Structures of the stator complex that drives rotation of the bacterial flagellum.

The bacterial flagellum is the prototypical protein nanomachine and comprises a rotating helical propeller attached to a membrane-embedded motor complex. The motor consists of a central rotor surrounded by stator units that couple ion flow across the cytoplasmic membrane to generate torque. Here, we present the structures of the stator complexes from Clostridium sporogenes, Bacillus subtilis and Vibrio mimicus, allowing interpretation of the extensive body of data on stator mechanism. The structures reveal an unexpected asymmetric A5B2 subunit assembly where the five A subunits enclose the two B subunits. Comparison to structures of other ion-driven motors indicates that this A5B2 architecture is fundamental to bacterial systems that couple energy from ion flow to generate mechanical work at a distance and suggests that such events involve rotation in the motor structures.

Novel Cholera Toxin Variant and ToxT Regulon in Environmental Vibrio mimicus Isolates: Potential Resources for the Evolution of Vibrio cholerae Hybrid Strains.

Atypical El Tor strains of Vibrio cholerae O1 harboring variant ctxB genes of cholera toxin (CT) have gradually become a major cause of recent cholera epidemics. Vibrio mimicus occasionally produces CT, encoded by ctxAB on CTXФ genome; toxin-coregulated pilus (TCP), a major intestinal colonization factor; and also the CTXФ-specific receptor. This study carried out extensive molecular characterization of CTXФ and ToxT regulon in V. mimicusctx-positive (ctx+) strains (i.e., V. mimicus strains containing ctx) isolated from the Bengal coast. Southern hybridization, PCR, and DNA sequencing of virulence-related genes revealed the presence of an El Tor type CTX prophage (CTXET) carrying a novel ctxAB, tandem copies of environmental type pre-CTX prophage (pre-CTXEnv), and RS1 elements, which were organized as an RS1-CTXET-RS1-pre-CTXEnv-pre-CTXEnv array. Additionally, novel variants of tcpA and toxT, respectively, showing phylogenetic lineage to a clade of V. cholerae non-O1 and to a clade of V. cholerae non-O139, were identified. The V. mimicus strains lacked the RTX (repeat in toxin) and TLC (toxin-linked cryptic) elements and lacked Vibrio seventh-pandemic islands of the El Tor strains but contained five heptamer (TTTTGAT) repeats in ctxAB promoter region similar to those seen with some classical strains of V. cholerae O1. Pulsed-field gel electrophoresis (PFGE) analysis showed that all the ctx+V. mimicus strains were clonally related. However, their in vitro CT production and in vivo toxigenicity characteristics were variable, which could be explainable by differential transcription of virulence genes along with the ToxR regulon. Taken together, our findings strongly suggest that environmental V. mimicus strains act as a potential reservoir of atypical virulence factors, including variant CT and ToxT regulons, and may contribute to the evolution of V. cholerae hybrid strains.IMPORTANCE Natural diversification of CTXФ and ctxAB genes certainly influences disease severity and shifting patterns in major etiological agents of cholera, e.g., the overwhelming emergence of hybrid El Tor variants, replacing the prototype El Tor strains of V. cholerae This report, showing the occurrence of CTXET comprising a novel variant of ctxAB in V. mimicus, points out a previously unnoticed evolutionary event that is independent of the evolutionary event associated with the El Tor strains of V. cholerae Identification and cluster analysis of the newly discovered alleles of tcpA and toxT suggest their horizontal transfer from an uncommon clone of V. cholerae The genomic contents of ToxT regulon and of tandemly arranged multiple pre-CTXФEnv and of a CTXФET in V. mimicus probably act as salient raw materials that induce natural recombination among the hallmark virulence genes of hybrid V. cholerae strains. This report provides valuable information to enrich our knowledge on the evolution of new variant CT and ToxT regulons.

Screening a Phage Display Library for Two Novel OmpU-Binding Peptides with Adhesion Antagonistic Activity against Vibrio mimicus.

