Identification of T6SS-dependent effector and immunity proteins by Tn-seq in Vibrio cholerae

Abstract

Type VI protein secretion system (T6SS) is important for bacterial competition through contact-dependent killing of competitors. T6SS delivers effectors to neighboring cells and corresponding antagonistic proteins confer immunity against effectors that are delivered by sister cells. Although T6SS has been found in more than 100 gram-negative bacteria including many important human pathogens, few T6SS-dependent effector and immunity proteins have been experimentally determined. Here we report a high-throughput approach using transposon mutagenesis and deep sequencing (Tn-seq) to identify T6SS immunity proteins in Vibrio cholerae . Saturating transposon mutagenesis was performed in wild type and a T6SS null mutant. Genes encoding immunity proteins were predicted to be essential in the wild type but dispensable in the T6SS mutant. By comparing the relative abundance of each transposon mutant in the mutant library using deep sequencing, we identified three immunity proteins that render protection against killing by T6SS predatory cells. We also identified their three cognate T6SS-secreted effectors and show these are important for not only antibacterial and antieukaryotic activities but also assembly of T6SS apparatus. The lipase and muramidase T6SS effectors identified in this study underscore the diversity of T6SS-secreted substrates and the distinctly different mechanisms that target these for secretion by the dynamic T6SS organelle.

Generation of reactive oxygen species by lethal attacks from competing microbes

“…We then determined whether T6SS-dependent effectors are important for inducing soxS. We have previously identified three T6SS antimicrobial effectors in V52 (11), one of which is VgrG3, a lysozymelike enzyme that attacks the cell wall of target species. Expression of VgrG3 in E. coli is highly toxic, whereas a point mutation in its catalytic residue D842A abolishes such toxicity (11).…”

“…We have previously identified three T6SS antimicrobial effectors in V52 (11), one of which is VgrG3, a lysozymelike enzyme that attacks the cell wall of target species. Expression of VgrG3 in E. coli is highly toxic, whereas a point mutation in its catalytic residue D842A abolishes such toxicity (11). We found that transcription levels of soxS were 12-fold higher in E. coli cells expressing VgrG3 than in cells expressing the catalytic mutant VgrG3 D842A (Fig.…”

“…Given that some cells can detect T6SS attack but not suffer any measurable loss in viability (8,9), it would seem that cell killing is likely due to the toxicity of effectors rather than membrane disruptions caused by insertion of the spear-like VgrG/PAAR/Hcp tube complex. T6SS-dependent effectors can attack a number of essential cellular targets, including the cell wall (10,11), membranes (11,12), and nucleic acids (13), and thus can mimic the actions of antibiotics and bacteriocins. As a model prey or target organism, Escherichia coli can be killed by the T6SS activities of a number of bacteria including Vibrio cholerae (14), Pseudomonas aeruginosa (10,15), and Acinetobacter baylyi ADP1 (7).…”

VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor–effector complex

“…A number of T6SS-secreted effectors were recently found to have contact-dependent antibacterial activity in various bacteria (1). The biochemical functions of characterized effectors include the cell wall-degrading enzymes (2-5), nuclease (6)(7)(8), and membrane lipid-degrading phospholipase (9)(10)(11). These diverse toxin effectors are widespread superfamilies in the bacterial kingdom and therefore likely have a general antibacterial strategy against competitor bacterial cells.…”

“…It may feature the effector function at the C-terminal domain. Such "evolved VgrGs" include Vibrio cholerae VgrG-1, with an actin cross-linking domain; Aeromonas hydrophila VgrG1, with an actin-ADP ribosylating VIP-2 domain; and V. cholerae VgrG-3, with glycoside hydrolase activity to target peptidoglycan (9,(25)(26)(27). Furthermore, VgrG may function as a carrier for effector delivery by binding directly with specific effectors carrying the proline-alanine-alanine-arginine (PAAR) domain (28,29).…”