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.

The type VI secretion system (T6SS) is recognized as an important virulence mechanism in several Gram-negative pathogens. In Vibrio cholerae, the causative agent of the diarrheal disease cholera, a minimum of three gene clusters-one main cluster and two auxiliary clusters-are required to form a functional T6SS apparatus capable of conferring virulence toward eukaryotic and prokaryotic hosts. Despite an increasing understanding of the components that make up the T6SS apparatus, little is known about the regulation of these genes and the gene products delivered by this nanomachine. VasH is an important regulator of the V. cholerae T6SS. Here, we present evidence that VasH regulates the production of a newly identified protein, VasX, which in turn requires a functional T6SS for secretion. Deletion of vasX does not affect export or enzymatic function of the structural T6SS proteins Hcp and VgrG-1, suggesting that VasX is dispensable for the assembly of the physical translocon complex. VasX localizes to the bacterial membrane and interacts with membrane lipids. We present VasX as a novel virulence factor of the T6SS, as a V. cholerae mutant lacking vasX exhibits a phenotype of attenuated virulence toward Dictyostelium discoideum.Vibrio cholerae is the marine bacterium responsible for the diarrheal disease cholera. Multiple cholera pandemics have been caused by the O1 serogroup of V. cholerae, and recently, O139 has emerged as a new pandemic strain (11). The principal virulence factors postulated to be essential for the pandemic spread of strains belonging to these serogroups are cholera toxin (CT) and the toxin-coregulated pilus (TCP) (13, 17). Non-O1 and non-O139 serogroups of V. cholerae are also capable of causing disease, in some cases in the absence of CT and TCP (10). In 1968, the O37 serogroup strain V52 was responsible for an outbreak of cholera-like diarrheal illness in Sudan, with 460 cases leading to 125 deaths (42). The genome of V52 carries both CT and TCP genes, but whether this strain produces CT and TCP in vivo has not yet been determined. V52 possesses a constitutively active type VI secretion system (T6SS) that confers virulence toward phagocytic cells, including the social amoeba Dictyostelium discoideum and murine macrophages (34). Pandemic strains of V. cholerae also possess the full complement of T6SS genes, but it is currently unclear how the T6SS is activated and how this system contributes to human disease.

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.

The Gram-negative bacterium Vibrio cholerae is the etiological agent of cholera, a disease characterized by the release of high volumes of watery diarrhea. Many medically important proteobacteria, including V. cholerae, carry one or multiple copies of the gene cluster that encodes the bacterial type VI secretion system (T6SS) to confer virulence or interspecies competitiveness. Structural similarity and sequence homology between components of the T6SS and the cell-puncturing device of T4 bacteriophage suggest that the T6SS functions as a molecular syringe to inject effector molecules into prokaryotic and eukaryotic target cells. Although our understanding of how the structural T6SS apparatus assembles is developing, little is known about how this system is regulated. Here, we report on the contribution of the activator of the alternative sigma factor 54, VasH, as a global regulator of the V. cholerae T6SS. Using bioinformatics and mutational analyses, we identified domains of the VasH polypeptide that are essential for its ability to initiate transcription of T6SS genes and established a universal role for VasH in endemic and pandemic V. cholerae strains.Vibrio cholerae, the etiological agent of the diarrheal disease cholera, remains a major health risk in developing countries, with approximately 120,000 deaths annually (49). The organism is classified into more than 200 serogroups based on its lipopolysaccharide O antigens, but only strains of the O1 serogroup have been associated with pandemics. The seventh, current pandemic O1 El Tor V. cholerae biotype (5) replaced the O1 classical biotype that was responsible for the sixth pandemic and most likely for the first five pandemics (4, 40). Since its appearance in southeastern India and the Bay of Bengal in 1992 (3), strains of the O139 serotype have spread to a large portion of the Asian subcontinent and are considered to have pandemic potential (17). O1 and O139 serogroup strains utilize two major virulence factors: toxin-coregulated pilus (TCP) and cholera toxin (CT). In contrast, many non-O1 and non-O139 strains of V. cholerae are capable of eliciting diarrheal or nonintestinal diseases in a CT/TCP-independent manner (33, 43). V. cholerae often employs accessory toxins such as hemolysin (HlyA) and actin cross-linking repeats in toxin (RtxA) (10). We believe that the type VI secretion system (T6SS) is one of the accessory factors used by V. cholerae to export effector molecules across its cell wall to confer cytotoxic effects on host cells.Three gene clusters-one large cluster (VCA0107 to VCA0124) and two auxiliary clusters (VCA0017 to VCA0022 and VC1415 to VC1416)-collectively encode the V. cholerae T6SS. Two copies of hcp and three copies of vgrG (vgrG1-3) gene are commonly found in T6SS gene clusters (29,36,42,46,50) and are believed to form the translocon conduit (35). Hcp is predicted to form the inner tube of the envelope translocon conduit which is decorated with a trimeric VgrG cap (20).Virulence of V. cholerae toward eukaryotic phagocytes, including D...

In cancer cells, the glycoprotein Mucin 1 (MUC1) undergoes abnormal, truncated glycosylation. The truncated glycosylation exposes cryptic peptide epitopes that can be recognized by antibodies. Since these immunogenic regions are cancer specific, they represent ideal targets for therapeutic antibodies. We investigated the role of tumor-specific glycosylation on antigen recognition by the therapeutic antibody AR20.5. We explored the affinity of AR20.5 to a synthetic cancer-specific MUC1 glycopeptide and peptide. The antibody bound to the glycopeptide with an order of magnitude stronger affinity than the naked peptide. Given these results, we postulated that AR20.5 must specifically bind the carbohydrate as well as the peptide. Using X-ray crystallography, we examined this hypothesis by determining the structure of AR20.5 in complex with both peptide and glycopeptide. Surprisingly, the structure revealed that the carbohydrate did not form any specific polar contacts with the antibody. The high affinity of AR20.5 for the glycopeptide and the lack of specific binding contacts support a hypothesis that glycosylation of MUC1 stabilizes an extended bioactive conformation of the peptide recognized by the antibody. Since high affinity binding of AR20.5 to the MUC1 glycopeptide may not driven by specific antibody-antigen contacts, but rather evidence suggests that glycosylation alters the conformational equilibrium of the antigen, which allows the antibody to select the correct conformation. This study suggests a novel mechanism of antibody-antigen interaction and also suggests that glycosylation of MUC1 is important for the generation of high affinity therapeutic antibodies.

causes listeriosis, a potentially fatal food-borne disease. The condition is especially harmful to pregnant women. outbreaks can originate from diverse foods, highlighting the need for novel strategies to improve food safety. The first step in invasion is internalization of the bacteria, which is mediated by the interaction of the internalin family of virulence factors with host cell receptors. A crucial interaction for invasion of the placenta, and thus a target for therapeutic intervention, is between internalin B (InlB) and the receptor c-Met. Single-domain antibodies (VH, also called nanobodies, or sdAbs) from camel heavy-chain antibodies are a novel solution for preventing infections. The VH R303, R330, and R326 all bind InlB with high affinity; however, the molecular mechanism behind their mode of action was unknown. We demonstrate that despite a high degree of sequence and structural diversity, the VH bind a single epitope on InlB. A combination of gentamicin protection assays and florescent microscopy establish that InlB-specific VH inhibit invasion of HeLa cells. A high-resolution X-ray structure of VH R303 in complex with InlB showed that the VH binds at the c-Met interaction site on InlB, thereby acting as a competitive inhibitor preventing bacterial invasion. These results point to the potential of VH as a novel class of therapeutics for the prevention of listeriosis.

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.