Vibrio mimicus is a pathogen that causes ascites disease in fish. We have previously demonstrated that the outer membrane protein U (OmpU) is an important adhesin in V. mimicus. Here eight specific OmpU-binding phage clones, which presented three different OmpU-binding peptides (designated P1, P2, P3), were screened from a commercially available phage displayed 12-mer peptide library using rOmpU protein as target. Then, synthetic OmpU-binding peptides were measured for their adhesion antagonistic activity and binding affinity via adhesion inhibition test and non-competitive ELISA, respectively. The results showed that after co-incubated with the mixture of rOmpU and P3, visible green fluorescence could be observed on the epithelioma papulosum cyprinidi (EPC) cells surface; while the EPC cells co-incubated with the mixture of rOmpU and P1/P2 exhibited little green fluorescence. The average adhesion number of V. mimicus 04-14 isolate before and after treatment with peptide was 21.4 ± 1.5, 20.8 ± 0.8 (irrelevant peptide), 20.2 ± 0.5 (P3), 5.1 ± 0.7 (P1) and 3.4 ± 0.8 (P2), respectively. There was a significant decrease in the adhesive level of 04-14 isolate treated with P1/ P2 compared to the untreated isolate (p<0.01). The affinity constants of P1 and P2 were (6.17 ± 0.19) × 108 L/mol and (1.24 ± 0.56) × 109 L/mol, respectively. Furthermore, protective effects of P1 and P2 on grass carps challenged with V. mimicus were preliminary detected. It was found there was delayed death of fish in the groups treated with P1/P2, and the survival rate of challenged fish improved with the increase of the dose of adhesion antagonistic peptide. Taken together, two novel OmpU-binding peptides, which possessed adhesion antagonistic activity, high affinity and a certain degree of antibacterial activity against V. mimicus, were screened and identified.

Regulation of Vibrio mimicus metalloprotease (VMP) production by the quorum-sensing master regulatory protein, LuxR.

Vibrio mimicus is an estuarine bacterium, while it can cause severe diarrhea, wound infection, and otitis media in humans. This pathogen secretes a relatively important toxin named V. mimicus metalloprotease (VMP). In this study, we clarified regulation of the VMP production according to the quorum-sensing master regulatory protein named LuxR. First, the full length of luxR gene, encoding LuxR, was detected in V. mimicus strain E-37, an environmental isolate. Next, the putative consensus binding sequence of LuxR protein could be detected in the upstream (promoter) region of VMP encoding gene, vmp. Finally, the effect of disruption of luxR gene on the expression of vmp and production of VMP was evaluated. Namely, the expression of vmp was significantly diminished by luxR disruption and the production of VMP was severely altered. Taken together, here we report that VMP production is under the positive regulation of the quorum-sensing master regulatory protein, LuxR.

Genome sequencing reveals unique mutations in characteristic metabolic pathways and the transfer of virulence genes between V. mimicus and V. cholerae.

Vibrio mimicus, the species most similar to V. cholerae, is a microbe present in the natural environmental and sometimes causes diarrhea and internal infections in humans. It shows similar phenotypes to V. cholerae but differs in some biochemical characteristics. The molecular mechanisms underlying the differences in biochemical metabolism between V. mimicus and V. cholerae are currently unclear. Several V. mimicus isolates have been found that carry cholera toxin genes (ctxAB) and cause cholera-like diarrhea in humans. Here, the genome of the V. mimicus isolate SX-4, which carries an intact CTX element, was sequenced and annotated. Analysis of its genome, together with those of other Vibrio species, revealed extensive differences within the Vibrionaceae. Common mutations in gene clusters involved in three biochemical metabolism pathways that are used for discrimination between V. mimicus and V. cholerae were found in V. mimicus strains. We also constructed detailed genomic structures and evolution maps for the general types of genomic drift associated with pathogenic characters in polysaccharides, CTX elements and toxin co-regulated pilus (TCP) gene clusters. Overall, the whole-genome sequencing of the V. mimicus strain carrying the cholera toxin gene provides detailed information for understanding genomic differences among Vibrio spp. V. mimicus has a large number of diverse gene and nucleotide differences from its nearest neighbor, V. cholerae. The observed mutations in the characteristic metabolism pathways may indicate different adaptations to different niches for these species and may be caused by ancient events in evolution before the divergence of V. cholerae and V. mimicus. Horizontal transfers of virulence-related genes from an uncommon clone of V. cholerae, rather than the seventh pandemic strains, have generated the pathogenic V. mimicus strain carrying cholera toxin genes.

