Search

Virstatin 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 virstatin-resistant 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 toxT(ENV) because of its occurrence in isolates of V. cholerae from the environment. We show that ToxT(ENV) 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.

Vibrio 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.

Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, has evolved signal transduction systems to control co-ordinately the expression of virulence determinants. It was previously shown that the presence of the bile salts glycocholate and taurocholate in the small intestine causes dimerization of the transmembrane transcription factor TcpP by inducing intermolecular disulphide bonds in the TcpP periplasmic domain. In this study, they further investigated the mechanism of how taurocholate affects V. cholerae virulence determinants. In vitro assay of TcpP oxidation by VcDsbA showed that VcDsbA induced TcpP dimerization in the presence of taurocholate. Taurocholate bound to VcDsbA with a KD of 40 ± 2.5 μM, and also bound other Dsb proteins, including EcDsbA, EcDsbC and VcDsbC. Taurocholate inhibited VcDsbA reductase activity without affecting VcDsbA secondary structure or thermostability. VcDsbA and its substrates were more extensively reduced in the presence of taurocholate, as compared with their redox state in the absence of taurocholate. The data presented here not only provide new insights into the mechanism by which bile salts induce V. cholerae virulence but also suggest a means by which to develop inhibitors against DsbA. [ABSTRACT FROM AUTHOR]

Type VI secretion is critical for V ibrio cholerae to successfully combat phagocytic eukaryotes and to survive in the presence of competing bacterial species. V . cholerae type VI secretion system genes are encoded in one large and two small clusters. In V . cholerae, type VI secretion is controlled by quorum sensing, the cell-cell communication process that enables bacteria to orchestrate group behaviours. The quorum-sensing response regulator LuxO represses type VI secretion genes at low cell density and the quorum-sensing regulator HapR activates type VI secretion genes at high cell density. We demonstrate that the quorum regulatory small RNAs ( Qrr sRNAs) that function between LuxO and HapR in the quorum-sensing cascade are required for these regulatory effects. The Qrr sRNAs control type VI secretion via two mechanisms: they repress expression of the large type VI secretion system cluster through base pairing and they repress HapR, the activator of the two small type VI secretion clusters. This regulatory arrangement ensures that the large cluster encoding many components of the secretory machine is expressed prior to the two small clusters that encode the secreted effectors. Qrr sRNA-dependent regulation of the type VI secretion system is conserved in pandemic and non-pandemic V . cholerae strains. [ABSTRACT FROM AUTHOR]

The multicomponent type VI secretion system ( T6SS) mediates the transport of effector proteins by puncturing target membranes. T6SSs are suggested to form a contractile nanomachine, functioning similar to the cell-puncturing device of tailed bacteriophages. The T6SS members VipA/ VipB form tubular complexes and are predicted to function in analogy to viral tail sheath proteins by providing the energy for secretion via contraction. The ATPase ClpV disassembles VipA/ VipB tubules in vitro, but the physiological relevance of tubule disintegration remained unclear. Here, we show that VipA/ VipB tubules localize near-perpendicular to the inner membrane of Vibrio cholerae cells and exhibit repetitive cycles of elongation, contraction and disassembly. VipA/ VipB tubules are decorated by ClpV in vivo and become static in Δ clpV cells, indicating that ClpV is required for tubule removal. VipA/ VipB tubules mislocalize in Δ clpV cells and exhibit a reduced frequency of tubule elongation, indicating that ClpV also suppresses the spontaneous formation of contracted, non-productive VipA/ VipB tubules. ClpV activity is restricted to the contracted state of VipA/ VipB, allowing formation of functional elongated tubules at a T6SS assembly. Targeting of an unrelated ATPase to VipA/ VipB is sufficient to replace ClpV function in vivo, suggesting that ClpV activity is autonomously regulated by VipA/ VipB conformation. [ABSTRACT FROM AUTHOR]

In Pseudomonas aeruginosa three type VI secretion systems ( T6SSs) coexist, called H1- to H3-T6SSs. Several T6SS components are proposed to be part of a macromolecular complex resembling the bacteriophage tail. The T6SS protein HsiE1 ( TagJ) is unique to the H1-T6SS and absent from the H2- and H3-T6SSs. We demonstrate that HsiE1 interacts with a predicted N-terminal α-helix in HsiB1 ( TssB) thus forming a novel subcomplex of the T6SS. HsiB1 is homologous to the Vibrio cholerae VipA component, which contributes to the formation of a bacteriophage tail sheath-like structure. We show that the interaction between HsiE1 and HsiB1 is specific and does not occur between HsiE1 and HsiB2. Proteins of the TssB family encoded in T6SS clusters lacking a gene encoding a TagJ-like component are often devoid of the predicted N-terminal helical region, which suggests co-evolution. We observe that a synthetic peptide corresponding to the N-terminal 20 amino acids of HsiB1 interacts with purified HsiE1 protein. This interaction is a common feature to other bacterial T6SSs that display a TagJ homologue as shown here with Serratia marcescens. We further show that hsiE1 is a non-essential gene for the T6SS and suggest that HsiE1 may modulate incorporation of HsiB1 into the T6SS. [ABSTRACT FROM AUTHOR]