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-O1͞non-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-O1͞non-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.Dictyostelium discoideum ͉ type VI secretion ͉ virulence-associated secretion C holera is a severe, life-threatening diarrheal disease caused by Vibrio cholerae strains of the O1 and O139 serogroups. In contrast, non-O1, non-O139 strains of V. cholerae are primarily associated with isolated cases of extra-intestinal infection or gastroenteritis. An exception to this pattern was a large outbreak of a cholera-like illness that occurred in 1968 in Sudan, where an O37 strain of V. cholerae caused 460 cases and 125 deaths (1). The virulence mechanisms of O1 and O139 strains involve the elaboration of extracellular factors such as cholera enterotoxin and toxin coregulated pili. In contrast, the virulence mechanisms used by non-O1, non-O139 strains remain poorly defined (2). Using the social amoeba Dictyostelium discoideum as a model host, we have developed an experimental system designed to identify novel virulence mechanisms from pathogenic non-O1, non-O139 strains.D. discoideum is a eukaryotic organism that seeks out and preys on bacteria through its phagocytic feeding behavior. As such, it has been used as a model eukaryotic cell that mimics a mammalian macrophage in aspects of its cell biology and interaction with microbes. Several environmental pathogenic bacteria, including Legionella pneumophila, Mycobacterium marinum, and Pseudomonas aeruginosa (3), resist Dictyostelium predation by producing factors that either kill amoebae or allow successful intracellular survival and multiplication. In these cases, the same virulence mechanisms operative against mammalian cells have also been implicated in resistance to Dictyosteli...

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 ''cellpuncturing device'' analogous to phage tail spikes to deliver effector protein domains through membranes of target host cells.bacteriophage ͉ cytotoxicity ͉ Vibrio cholerae ͉ virulence

Non-O1, non-O139 Vibrio cholerae can cause gastroenteritis and extraintestinal infections, but, unlike O1 and O139 strains of V. cholerae, little is known about the virulence gene content of non-O1, non-O139 strains and their phylogenetic relationship to other pathogenic V. cholerae. Comparative genomic microarray analysis of four pathogenic non-O1, non-O139 strains indicates that these strains are quite divergent from O1 and O139 strains. Genomic sequence analysis of a non-O1, non-O139 strain (AM-19226) that appeared particularly pathogenic in experimental animals suggests that this strain carries a type III secretion system (TTSS) that is related to the TTSS2 gene cluster found in a pandemic clone of Vibrio parahaemolyticus. The genes for this V. cholerae TTSS system appear to be present in many clinical and environmental non-O1, non-O139 strains, including at least one clone that is globally distributed. We hypothesize that the TTSS present in some pathogenic strains of non-O1, non-O139

SummaryVibrio cholerae is a Gram-negative bacterium that causes the acute diarrhoeal disease cholera. After the bacterium is ingested, it passes through the digestive tract, encountering various environmental stresses including the acidic milieu of the stomach and the toxic effects of bile in the duodenum. While these stresses serve as part of a host defence system, V. cholerae has evolved resistance mechanisms that allow it to evade these defences and establish infection. We examined the expression profiles of V. cholerae in response to bile or bile acids and found an induction of biofilm genes. We found that V. cholerae shows significantly enhanced biofilm formation in response to bile acids, and that bacteria within the biofilm are more resistant to the toxicity of bile acids compared with planktonic cells. Bile acid induction of biofilms was found to be dependent on the vps genes ( Vibrio polysaccharide synthesis) and their transcriptional activator VpsR, but VpsT is not required. These results contribute to the developing picture of a complex relationship between V. cholerae and its environment within the host during infection.

Inteins are intervening protein domains with self-splicing ability that can be used as molecular switches to control activity of their host protein. Successfully engineering an intein into a host protein requires identifying an insertion site that permits intein insertion and splicing while allowing for proper folding of the mature protein post-splicing. By analyzing sequence and structure based properties of native intein insertion sites we have identified four features that showed significant correlation with the location of the intein insertion sites, and therefore may be useful in predicting insertion sites in other proteins that provide native-like intein function. Three of these properties, the distance to the active site and dimer interface site, the SVM score of the splice site cassette, and the sequence conservation of the site showed statistically significant correlation and strong predictive power, with area under the curve (AUC) values of 0.79, 0.76, and 0.73 respectively, while the distance to secondary structure/loop junction showed significance but with less predictive power (AUC of 0.54). In a case study of 20 insertion sites in the XynB xylanase, two features of native insertion sites showed correlation with the splice sites and demonstrated predictive value in selecting non-native splice sites. Structural modeling of intein insertions at two sites highlighted the role that the insertion site location could play on the ability of the intein to modulate activity of the host protein. These findings can be used to enrich the selection of insertion sites capable of supporting intein splicing and hosting an intein switch.

SummaryVirstatin is a previously described small molecule inhibitor of Vibrio cholerae virulence. We have demonstrated that the molecule inhibits the activity of the transcriptional activator ToxT, thereby preventing elaboration of the toxin co-regulated pilus (TCP) and cholera toxin in vitro and in vivo in O1 strains of V. cholerae. While strains of the O1 and O139 serogroups are the cause of most epidemic and endemic cholera currently seen globally, sporadic disease caused by strains of non-O1/non-O139 serogroups suggests that understanding the pathogenic mechanisms of these unusual strains is relevant for disease. Although some non-O1/non-O139 strains have acquired the pathogenicity island that encodes the TCP, the role that this essential colonization factor of O1/O139 strains plays in the virulence of non-O1/ non-O139 strains has not been determined. In this study, we utilize virstatin in a 'chemical genetic approach' to examine the role of ToxT, and thus by inference TCP, in the colonization of a panel of predominantly non-O1/non-O139 tcp+ strains. We identified nine strains whose colonization was resistant to virstatin inhibition in the infant mouse model. These strains presumably colonize by a TCP-independent mechanism or contain a naturally occurring virstatinresistant ToxT. Four strains contained the typical toxT gene found in O1/O139 strains (toxT EPI) isolated from cholera epidemics. Interruption of toxT in one of these strains did not affect colonization of the infant mouse small intestine. The remaining five strains were found to contain a sequence divergent toxT gene that has been previously designated toxTENV because of its occurrence in isolates of V. cholerae from the environment. We show that ToxTENV is resistant to virstatin in two separate heterologous systems and is necessary for efficient colonization of the infant mouse small intestine. These results support the new concept that chemical genetic probes for the in vivo function or expression of virulence genes can be used to identify strains that express alternative virulence factors or novel regulatory systems that are functional in vivo.