Presence of genes for type III secretion system 2 in Vibrio mimicus strains.

BACKGROUND: Vibrios, which include more than 100 species, are ubiquitous in marine and estuarine environments, and several of them e.g. Vibrio cholerae, V. parahaemolyticus, V. vulnificus and V. mimicus, are pathogens for humans. Pathogenic V. parahaemolyticus strains possess two sets of genes for type III secretion system (T3SS), T3SS1 and T3SS2. The latter are critical for virulence of the organism and be classified into two distinct phylogroups, T3SS2α and T3SS2ß, which are reportedly also found in pathogenic V. cholerae non-O1/non-O139 serogroup strains. However, whether T3SS2-related genes are present in other Vibrio species remains unclear. RESULTS: We therefore examined the distribution of the genes for T3SS2 in vibrios other than V. parahaemolyticus by using a PCR assay targeting both T3SS2α and T3SS2ß genes. Among the 32 Vibrio species tested in our study, several T3SS2-related genes were detected in three species, V. cholerae, V. mimicus and V. hollisae, and most of the essential genes for type III secretion were present in T3SS2-positive V. cholerae and V. mimicus strains. Moreover, both V. mimicus strains possessing T3SS2α and T3SS2ß were identified. The gene organization of the T3SS2 gene clusters in V. mimicus strains was fundamentally similar to that of V. parahaemolyticus and V. cholerae in both T3SS2α- and T3SS2ß-possessing strains. CONCLUSIONS: This study is the first reported evidence of the presence of T3SS2 gene clusters in V. mimicus strains. This finding thus provides a new insight into the pathogenicity of the V. mimicus species.

Modulation of Vibrio mimicus hemolysin through limited proteolysis by an endogenous metalloprotease.

Vibrio mimicus is a causative agent of human gastroenteritis and food poisoning, and this species produces an enterotoxic hemolysin (V. mimicus hemolysin) as a virulence determinant. Vibrio mimicus hemolysin is secreted as an 80 kDa precursor, which is later converted to the 66 kDa mature toxin through removal of an N-terminal propeptide via cleavage of the Arg151-Ser152 bond. In this article, we investigate the role of the endogenous metalloprotease (V. mimicus protease) in the maturation of V. mimicus hemolysin. In vitro experiments using purified proteins showed that, although it activated the precursor at the early stage via cleavage of the Asn157-Val158 bond, V. mimicus protease finally converted the activated and physiologically maturated toxin to a 51 kDa protein through removal of the C-terminal polypeptide. This 51 kDa derivative was unable to lyse erythrocytes because of its inability to bind to the erythrocyte membrane. Vibrio mimicus protease-negative strains were found to produce high levels of V. mimicus hemolysin at the logarithmic phase of bacterial growth and maintained high hemolytic activity even at the stationary phase. These findings indicate that, although it is not directly related to toxin maturation in vivo, V. mimicus protease can modulate the activity of V. mimicus hemolysin and/or its precursor through limited proteolysis.

Haemolysin produced by Vibrio mimicus activates two Cl- secretory pathways in cultured intestinal-like Caco-2 cells.

Haemolysin (VMH) is a virulent factor produced by Vibrio mimicus, a human pathogen that causes diarrhoea. As intestinal epithelial cells are the primary targets of haemolysin, we investigated its effects on ion transport in human colonic epithelial Caco-2 cells. VMH increased the cellular short circuit current (Isc), used to estimated ion fluxes, and 125I efflux of the cells. The VMH-induced increases in Isc and 125I efflux were suppressed by depleting Ca2+ from the medium or by pretreating the cells with BAPTA-AM or by Rp-adenosin 3',5'-cyclic monophosphorothioate triethylammonium salt (Rp-cAMPS). The Cl- channel inhibitors 4,4'-disothiocyanatostibene-2,2'-disulfonic acid (DIDS), glybenclamide, and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) suppressed the VMH-induced increases in Isc and 125I efflux. Moreover, VMH increased the intracellular concentrations of Ca2+ and cAMP. Thus, VMH stimulates Caco-2 cells to secrete Cl- by activating both Ca2+ -dependent and cAMP-dependent Cl- secretion mechanisms. VMH forms ion-permeable pores in the lipid bilayer that are non-selectively permeable to small ions. However, the ion permeability of these pores was not inhibited by glybenclamide and DIDS, and VMH did not change the cell membrane potential. These observations indicate that the pores formed on the cell membrane by VMH are unlikely to be involved in VMH-induced Cl- secretion. Notably, VMH stimulated fluid accumulation in the iliac loop test that was fully suppressed by a combination of DIDS and glybenclamide. Thus, Ca2+-dependent and cAMP-dependent Cl- secretion may be important therapeutic targets with regard to the diarrhoea that is induced by Vibrio mimicus.

