Abstract
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.
Highlights
Vibrio cholerae is considered a deadly pathogen that still poses a major health threat in developing and underdeveloped regions of the world [1]
These results indicated that V. cholerae cytolysin (VCC) is a secreted protein that can localize in the periplasm during export to the extracellular space
Our results suggest that the presence of VCC in the Outer membrane vesicles (OMVs) from the wild type V. cholerae strain V:5/04 occurred as a result of localization during OMV formation and was not due to the binding of VCC on the surface of the OMVs or coprecipitation of VCC during the vesicle isolation procedure
Summary
Vibrio cholerae is considered a deadly pathogen that still poses a major health threat in developing and underdeveloped regions of the world [1]. V. cholerae pathogenesis has been primarily attributed to expression of two major virulence factors, cholera toxin (CT) and the toxin-coregulated pilus (TCP) [2]. These major virulence factors are expressed by only two serogroups of V. cholerae, namely O1 and O139, which are the only serogroups known to cause epidemic cholera outbreaks [3]. O139 (NOVC) serogroups, lack CT and TCP and are considered non-pathogenic [4,5]. Recent decades have seen an upsurge in sporadic outbreaks of non-cholera-causing V. cholerae infections involving NOVC strains [6]. Vibrio cholerae produce OMVs but their putative role in translocation of effectors involved in pathogenesis has not been well elucidated. The mechanisms involved in the secretion and delivery of the VCC have not been extensively studied
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