Pesticin-Like Effector VgrG3^cp Targeting Peptidoglycan Delivered by the Type VI Secretion System Contributes to Vibrio cholerae Interbacterial Competition

Vibrio cholerae can utilize a type VI secretion system (T6SS) to increase its intra- and interspecies competition. However, much still remains to be understood about the underlying mechanism of this intraspecies competition. In this study, we isolated an environmental V. cholerae strain E1 that lacked the typical virulence factors toxin-coregulated pilus and cholera toxin and that encoded a functional T6SS. We identified an evolved VgrG3 variant with a predicted C-terminal pesticin-like domain in V. cholerae E1, designated VgrG3(cp). Using heterologous expression, protein secretion, and peptidoglycan-degrading assays, we demonstrated that VgrG3(cp) is a T6SS-dependent effector harboring cell wall muramidase activity and that its toxicity can be neutralized by cognate immunity protein TsiV3(cp). Site-directed mutagenesis proved that the aspartic acid residue at position 867 is crucial for VgrG3(cp)-mediated antibacterial activity. Bioinformatic analysis showed that genes encoding VgrG3(cp)-like homologs are distributed in Vibrio species, are linked with T6SS structural genes and auxiliary genes, and the vgrG3(cp)-tsiV3(cp) gene pair of V. cholerae probably evolved from Vibrio anguillarum and Vibrio fluvialis via homologous recombination. Through a time-lapse microscopy assay, we directly determined that cells accumulating VgrG3(cp) disrupted bacterial division, while the cells continued to increase in size until the loss of membrane potential and cell wall breakage and finally burst. The results of the competitive killing assay showed that VgrG3(cp) contributes to V. cholerae interspecies competition. Collectively, our study revealed a novel T6SS E-I pair representing a new T6SS toxin family which allows V. cholerae to gain dominance within polymicrobial communities by T6SS.IMPORTANCE The type VI secretion system used by a broad range of Gram-negative bacteria delivers toxic proteins to target adjacent eukaryotic and prokaryotic cells. Diversification of effector proteins determines the complex bacterium-bacterium interactions and impacts the health of hosts and environmental ecosystems in which bacteria reside. This work uncovered an evolved valine-glycine repeat protein G3, carrying a C-terminal pesticin-like domain (VgrG3(cp)), which has been suggested to harbor cell wall hydrolase activity and is able to affect cell division and the integrity of cell wall structure. Pesticin-like homologs constitute a family of T6SS-associated effectors targeting bacterial peptidoglycan which are distributed in Vibrio species, and genetic loci of them are linked with T6SS structural genes and auxiliary genes. T6SS-delivered VgrG3(cp) mediated broad-spectrum antibacterial activity for several microorganisms tested, indicating that VgrG3(cp)-mediated antimicrobial activity is capable of conferring bacteria a competitive advantage over competitors in the same niches. The type VI secretion system used by a broad range of Gram-negative bacteria delivers toxic proteins to target adjacent eukaryotic and prokaryotic cells. Diversification of effector proteins determines the complex bacterium-bacterium interactions and impacts the health of hosts and environmental ecosystems in which bacteria reside.