Proteolytic activation of Vibrio mimicus (Vm) major outer membrane protein haemagglutinin (HA) with Vm-HA/protease: Implication for understanding bacterial adherence.

Vibrio mimicus (Vm) haemagglutinins (HAs), such as an extracellular HA/protease (Vm-HA/protease) and a major outer membrane protein-HA (Vm-OMPHA), have been recognized as the putative adherence factors for the bacterium. However, the mechanism by which HAs coordinate the adherence function of the bacterium remains as yet unknown. We report herein the positive interaction between Vm-HA/protease and Vm-OMPHA resulting in significant enhancement of the haemagglutinating ability. In this interaction, no cleaved polypeptide was detected; however, limited proteolysis of Vm-OMPHA was confirmed by SDS-PAGE. The proteolytic activation of the native cell-associated Vm-OMPHA by limited proteolysis was also demonstrated in several V. mimicus strains. Proteolytic activation of OMPHA was also achieved with various proteases from bacterial and eukaryotic sources. These findings may indicate a novel coordination of V. mimicus HAs in the adherence of the bacterium.

Presence of LuxS/AI-2 based quorum-sensing system in Vibrio mimicus : luxO controls protease activity.

Presence of the quorum-sensing regulation system in Vibrio mimicus was investigated. The culture supernatants of V. mimicus strains were found to possess AI-2 autoinducer like activity, and the strains were found to harbor the genes which are homologous to luxS, luxO, and luxR of V. harveyi. These genes of V. harveyi have been shown to be important components of V. harveyi-like quorum-sensing system. The luxO gene homologue known to encode LuxO, the central component of the regulation system, was disrupted, and effects on protease and hemolysin activity were studied. Disruption of luxO gene resulted in the increased protease activity, but the hemolysin activity did not vary considerably.

A hemolysin of Vibrio mimicus (VMH) stimulates cells to produce ATP and cyclic AMP which appear to be secretory mediators.

The hemolysin of Vibrio mimicus(VMH) is a pore-forming toxin with both enterotoxic and hemolytic activity. The hemolysis by VMH is induced by creation of pores in the membrane of erythrocyte; however, the mechanism for the enterotoxic action of VMH has remained unclear. In order to clarify the mechanism, we incubated T84 cells (a human colon carcinoma cell line) with VMH and found that the levels of ATP and cyclic AMP of culture medium increased after exposure of the cells to VMH. Subsequently, we found that the fluid accumulating activity of VMH in a mouse internal loop assay was reduced by administration of glibenclamide, an inhibitor of cyclic AMP-dependent chloride channels, into the intestinal loop. These results suggest that the stimulation of cells to produce nucleotides by VMH is linked to the enterotoxic activity of the toxin.

Identification and characterization of two contiguous operons required for aerobactin transport and biosynthesis in Vibrio mimicus.

In response to iron deprivation, Vibrio mimicus produces aerobactin as a major siderophore. Application of the Fur titration assay to a V. mimicus genomic DNA library followed by further cloning of the surrounding regions led to the identification of two adjacent, iron-regulated operons. One contains three genes encoding homologs of the Escherichia coli FhuCDB and the other, five genes encoding homologs of the E. coli IucABCD IutA. Construction of the V. mimicus polar disruptants in the respective operons allowed us to confirm their functions. The genetic arrangement of the aerobactin-mediated iron acquisition system in V. mimicus is unique in that the aerobactin operon (iucABCD iutA ) is contiguous to the operon (matCDB ) encoding components of an ATP-binding cassette transport system for ferric aerobactin. This is the first report demonstrating that aerobactin transport and biosynthesis genes are present in a species outside the family Enterobacteriaceae.