Vibrio cholerae is a Gram-negative bacterial pathogen that consists of over 200 serogroups with differing pathogenic potential. Only strains that express the virulence factors cholera toxin (CT) and toxin-coregulated pilus (TCP) are capable of pandemic spread of cholera diarrhoea. Regardless, all V. cholerae strains sequenced to date harbour genes for the type VI secretion system (T6SS) that translocates effectors into neighbouring eukaryotic and prokaryotic cells. Here we report that the effectors encoded within these conserved gene clusters differ widely among V. cholerae strains, and that immunity proteins encoded immediately downstream from the effector genes protect their host from neighbouring bacteria producing corresponding effectors. As a consequence, strains with matching effector-immunity gene sets can coexist, while strains with different sets compete against each other. Thus, the V. cholerae T6SS contributes to the competitive behaviour of this species.

Background:The type VI secretion system provides Gram-negative bacteria with a competitive advantage. Results: The V. cholerae T6SS component VgrG-3 has lysozyme-like activity that is inhibited by the product of the downstream gene tsaB. Conclusion: VgrG-3 and TsaB are a toxin-antitoxin complex of the V. cholerae T6SS. Significance: A T6SS effector is characterized along with its cognate antitoxin.

Vibrio cholerae is a diverse species of Gram-negative bacteria, commonly found in the aquatic environment and the causative agent of the potentially deadly disease cholera. These bacteria employ a type VI secretion system (T6SS) when they encounter prokaryotic and eukaryotic competitors. This contractile puncturing device translocates a set of effector proteins into neighboring cells. Translocated effectors are toxic unless the targeted cell produces immunity proteins that bind and deactivate incoming effectors. Comparison of multiple V. cholerae strains indicates that effectors are encoded in T6SS effector modules on mobile genetic elements. We identified a diverse group of chimeric T6SS adaptor proteins required for the translocation of diverse effectors encoded in modules. An example for a T6SS effector that requires T6SS adaptor protein 1 (Tap-1) is TseL found in pandemic V. cholerae O1 serogroup strains and other clinical isolates. We propose a model in which Tap-1 is required for loading TseL onto the secretion apparatus. After T6SS-mediated TseL export is completed, Tap-1 is retained in the bacterial cell to load other T6SS machines.

As the causative agent of cholera, the bacterium Vibrio cholerae represents an enormous public health burden, especially in developing countries around the world. Cholera is a self-limiting illness; however, antibiotics are commonly administered as part of the treatment regimen. Here we review the initial identification and subsequent evolution of antibiotic-resistant strains of V. cholerae. Antibiotic resistance mechanisms, including efflux pumps, spontaneous chromosomal mutation, conjugative plasmids, SXT elements and integrons, are also discussed. Numerous multidrug-resistant strains of V. cholerae have been isolated from both clinical and environmental settings, indicating that antibiotic use has to be restricted and alternative methods for treating cholera have to be implemented.

The Vibrio cholerae type VI secretion system (T6SS) assembles as a molecular syringe that injects toxic protein effectors into both eukaryotic and prokaryotic cells. We previously reported that the V. cholerae O37 serogroup strain V52 maintains a constitutively active T6SS to kill other Gram-negative bacteria while being immune to attack by kin bacteria. The pandemic O1 El Tor V. cholerae strain C6706 is T6SS-silent under laboratory conditions as it does not produce T6SS structural components and effectors, and fails to kill Escherichia coli prey. Yet, C6706 exhibits full resistance when approached by T6SS-active V52. These findings suggested that an active T6SS is not required for immunity against T6SS-mediated virulence. Here, we describe a dual expression profile of the T6SS immunity protein-encoding genes tsiV1, tsiV2, and tsiV3 that provides pandemic V. cholerae strains with T6SS immunity and allows T6SS-silent strains to maintain immunity against attacks by T6SS-active bacterial neighbors. The dual expression profile allows transcription of the three genes encoding immunity proteins independently of other T6SS proteins encoded within the same operon. One of these immunity proteins, TsiV2, protects against the T6SS effector VasX which is encoded immediately upstream of tsiV2. VasX is a secreted, lipid-binding protein that we previously characterized with respect to T6SS-mediated virulence towards the social amoeba Dictyostelium discoideum. Our data suggest the presence of an internal promoter in the open reading frame of vasX that drives expression of the downstream gene tsiV2. Furthermore, VasX is shown to act in conjunction with VasW, an accessory protein to VasX, to compromise the inner membrane of prokaryotic target cells. The dual regulatory profile of the T6SS immunity protein-encoding genes tsiV1, tsiV2, and tsiV3 permits V. cholerae to tightly control T6SS gene expression while maintaining immunity to T6SS activity.

