Dataset Information

ATP-Binding Cassette Transporter VcaM from Vibrio cholerae is Dependent on the Outer Membrane Factor Family for Its Function.

ABSTRACT: Vibrio cholerae ATP-binding cassette transporter VcaM (V. cholerae ABC multidrug resistance pump) has previously been shown to confer resistance to a variety of medically important drugs. In this study, we set to analyse its properties both in vitro in detergent-solubilised state and in vivo to differentiate its dependency on auxiliary proteins for its function. We report the first detailed kinetic parameters of purified VcaM and the rate of phosphate (Pi) production. To determine the possible functional dependencies of VcaM on the tripartite efflux pumps we then utilized different E. coli strains lacking the principal secondary transporter AcrB (Acriflavine resistance protein), as well as cells lacking the outer membrane factor (OMF) TolC (Tolerance to colicins). Consistent with the ATPase function of VcaM we found it to be susceptible to sodium orthovanadate (NaOV), however, we also found a clear dependency of VcaM function on TolC. Inhibitors targeting secondary active transporters had no effects on either VcaM-conferred resistance or Hoechst 33342 accumulation, suggesting that VcaM might be capable of engaging with the TolC-channel without periplasmic mediation by additional transporters. Our findings are indicative of VcaM being capable of a one-step substrate translocation from cytosol to extracellular space utilising the TolC-channel, making it the only multidrug ABC-transporter outside of the MacB-family with demonstrable TolC-dependency.

SUBMITTER: Lu WJ

PROVIDER: S-EPMC5979437 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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ATP-Binding Cassette Transporter VcaM from Vibrio cholerae is Dependent on the Outer Membrane Factor Family for Its Function.

Lu Wen-Jung WJ Lin Hsuan-Ju HJ Janganan Thamarai K TK Li Cheng-Yi CY Chin Wei-Chiang WC Bavro Vassiliy N VN Lin Hong-Ting Victor HV

International journal of molecular sciences 20180327 4

<i>Vibrio cholerae</i> ATP-binding cassette transporter VcaM (<i>V. cholerae</i> ABC multidrug resistance pump) has previously been shown to confer resistance to a variety of medically important drugs. In this study, we set to analyse its properties both in vitro in detergent-solubilised state and in vivo to differentiate its dependency on auxiliary proteins for its function. We report the first detailed kinetic parameters of purified VcaM and the rate of phosphate (Pi) production. To determine ...[more]

PMID: 29584668

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Project description:Biofilms are matrix-encased microbial communities that increase the environmental fitness and infectivity of many human pathogens including Vibrio cholerae. Biofilm matrix assembly is essential for biofilm formation and function. Known components of the V. cholerae biofilm matrix are the polysaccharide Vibrio polysaccharide (VPS), matrix proteins RbmA, RbmC, Bap1, and extracellular DNA, but the majority of the protein composition is uncharacterized. This study comprehensively analyzed the biofilm matrix proteome and revealed the presence of outer membrane proteins (OMPs). Outer membrane vesicles (OMVs) were also present in the V. cholerae biofilm matrix and were associated with OMPs and many biofilm matrix proteins suggesting that they participate in biofilm matrix assembly. Consistent with this, OMVs had the capability to alter biofilm structural properties depending on their composition. OmpU was the most prevalent OMP in the matrix, and its absence altered biofilm architecture by increasing VPS production. Single-cell force spectroscopy revealed that proteins critical for biofilm formation, OmpU, the matrix proteins RbmA, RbmC, Bap1, and VPS contribute to cell-surface adhesion forces at differing efficiency, with VPS showing the highest efficiency whereas Bap1 showing the lowest efficiency. Our findings provide new insights into the molecular mechanisms underlying biofilm matrix assembly in V. cholerae, which may provide new opportunities to develop inhibitors that specifically alter biofilm matrix properties and, thus, affect either the environmental survival or pathogenesis of V. cholerae.IMPORTANCECholera remains a major public health concern. Vibrio cholerae, the causative agent of cholera, forms biofilms, which are critical for its transmission, infectivity, and environmental persistence. While we know that the V. cholerae biofilm matrix contains exopolysaccharide, matrix proteins, and extracellular DNA, we do not have a comprehensive understanding of the majority of biofilm matrix components. Here, we discover outer membrane vesicles (OMVs) within the biofilm matrix of V. cholerae. Proteomic analysis of the matrix and matrix-associated OMVs showed that OMVs carry key matrix proteins and Vibrio polysaccharide (VPS) to help build biofilms. We also characterize the role of the highly abundant outer membrane protein OmpU in biofilm formation and show that it impacts biofilm architecture in a VPS-dependent manner. Understanding V. cholerae biofilm formation is important for developing a better prevention and treatment strategy framework.

Project description:BackgroundOuter membrane vesicles (OMVs) released from Gram-negative bacteria can serve as vehicles for the translocation of virulence factors. Vibrio cholerae produce OMVs but their putative role in translocation of effectors involved in pathogenesis has not been well elucidated. The V. cholerae cytolysin (VCC), is a pore-forming toxin that lyses target eukaryotic cells by forming transmembrane oligomeric β-barrel channels. It is considered a potent toxin that contributes to V. cholerae pathogenesis. The mechanisms involved in the secretion and delivery of the VCC have not been extensively studied.Methodology/principal findingsOMVs from V. cholerae strains were isolated and purified using a differential centrifugation procedure and Optiprep centrifugation. The ultrastructure and the contents of OMVs were examined under the electron microscope and by immunoblot analyses respectively. We demonstrated that VCC from V. cholerae strain V:5/04 was secreted in association with OMVs and the release of VCC via OMVs is a common feature among V. cholerae strains. The biological activity of OMV-associated VCC was investigated using contact hemolytic assay and epithelial cell cytotoxicity test. It showed toxic activity on both red blood cells and epithelial cells. Our results indicate that the OMVs architecture might play a role in stability of VCC and thereby can enhance its biological activities in comparison with the free secreted VCC. Furthermore, we tested the role of OMV-associated VCC in host cell autophagy signalling using confocal microscopy and immunoblot analysis. We observed that OMV-associated VCC triggered an autophagy response in the target cell and our findings demonstrated for the first time that autophagy may operate as a cellular defence mechanism against an OMV-associated bacterial virulence factor.Conclusion/significanceBiological assays of OMVs from the V. cholerae strain V:5/04 demonstrated that OMV-associated VCC is indeed biologically active and induces toxicity on mammalian cells and furthermore can induce autophagy.

Dataset Information

ATP-Binding Cassette Transporter VcaM from Vibrio cholerae is Dependent on the Outer Membrane Factor Family for Its Function.

Publications

ATP-Binding Cassette Transporter VcaM from Vibrio cholerae is Dependent on the Outer Membrane Factor Family for Its Function.

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