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Crystal structure of the Vibrio cholerae cytolysin heptamer reveals common features among disparate pore-forming toxins.

ABSTRACT: Pore-forming toxins (PFTs) are potent cytolytic agents secreted by pathogenic bacteria that protect microbes against the cell-mediated immune system (by targeting phagocytic cells), disrupt epithelial barriers, and liberate materials necessary to sustain growth and colonization. Produced by gram-positive and gram-negative bacteria alike, PFTs are released as water-soluble monomeric or dimeric species, bind specifically to target membranes, and assemble transmembrane channels leading to cell damage and/or lysis. Structural and biophysical analyses of individual steps in the assembly pathway are essential to fully understanding the dynamic process of channel formation. To work toward this goal, we solved by X-ray diffraction the 2.9-Å structure of the 450-kDa heptameric Vibrio cholerae cytolysin (VCC) toxin purified and crystallized in the presence of detergent. This structure, together with our previously determined 2.3-Å structure of the VCC water-soluble monomer, reveals in detail the architectural changes that occur within the channel region and accessory lectin domains during pore formation including substantial rearrangements of hydrogen-bonding networks in the pore-forming amphipathic loops. Interestingly, a ring of tryptophan residues forms the narrowest constriction in the transmembrane channel reminiscent of the phenylalanine clamp identified in anthrax protective antigen [Krantz BA, et al. (2005) Science 309:777-781]. Our work provides an example of a ?-barrel PFT (?-PFT) for which soluble and assembled structures are available at high-resolution, providing a template for investigating intermediate steps in assembly.

SUBMITTER: De S

PROVIDER: S-EPMC3088620 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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Crystal structure of the Vibrio cholerae cytolysin heptamer reveals common features among disparate pore-forming toxins.

De Swastik S Olson Rich R

Proceedings of the National Academy of Sciences of the United States of America 20110418 18

Pore-forming toxins (PFTs) are potent cytolytic agents secreted by pathogenic bacteria that protect microbes against the cell-mediated immune system (by targeting phagocytic cells), disrupt epithelial barriers, and liberate materials necessary to sustain growth and colonization. Produced by gram-positive and gram-negative bacteria alike, PFTs are released as water-soluble monomeric or dimeric species, bind specifically to target membranes, and assemble transmembrane channels leading to cell dama ...[more]

PMID: 21502531

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Project description:Alpha-pore-forming toxins (α-PFTs) are secreted by many species of bacteria, including Escherichia coli, Aeromonas hydrophila, and Bacillus thuringiensis, as part of their arsenal of virulence factors, and are often cytotoxic. In particular, for α-PFTs, the membrane-spanning channel they form is composed of hydrophobic α-helices. These toxins oligomerize at the surface of target cells and transition from a soluble to a protomer state in which they expose their hydrophobic regions and insert into the membrane to form a pore. The pores may be composed of homooligomers of one component or heterooligomers with two or three components, resulting in bi- or tripartite toxins. The multicomponent α-PFTs are often expressed from a single operon. Recently, motility-associated killing factor A (MakA), an α-PFT, was discovered in Vibrio cholerae. We report that makA is found on the V. cholerae GI-10 genomic island within an operon containing genes for two other potential α-PFTs, MakB and MakE. We determined the X-ray crystal structures for MakA, MakB, and MakE and demonstrated that all three are structurally related to the α-PFT family in the soluble state, and we modeled their protomer state based on the α-PFT AhlB from A. hydrophila. We found that MakA alone is cytotoxic at micromolar concentrations. However, combining MakA with MakB and MakE is cytotoxic at nanomolar concentrations, with specificity for J774 macrophage cells. Our data suggest that MakA, -B, and -E are α-PFTs that potentially act as a tripartite pore-forming toxin with specificity for phagocytic cells. IMPORTANCE The bacterium Vibrio cholerae causes gastrointestinal, wound, and skin infections. The motility-associated killing factor A (MakA) was recently shown to be cytotoxic against colon, prostate, and other cancer cells. However, at the outset of this study, the capacity of MakA to damage cells in combination with other Mak proteins encoded in the same operon had not been elucidated. We determined the structures of three Mak proteins and established that they are structurally related to the α-PFTs. Compared to MakA alone, the combination of all three toxins was more potent specifically in mouse macrophages. This study highlights the idea that the Mak toxins are selectively cytotoxic and thus may function as a tripartite toxin with cell type specificity.

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

Crystal structure of the Vibrio cholerae cytolysin heptamer reveals common features among disparate pore-forming toxins.

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Crystal structure of the Vibrio cholerae cytolysin heptamer reveals common features among disparate pore-forming toxins.

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