Type VI secretion systems (T6SS) enable bacteria to engage neighboring cells in contact-dependent competition. In Vibrio cholerae, three chromosomal clusters each encode a pair of effector and immunity genes downstream of those encoding the T6SS structural machinery for effector delivery. Different combinations of effector-immunity proteins lead to competition between strains of V. cholerae, which are thought to be protected only from the toxicity of their own effectors. Screening of all publically available V. cholerae genomes showed that numerous strains possess long arrays of orphan immunity genes encoded in the 3′ region of their T6SS clusters. Phylogenetic analysis reveals that these genes are highly similar to those found in the effector-immunity pairs of other strains, indicating acquisition by horizontal gene transfer. Extensive genomic comparisons also suggest that successive addition of effector-immunity gene pairs replaces ancestral effectors, yet retains the cognate immunity genes. The retention of old immunity genes perhaps provides protection against nearby kin bacteria in which the old effector was not replaced. This mechanism, combined with frequent homologous recombination, is likely responsible for the high diversity of T6SS effector-immunity gene profiles observed for V. cholerae and closely related species.

HIV-1 groups M and N emerged within the last century following two independent cross-species transmissions of SIVcpz from chimpanzees to humans. In contrast to pandemic group M strains, HIV-1 group N viruses are exceedingly rare, with only about a dozen infections identified, all but one in individuals from Cameroon. Poor adaptation to the human host may be responsible for this limited spread of HIV-1 group N in the human population. Here, we analyzed the function of Vpu proteins from seven group N strains from Cameroon, the place where this zoonosis originally emerged. We found that these N-Vpus acquired four amino acid substitutions (E15A, V19A and IV25/26LL) in their transmembrane domain (TMD) that allow efficient interaction with human tetherin. However, despite these adaptive changes, most N-Vpus still antagonize human tetherin only poorly and fail to down-modulate CD4, the natural killer (NK) cell ligand NTB-A as well as the lipid-antigen presenting protein CD1d. These functional deficiencies were mapped to amino acid changes in the cytoplasmic domain that disrupt putative adaptor protein binding sites and an otherwise highly conserved ßTrCP-binding DSGxxS motif. As a consequence, N-Vpus exhibited aberrant intracellular localization and/or failed to recruit the ubiquitin-ligase complex to induce tetherin degradation. The only exception was the Vpu of a group N strain recently discovered in France, but originally acquired in Togo, which contained intact cytoplasmic motifs and counteracted tetherin as effectively as the Vpus of pandemic HIV-1 M strains. These results indicate that HIV-1 group N Vpu is under strong host-specific selection pressure and that the acquisition of effective tetherin antagonism may lead to the emergence of viral variants with increased transmission fitness.

The type VI secretion system (T6SS) mediates protein translocation across the cell membrane of Gram-negative bacteria, including Vibrio cholerae – the causative agent of cholera. All V. cholerae strains examined to date harbor gene clusters encoding a T6SS. Structural similarity and sequence homology between components of the T6SS and the T4 bacteriophage cell-puncturing device suggest that the T6SS functions as a contractile molecular syringe to inject effector molecules into prokaryotic and eukaryotic target cells. Regulation of the T6SS is critical. A subset of V. cholerae strains, including the clinical O37 serogroup strain V52, express T6SS constitutively. In contrast, pandemic strains impose tight control that can be genetically disrupted: mutations in the quorum sensing gene luxO and the newly described regulator gene tsrA lead to constitutive T6SS expression in the El Tor strain C6706. In this report, we examined environmental V. cholerae isolates from the Rio Grande with regard to T6SS regulation. Rough V. cholerae lacking O-antigen carried a nonsense mutation in the gene encoding the global T6SS regulator VasH and did not display virulent behavior towards Escherichia coli and other environmental bacteria. In contrast, smooth V. cholerae strains engaged constitutively in type VI-mediated secretion and displayed virulence towards prokaryotes (E. coli and other environmental bacteria) and a eukaryote (the social amoeba Dictyostelium discoideum). Furthermore, smooth V. cholerae strains were able to outcompete each other in a T6SS-dependent manner. The work presented here suggests that constitutive T6SS expression provides V. cholerae with an advantage in intraspecific and interspecific competition.