title: Outer membrane vesicles mediate transport of biologically active Vibrio cholerae cytolysin (VCC) from V. cholerae strains.
abstract: BACKGROUND
  Outer 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.
  
  
  RESULTS
  OMVs 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.
  
  
  CONCLUSIONS
  Biological 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.
authors.affiliation: Department of Molecular Biology, The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, Umeå, Sweden; Division of Pathophysiology, National Institute of Cholera and Enteric Diseases, Kolkata, West Bengal, India.
authors.forename: Sridhar
authors.initials: S
authors.lastname: Elluri
authors.affiliation: Department of Molecular Biology, The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, Umeå, Sweden.
authors.forename: Constance
authors.initials: C
authors.lastname: Enow
authors.affiliation: Department of Molecular Biology, The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, Umeå, Sweden.
authors.forename: Svitlana
authors.initials: S
authors.lastname: Vdovikova
authors.affiliation: Department of Molecular Biology, The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, Umeå, Sweden.
authors.forename: Pramod K
authors.initials: PK
authors.lastname: Rompikuntal
authors.affiliation: Department of Molecular Biology, The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, Umeå, Sweden.
authors.forename: Mitesh
authors.initials: M
authors.lastname: Dongre
authors.affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden.
authors.forename: Sven
authors.initials: S
authors.lastname: Carlsson
authors.affiliation: Division of Pathophysiology, National Institute of Cholera and Enteric Diseases, Kolkata, West Bengal, India.
authors.forename: Amit
authors.initials: A
authors.lastname: Pal
authors.affiliation: Department of Molecular Biology, The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, Umeå, Sweden.
authors.forename: Bernt Eric
authors.initials: BE
authors.lastname: Uhlin
authors.affiliation: Department of Molecular Biology, The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, Umeå, Sweden.
authors.forename: Sun Nyunt
authors.initials: SN
authors.lastname: Wai
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000633:toxicity*; D003603:Cytotoxins / Q000096:biosynthesis / Q000302:isolation & purification / Q000633:toxicity*; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D000067128:Extracellular Vesicles / Q000737:chemistry*; D015870:Gene Expression; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D052899:Pore Forming Cytotoxic Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000633:toxicity*; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000633:toxicity; D014734:Vibrio cholerae / Q000737:chemistry* / Q000378:metabolism / Q000472:pathogenicity; D037521:Virulence Factors / Q000096:biosynthesis / Q000302:isolation & purification / Q000633:toxicity*
chemical_list: D001426:Bacterial Proteins; D003603:Cytotoxins; D052899:Pore Forming Cytotoxic Proteins; D011994:Recombinant Proteins; D037521:Virulence Factors