Background Like many bacteria, Vibrio cholerae deploys a harpoon-like type VI secretion system (T6SS) to compete against other microbes in environmental and host settings. The T6SS punctures adjacent cells and delivers toxic effector proteins that are harmless to bacteria carrying cognate immunity factors. Only four effector/immunity pairs encoded on one large and three auxiliary gene clusters have been characterized from largely clonal, patient-derived strains of V. cholerae . Results We sequence two dozen V. cholerae strain genomes from diverse sources and develop a novel and adaptable bioinformatics tool based on hidden Markov models. We identify two new T6SS auxiliary gene clusters and describe Aux 5 here. Four Aux 5 loci are present in the host strain, each with an atypical effector/immunity gene organization. Structural prediction of the putative effector indicates it is a lipase, which we name TleV1 (type VI lipase effector Vibrio ). Ectopic TleV1 expression induces toxicity in Escherichia coli , which is rescued by co-expression of the TliV1a immunity factor. A clinical V. cholerae reference strain expressing the Aux 5 cluster uses TleV1 to lyse its parental strain upon contact via its T6SS but is unable to kill parental cells expressing the TliV1a immunity factor. Conclusion We develop a novel bioinformatics method and identify new T6SS gene clusters in V. cholerae . We also show the TleV1 toxin is delivered in a T6SS manner by V. cholerae and can lyse other bacterial cells. Our web-based tool can be modified to identify additional novel T6SS genomic loci in diverse bacterial species. Electronic supplementary material The online version of this article (10.1186/s13059-019-1765-5) contains supplementary material, which is available to authorized users.

The type VI secretion system (T6SS) is a proteinaceous weapon used by many Gram-negative bacteria to deliver toxins into adjacent target cells. Vibrio cholerae, the bacterium responsible for the fatal water-borne cholera disease, uses the T6SS to evade phagocytic eukaryotes, cause intestinal inflammation, and compete against other bacteria with toxins that disrupt lipid membranes, cell walls and actin cytoskeletons. The control of T6SS genes varies among V. cholerae strains and typically includes inputs from external signals and cues, such as quorum sensing and chitin availability. In the following review, we highlight the repertoire of toxic T6SS effectors and the diverse genetic regulation networks among different isolates of V. cholerae. Finally, we discuss the roles played by the T6SS of V. cholerae in both natural environments and hosts.

Evolutionary arms races are broadly prevalent among organisms including bacteria, which have evolved defensive strategies against various attackers. A common microbial aggression mechanism is the type VI secretion system (T6SS), a contact-dependent bacterial weapon used to deliver toxic effector proteins into adjacent target cells. Sibling cells constitutively express immunity proteins that neutralize effectors. However, less is known about factors that protect non-sibling bacteria from T6SS attacks independently of cognate immunity proteins. In this study, we observe that human Escherichia coli commensal strains sensitive to T6SS attacks from Vibrio cholerae are protected when co-cultured with glucose. We confirm that glucose does not impair V. cholerae T6SS activity. Instead, we find that cells lacking the cAMP receptor protein (CRP), which regulates expression of hundreds of genes in response to glucose, survive significantly better against V. cholerae T6SS attacks even in the absence of glucose. Finally, we show that the glucose-mediated T6SS protection varies with different targets and killers. Our findings highlight the first example of an extracellular small molecule modulating a genetically controlled response for protection against T6SS attacks. This discovery may have major implications for microbial interactions during pathogen-host colonization and survival of bacteria in environmental communities.

Bacterial communities are governed by a wide variety of social interactions, some of which are antagonistic with potential significance for bacterial warfare. Several antagonistic mechanisms, such as killing via the type VI secretion system (T6SS), require killer cells to directly contact target cells. The T6SS is hypothesized to be a highly potent weapon, capable of facilitating the invasion and defence of bacterial populations. However, we find that the efficacy of contact killing is severely limited by the material consequences of cell death. Through experiments with Vibrio cholerae strains that kill via the T6SS, we show that dead cell debris quickly accumulates at the interface that forms between competing strains, preventing physical contact and thus preventing killing. While previous experiments have shown that T6SS killing can reduce a population of target cells by as much as 10 6 -fold, we find that, as a result of the formation of dead cell debris barriers, the impact of contact killing depends sensitively on the initial concentration of killer cells. Killer cells are incapable of invading or eliminating competitors on a community level. Instead, bacterial warfare itself can facilitate coexistence between nominally antagonistic strains. While a variety of defensive strategies against microbial warfare exist, the material consequences of cell death provide target cells with their first line of defence.

