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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.

Cystic fibrosis (CF) is a genetic disease that affects almost 100 000 people worldwide. CF patients suffer from chronic bacterial airway infections that are often polymicrobial and are the leading cause of mortality. Interactions between pathogens modulate expression of genes responsible for virulence and antibiotic resistance. One of the ways bacteria can interact is through contact-dependent systems, which secrete antibacterial proteins (effectors) that confer advantages to cells that harbor them. Here, we highlight recent work that describes effectors used by Gram-negative CF pathogens to eliminate competitor bacteria. Understanding the mechanisms of secreted effectors may lead to novel insights into the ecology of bacteria that colonize respiratory tracts and could also pave the way for the design of new therapeutics.

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.

Vibrio cholerae is an aquatic Gram-negative bacterium that causes severe diarrheal cholera disease when ingested by humans. To eliminate competitor cells in both the external environment and inside hosts, V. cholerae uses the Type VI Secretion System (T6SS). The T6SS is a macromolecular weapon employed by many Gram-negative bacteria to deliver cytotoxic proteins into adjacent cells. In addition to canonical T6SS gene clusters encoded by all sequenced V. cholerae isolates, strain BGT49 encodes an additional locus, which we named auxiliary cluster 4 (Aux 4). The Aux 4 cluster is located on a mobile genetic element and can be used by killer cells to eliminate both V. cholerae and Escherichia coli cells in a T6SS-dependent manner. A putative toxin encoded in the cluster, which we name TpeV (Type VI Permeabilizing Effector Vibrio), shares no homology to known proteins and does not contain motifs or domains indicative of function. Ectopic expression of TpeV in the periplasm of E. coli permeabilizes cells and disrupts the membrane potential. Using confocal microscopy, we confirm that susceptible target cells become permeabilized when competed with killer cells harboring the Aux 4 cluster. We also determine that tpiV, the gene located immediately downstream of tpeV, encodes an immunity protein that neutralizes the toxicity of TpeV. Finally, we show that TpeV homologs are broadly distributed across important animal and plant pathogens and are localized in proximity to other T6SS genes. Our results suggest that TpeV is a toxin that belongs to a large family of T6SS proteins.IMPORTANCEBacteria 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. However, the mechanisms by which many T6SS toxins kill or inhibit susceptible target cells are poorly understood. The sequence of the TpeV toxin we describe here is unlike any previously described protein. We demonstrate that it has antimicrobial activity by permeabilizing cells, eliminating membrane potentials and causing severe cytotoxicity. TpeV homologs are found near known T6SS genes in human, animal and plant bacterial pathogens, indicating that the toxin is a representative member of a broadly distributed protein family. We propose that TpeV-like toxins contribute to the fitness and pathogenicity of many bacteria. Finally, since antibiotic resistance is a critical global health threat, the discovery of new antimicrobial mechanisms could lead to the development of new treatments against resistant strains.

Biofilms are highly structured, densely packed bacterial consortia where many different species can coexist. During biofilm development and growth, the different species often form spatial distribution patterns that govern biofilm composition and function. In some cases, emerging structures have been explained as the result of social interactions between bacteria, e.g. cooperation and competition. Others emphasize the role of local mechanics, where spatial structuring arises from forces exerted between cells or between cells and their environment. Typically, these two lines of argumentation are treated separately. Here, we show that mechanics and social interactions can be strongly interrelated and their combination can crucially impact biofilm formation and dynamics. Using confocal microscopy and bacterial co-culture assays, we examine how bacterial antagonism impacts biofilm mechanics, and vice versa. We study competing Vibrio cholerae strains that kill on contact using the Type 6 secretion system. In case of mutual killers, i.e. two V. cholerae strains that can kill each other on contact, this social interaction leads to the formation of clonal domains of the competing strains (Mc Nally et al., Nat Commun, 2017). Intuitively, an unequal fight may enable a superior killer to invade and quickly eliminate a much weaker competitor. However, we observe that killer cells can coexist with killing-deficient target cells for very long times, and find that this results from the mechanical consequences of the deadly competition. Killing produces dead cells, which accumulates between domains of competing cells and prevents subsequent killing. Counterintuitively, our results suggest that antagonistic interactions stabilize coexistence in diverse communities. The findings demonstrate that the impact of social interactions in bacterial consortia is complex, requiring the understanding of the structural and the statistical-mechanical processes in biofilms.

Evolutionary arms races among organisms are broadly prevalent and bacteria 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 mechanisms that allow non-sibling bacteria to respond to external cues and survive T6SS attacks independently of immunity proteins. In this study, we show that resistance to T6SS attacks is promoted by a genetically controlled response to exogenous glucose. We observe that multiple human Escherichia coli commensal strains lacking immunity proteins are sensitive to T6SS attacks from pandemic Vibrio cholerae on nutrient-rich media. By contrast, E. coli cells become resistant to attacks when co-cultured on the same media with glucose. We confirm that glucose does not impair V. cholerae T6SS activity. Instead, we find that cAMP receptor protein (CRP), which alters expression of hundreds of genes in response to glucose, controls resistance to T6SS attacks in E. coli cells. Consistent with the observed resistance on media with glucose, an E. coli crp disruption mutant survives significantly better against V. cholerae T6SS attacks even in the absence of glucose. Finally, we also show that resistance to T6SS attacks depends on the pH of the medium and varies based on the target and killer strains.IMPORTANCEMany Gram-negative bacteria, including important pathogens, encode T6SS genes to deliver toxic effectors and eliminate competitors. Our results uncover a novel defense mechanism against T6SS attacks that is triggered by an external stimulus and mediated by a metabolic response in non-kin target cells. In microbiomes such as those in gastrointestinal tracts where T6SS activity is known to occur, signaling by metabolites like glucose may affect the efficacy of T6SS attacks and alter microbial community composition. Our findings could have vast implications for microbial interactions during pathogen colonization of hosts and survival of bacterial cells in environmental communities. Furthermore, the glucose-mediated resistance observed here might provide a novel example of an evolutionary arms race between killer T6SS cells and target bacteria.

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 withVibrio choleraestrains 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 106-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.

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.

BackgroundLike 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.ResultsWe 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.ConclusionWe 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.