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 ß-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 FINDINGS: 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. CONCLUSION/SIGNIFICANCE: 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.
Subject(s)
Bacterial Proteins/toxicity , Cytotoxins/toxicity , Extracellular Vesicles/chemistry , Pore Forming Cytotoxic Proteins/toxicity , Vibrio cholerae/chemistry , Virulence Factors/toxicity , Animals , Bacterial Proteins/biosynthesis , Bacterial Proteins/isolation & purification , Cytotoxins/biosynthesis , Cytotoxins/isolation & purification , Erythrocytes/cytology , Erythrocytes/drug effects , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , HEK293 Cells , HeLa Cells , Humans , Pore Forming Cytotoxic Proteins/biosynthesis , Pore Forming Cytotoxic Proteins/isolation & purification , Rabbits , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/toxicity , Vibrio cholerae/metabolism , Vibrio cholerae/pathogenicity , Virulence Factors/biosynthesis , Virulence Factors/isolation & purificationABSTRACT
BACKGROUND: Analysis of the Escherichia coli collection of reference strains (ECOR) for the presence of the gene locus clyA, which encodes the pore-forming protein ClyA (cytolysin A), revealed that a non-functional clyA locus is common among certain extraintestinal pathogenic E. coli (ExPEC). In fact, all 15 ECOR group B2 strains and several additionally examined extraintestinal pathogenic (uropathogenic (UPEC) and neonatal meningitis (NBM)) E. coli strains contained various ΔclyA alleles. RESULTS: There are at least four different variants of ΔclyA, suggesting that such deletions in clyA have arisen at more than one occasion. On the basis of this occurrence of the truncated clyA genes, we considered that there may be a patho-adaptive selection for deletions in clyA in extraintestinal pathogenic E. coli. In E. coli K-12 the clyA gene has been viewed as "cryptic" since it is tightly silenced by the nucleoid structuring protein H-NS. We constructed a restored clyA+ locus in derivatives of the UPEC strain 536 for further investigation of this hypothesis and, in particular, how the gene would be expressed. Our results show that the level of clyA+ expression is highly increased in the UPEC derivatives in comparison with the non-pathogenic E. coli K-12. Transcription of the clyA+ gene was induced to even higher levels when the SfaX regulatory protein was overproduced. The derivative with a restored clyA+ locus displayed a somewhat slower growth than the parental UPEC strain 536 when a sub-inhibitory concentration of the antimicrobial peptide Polymyxin B was added to the growth medium. CONCLUSIONS: Taken together, our findings show that the clyA+ locus is expressed at an elevated level in the UPEC strain and we conclude that this is at least in part due to the effect of the SfaX/PapX transcriptional regulators.
