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.

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