgrG1^E1. Tle1^Vc contains a DUF2235 domain and belongs to the Tle1 (type VI lipase effector) family. Bacterial toxicity assays, lipase activity assays and site-directed mutagenesis revealed that Tle1^Vc possessed phospholipase A₁ activity and phospholipase A₂ activity, and that Tle1^Vc-induced toxicity required a serine residue (S356) and two aspartic acid residues (D417 and D496). Cells intoxication with Tle1^Vc lead to membrane depolarization and alter membrane permeability. Tli1^tox-, a cognate immunity protein, directly interacts with Tle1^Vc to neutralize its toxicity. Moreover, Tle1^Vc can kill multiple microorganisms by T6SS and promote in vivo fitness of V. cholerae through mediating antibacterial activity. Tle1^Vc induces bacterial motility by increasing the expression of flagellar-related genes independently of functional T6SS and the tit-for-tat (TFT) response, where Pseudomonas aeruginosa uses its T6SS-H1 cluster to counterattack other offensive attackers. Our study also demonstrated that the physical puncture of E1 T6SS can induce a moderate TFT response, which is essential to the Tle1^Vc-mediated strong TFT response, maximizing effector functions. Overall, our study characterized the antibacterial mechanism of phospholipase effector Tle1^Vc and its multiple physiological significance.

Keywords: T6SS; Vibrio cholerae; motility; phospholipase effector; tit-for-tat.

Figure 1.

Tle1^Vc is a lipase effector with antibacterial activity.

Figure 2.

Tle1^Vc effector impairs the integrity of bacterial membrane.

Figure 3.

Tli1^tox- specifically neutralizes the toxicity of Tle1^Vc.

Figure 4.

Tle1^Vc delivery requires functional T6SS and VgrG1^E1.

Figure 5.

Tle1^Vc contributes to Vibrio cholerae intra- and interspecies antagonism.

Figure 6.

Tle1^vc-induced strong tit-for-tat response requires the T6SS-H1 of PAO1, catalytic activity of effector and the physical puncture of T6SS.

Figure 7.

Phospholipase effector Tle1^Vc mediates bacterial motility through modulating flagellar gene expression.

Figure 8.

The effect of Tle1^Vc on E1’s in vivo fitness and survival of immunity mutant.