The bacterium Vibrio cholerae, like other human pathogens that reside in environmental reservoirs, survives predation by unicellular eukaryotes. Strains of the O1 and O139 serogroups cause cholera, ...whereas non-O1/non-O139 strains cause human infections through poorly defined mechanisms. Using Dictyostelium discoideum as a model host, we have identified a virulence mechanism in a non-O1/non-O139 V. cholerae strain that involves extracellular translocation of proteins that lack N-terminal hydrophobic leader sequences. Accordingly, we have named these genes "VAS" genes for virulence-associated secretion, and we propose that these genes encode a prototypic "type VI" secretion system. We show that vas genes are required for cytotoxicity of V. cholerae cells toward Dictyostelium amoebae and mammalian J774 macrophages by a contact-dependent mechanism. A large number of Gram-negative bacterial pathogens carry genes homologous to vas genes and potential effector proteins secreted by this pathway (i.e., hemolysin-coregulated protein and VgrG). Mutations in vas homologs in other bacterial species have been reported to attenuate virulence in animals and cultured macrophages. Thus, the genes encoding the VAS-related, type VI secretion system likely play an important conserved function in microbial pathogenesis and represent an additional class of targets for vaccine and antimicrobial drug-based therapies.
The alternative sigma factor RpoN is an essential colonization factor of Vibrio cholerae and controls important cellular functions including motility and type VI secretion (T6SS). The RpoN regulon ...has yet to be clearly defined in T6SS-active V. cholerae isolates, which use T6SS to target both bacterial competitors and eukaryotic cells. We hypothesize that T6SS-dependent secreted effectors are co-regulated by RpoN. To systemically identify RpoN-controlled genes, we used chromatin immunoprecipitation coupled with sequencing (ChIP-Seq) and transcriptome analysis (RNA-Seq) to determine RpoN-binding sites and RpoN-controlled gene expression. There were 68 RpoN-binding sites and 82 operons positively controlled by RpoN, among which 37 operons had ChIP-identified binding sites. A consensus RpoN-binding motif was identified with a highly conserved thymine (-14) and an AT-rich region in the middle between the hallmark RpoN-recognized motif GG(-24)/GC(-12). There were seven new RpoN-dependent promoters in the flagellar regions. We identified a small RNA, flaX, downstream of the major flagellin gene flaA. Mutation of flaX substantially reduced motility. In contrast to previous results, we report that RpoN positively regulates the expression of hcp operons and vgrG3 that encode T6SS secreted proteins but has no effect on the expression of the main T6SS cluster encoding sheath and other structural components.
Genes encoding type VI secretion systems (T6SS) are widely distributed in pathogenic Gram-negative bacterial species. In Vibrio cholerae, T6SS have been found to secrete three related proteins ...extracellularly, VgrG-1, VgrG-2, and VgrG-3. VgrG-1 can covalently cross-link actin in vitro, and this activity was used to demonstrate that V. cholerae can translocate VgrG-1 into macrophages by a T6SS-dependent mechanism. Protein structure search algorithms predict that VgrG-related proteins likely assemble into a trimeric complex that is analogous to that formed by the two trimeric proteins gp27 and gp5 that make up the baseplate "tail spike" of Escherichia coli bacteriophage T4. VgrG-1 was shown to interact with itself, VgrG-2, and VgrG-3, suggesting that such a complex does form. Because the phage tail spike protein complex acts as a membrane-penetrating structure as well as a conduit for the passage of DNA into phage-infected cells, we propose that the VgrG components of the T6SS apparatus may assemble a "cell-puncturing device" analogous to phage tail spikes to deliver effector protein domains through membranes of target host cells.
Vibrio cholerae is the causative agent of the diarrheal disease cholera. Although many V. cholerae virulence factors have been studied, the role of interbacterial interactions within the host gut and ...their influence on colonization are poorly understood. Here, we utilized the conjugative properties of a Vibrio-specific plasmid to serve as a quantifiable genetic marker for direct contact among V. cholerae cells in the infant rabbit model for cholera. In conjunction, we also quantified contact-dependent type 6 secretion system (T6SS)-mediated killing of co-infecting V. cholerae strains. Tracking these interbacterial interactions revealed that most contact-dependent cell-cell interactions among V. cholerae occur in specific intestinal microenvironments, notably the distal small intestine and cecum, and that the T6SS confers a competitive advantage within the middle small intestine. These results support a model for V. cholerae gut colonization, which includes microenvironments where critical microbial-host and bacterial-bacterial interactions occur to facilitate colonization by this pathogen.
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•DNA conjugation can be used to track bacterial cell-cell contacts during infection•Sites of in vivo cell-cell contact correspond to sites of T6SS-mediated killing•V. cholerae T6SS contributes to colonization of the middle small intestine•DNA conjugation among V. cholerae occurs more efficiently in vivo than in vitro
To examine V. cholerae bacterial population structure within the host intestine, Fu et al. use DNA conjugation as a genetic reporter for bacterial cell-cell contacts along with measurements of antagonistic T6SS-mediated interactions. These observations reveal that contact-dependent cell-cell interactions and the T6SS play specific roles in distinct intestinal microenvironments.