word count: 229 18 Text word count: 3540 19 20 21 22 23 24 25 26 27 28 2 ABSTRACT 29Evolutionary arms races among organisms are broadly prevalent and bacteria have evolved 30 defensive strategies against various attackers. A common microbial aggression mechanism is the 31 Type VI Secretion System (T6SS), a contact-dependent bacterial weapon used to deliver toxic 32 effector proteins into adjacent target cells. Sibling cells constitutively express immunity proteins 33 that neutralize effectors. However, less is known about mechanisms that allow non-sibling bacteria 34 to respond to external cues and survive T6SS attacks independently of immunity proteins. In this 35 study, we show that resistance to T6SS attacks is promoted by a genetically controlled response to 36 exogenous glucose. We observe that multiple human Escherichia coli commensal strains lacking 37 immunity proteins are sensitive to T6SS attacks from pandemic Vibrio cholerae on nutrient-rich 38 media. By contrast, E. coli cells become resistant to attacks when co-cultured on the same media 39 with glucose. We confirm that glucose does not impair V. cholerae T6SS activity. Instead, we find 40 that cAMP receptor protein (CRP), which alters expression of hundreds of genes in response to 41 glucose, controls resistance to T6SS attacks in E. coli cells. Consistent with the observed resistance 42 on media with glucose, an E. coli crp disruption mutant survives significantly better against V. 43 cholerae T6SS attacks even in the absence of glucose. Finally, we also show that resistance to 44 T6SS attacks depends on the pH of the medium and varies based on the target and killer strains. 45 IMPORTANCE 46Many Gram-negative bacteria, including important pathogens, encode T6SS genes to deliver toxic 47 effectors and eliminate competitors. Our results uncover a novel defense mechanism against T6SS 48 attacks that is triggered by an external stimulus and mediated by a metabolic response in non-kin 49 target cells. In microbiomes such as those in gastrointestinal tracts where T6SS activity is known 50 to occur, signaling by metabolites like glucose may affect the efficacy of T6SS attacks and alter 51 3 microbial community composition. Our findings could have vast implications for microbial 52 interactions during pathogen colonization of hosts and survival of bacterial cells in environmental 53 communities. Furthermore, the glucose-mediated resistance observed here might provide a novel 54 example of an evolutionary arms race between killer T6SS cells and target bacteria. 55 INTRODUCTION 56 Vibrio cholerae is the waterborne enteric pathogen that causes serious, often fatal cholera diarrheal 57 disease when ingested by humans. This ubiquitous microbe is found in dense polymicrobial marine 58 communities on chitinous surfaces and in animal reservoirs like fish or zooplankton (1-3). To 59 compete with other cells in densely-populated microbial environments, V. cholerae employs a 60 harpoon-like structure called the Type VI Secretion System (T6SS) (4-7). The T6SS pu...

Bacterial communities govern their composition using a wide variety of social interactions, some of which are antagonistic. Many antagonistic mechanisms, such as the Type VI Secretion System (T6SS), require killer cells to directly contact target cells. The T6SS is hypothesized to be a highly potent weapon, capable of facilitating the invasion and defense of bacterial populations. However, we find that the efficacy of the T6SS is severely limited by the material consequences of cell death. Through experiments with Vibrio cholerae strains that kill via the T6SS, we show that dead cell debris quickly accumulates at the interface that forms between competing strains, preventing contact and thus preventing killing. While previous experiments have shown that T6SS killing can reduce a population of target cells by as much as one-million-fold, we find that as a result of the formation of dead cell debris barriers, the impact of T6SS killing depends sensitively on the initial concentrations of killer and target cells. Therefore, while the T6SS provides defense against contacting competitors on a single cell level, it is incapable of facilitating invasion or the elimination of competitors on a community level.

Background: Like many bacteria, Vibrio cholerae, which causes fatal cholera, deploys a harpoon-like Type VI Secretion System (T6SS) to compete against other microbes in environmental and host settings. The T6SS punctures adjacent cells and delivers toxic effector proteins that are harmless to bacteria carrying cognate immunity factors. Only four effector/immunity pairs encoded on one large and three auxiliary gene clusters have been characterized from largely clonal, patient-derived strains of V. cholerae. Results:We sequenced two dozen V. cholerae strain genomes from diverse sources and developed a novel and adaptable bioinformatic tool based on Hidden Markov Models. We identified two new T6SS auxiliary gene clusters; one, Aux 5, is described here. Four Aux 5 loci are present in the host strain, each with an atypical effector/immunity gene organization. Structural prediction of the putative effector indicated it is a lipase, which we name TleV1 (Type VI lipase effector Vibrio, TleV1). Ectopic TleV1 expression induced toxicity in E. coli, which was rescued by co-expression of the TleV1 immunity factor. A clinical V. cholerae reference strain expressing the Aux 5 cluster used TleV1 to lyse its parental strain upon contact via its T6SS but was unable to kill parental cells expressing TleV1's immunity factor. Conclusion:We developed a novel bioinformatic method and identified new T6SS gene clusters in V. cholerae. We also showed the TleV1 toxin is delivered in a T6SS-manner by V. cholerae and can lyse other bacterial cells. Our web-based tool may be modified to identify additional novel T6SS genomic loci in diverse bacterial species.

Bacteria live in polymicrobial communities where competition for resources and space is essential for survival. Proteobacteria use the T6SS to eliminate neighboring cells and cause disease.