Cyclic dinucleotides (CDNs) have central roles in bacterial homeostasis and virulence by acting as nucleotide second messengers. Bacterial CDNs also elicit immune responses during infection when they ...are detected by pattern-recognition receptors in animal cells. Here we perform a systematic biochemical screen for bacterial signalling nucleotides and discover a large family of cGAS/DncV-like nucleotidyltransferases (CD-NTases) that use both purine and pyrimidine nucleotides to synthesize a diverse range of CDNs. A series of crystal structures establish CD-NTases as a structurally conserved family and reveal key contacts in the enzyme active-site lid that direct purine or pyrimidine selection. CD-NTase products are not restricted to CDNs and also include an unexpected class of cyclic trinucleotide compounds. Biochemical and cellular analyses of CD-NTase signalling nucleotides demonstrate that these cyclic di- and trinucleotides activate distinct host receptors and thus may modulate the interaction of both pathogens and commensal microbiota with their animal and plant hosts.
Gram-negative bacteria use the type VI secretion system (T6SS) to translocate toxic effector proteins into adjacent cells. The Pseudomonos aeruginosa H1-locus T6SS assembles in response to exogenous ...T6SS attack by other bacteria. We found that this lethal T6SS counterattack also occurs in response to the mating pair formation (Mpf) system encoded by broad-host-range IncPα conjugative plasmid RP4 present in adjacent donor cells. This T6SS response was eliminated by disruption of Mpf structural genes but not components required only for DNA transfer. Because T6SS activity was also strongly induced by membrane-disrupting natural product polymyxin B, we conclude that RP4 induces "donor-directed T6SS attacks" at sites corresponding to Mpf-mediated membrane perturbations in recipient P. aeruginosa cells to potentially block acquisition of parasitic foreign DNA.
Protein secretion is a common property of pathogenic microbes. Gram-negative bacterial pathogens use at least 6 distinct extracellular protein secretion systems to export proteins through their ...multilayered cell envelope and in some cases into host cells. Among the most widespread is the newly recognized Type VI secretion system (T6SS) which is composed of 15-20 proteins whose biochemical functions are not well understood. Using crystallographic, biochemical, and bioinformatic analyses, we identified 3 T6SS components, which are homologous to bacteriophage tail proteins. These include the tail tube protein; the membrane-penetrating needle, situated at the distal end of the tube; and another protein associated with the needle and tube. We propose that T6SS is a multicomponent structure whose extracellular part resembles both structurally and functionally a bacteriophage tail, an efficient machine that translocates proteins and DNA across lipid membranes into cells.
Vibrio cholerae is the causative agent of the diarrheal disease cholera. By an incompletely understood developmental process,
V. cholerae forms complex surface-associated communities called biofilms. ...Here we show that quorum sensing-deficient mutants of
V. cholerae produce thicker biofilms than those formed by wild-type bacteria. Microarray analysis of biofilm-associated bacteria shows that expression of the
Vibrio polysaccharide synthesis (
vps) operons is enhanced in
hapR mutants. CqsA, one of two known autoinducer synthases in
V. cholerae, acts through HapR to repress
vps gene expression.
Vibrio biofilms are more acid resistant than planktonic cells. However, quorum sensing-deficient biofilms have lower colonization capacities than those of wild-type biofilms, suggesting that quorum sensing may promote cellular exit from the biofilm once the organisms have traversed the gastric acid barrier of the stomach. These results shed light on the relationships among biofilm development, quorum sensing, infectivity, and pathogenesis in
V. cholerae.
The Vibrio cholerae lipopolysaccharide O1 antigen is a major target of bacteriophages and the human immune system and is of critical importance for vaccine design. We used an O1-specific lytic ...bacteriophage as a tool to probe the capacity of V. cholerae to alter its O1 antigen and identified a novel mechanism by which this organism can modulate O antigen expression and exhibit intra-strain heterogeneity. We identified two phase variable genes required for O1 antigen biosynthesis, manA and wbeL. manA resides outside of the previously recognized O1 antigen biosynthetic locus, and encodes for a phosphomannose isomerase critical for the initial step in O1 antigen biosynthesis. We determined that manA and wbeL phase variants are attenuated for virulence, providing functional evidence to further support the critical role of the O1 antigen for infectivity. We provide the first report of phase variation modulating O1 antigen expression in V. cholerae, and show that the maintenance of these phase variable loci is an important means by which this facultative pathogen can generate the diverse subpopulations of cells needed for infecting the host intestinal tract and for escaping predation by an O1-specific phage.
The bacterial type VI secretion system (T6SS) is a large multicomponent, dynamic macromolecular machine that has an important role in the ecology of many Gram-negative bacteria. T6SS is responsible ...for translocation of a wide range of toxic effector molecules, allowing predatory cells to kill both prokaryotic as well as eukaryotic prey cells. The T6SS organelle is functionally analogous to contractile tails of bacteriophages and is thought to attack cells by initially penetrating them with a trimeric protein complex called the VgrG spike. Neither the exact protein composition of the T6SS organelle nor the mechanisms of effector selection and delivery are known. Here we report that proteins from the PAAR (proline-alanine-alanine-arginine) repeat superfamily form a sharp conical extension on the VgrG spike, which is further involved in attaching effector domains to the spike. The crystal structures of two PAAR-repeat proteins bound to VgrG-like partners show that these proteins sharpen the tip of the T6SS spike complex. We demonstrate that PAAR proteins are essential for T6SS-mediated secretion and target cell killing by Vibrio cholerae and Acinetobacter baylyi. Our results indicate a new model of the T6SS organelle in which the VgrG-PAAR spike complex is decorated with multiple effectors that are delivered simultaneously into target cells in a single contraction-driven translocation event.
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