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Quantifying transient interactions between Bacillus phosphatidylinositol-specific phospholipase-C and phosphatidylcholine-rich vesicles.

ABSTRACT: Bacillus thuringiensis secretes the virulence factor phosphatidylinositol-specific phospholipase C (BtPI-PLC), which specifically binds to phosphatidylcholine (PC) and cleaves GPI-anchored proteins off eukaryotic plasma membranes. To elucidate how BtPI-PLC searches for GPI-anchored proteins on the membrane surface, we measured residence times of single fluorescently labeled proteins on PC-rich small unilamellar vesicles (SUVs). BtPI-PLC interactions with the SUV surface are transient with a lifetime of 379 ± 49 ms. These data also suggest that BtPI-PLC does not directly sense curvature, but rather prefers to bind to the numerous lipid packing defects in SUVs. Despite this preference for defects, all-atom molecular dynamics simulations of BtPI-PLC interacting with PC-rich bilayers show that the protein is shallowly anchored with the deepest insertions ?18 Å above the bilayer center. Membrane partitioning is mediated, on average, by 41 hydrophobic, 8 hydrogen-bonding, and 2 cation-? (between PC choline headgroups and Tyr residues) transient interactions with phospholipids. These results lead to a quantitative model for BtPI-PLC interactions with cell membranes where protein binding is mediated by lipid packing defects, possibly near GPI-anchored proteins, and the protein diffuses on the membrane for ?100-380 ms, during which time it may cleave ?10 GPI-anchored proteins before dissociating. This combination of short two-dimensional scoots followed by three-dimensional hops may be an efficient search strategy on two-dimensional surfaces with obstacles.

SUBMITTER: Yang B

PROVIDER: S-EPMC4304437 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Quantifying transient interactions between Bacillus phosphatidylinositol-specific phospholipase-C and phosphatidylcholine-rich vesicles.

Yang Boqian B Pu Mingming M Khan Hanif M HM Friedman Larry L Reuter Nathalie N Roberts Mary F MF Gershenson Anne A

Journal of the American Chemical Society 20141223 1

Bacillus thuringiensis secretes the virulence factor phosphatidylinositol-specific phospholipase C (BtPI-PLC), which specifically binds to phosphatidylcholine (PC) and cleaves GPI-anchored proteins off eukaryotic plasma membranes. To elucidate how BtPI-PLC searches for GPI-anchored proteins on the membrane surface, we measured residence times of single fluorescently labeled proteins on PC-rich small unilamellar vesicles (SUVs). BtPI-PLC interactions with the SUV surface are transient with a life ...[more]

PMID: 25517221

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Project description:BackgroundVibrio anguillarum is the causative agent of vibriosis in fish. Several extracellular proteins secreted by V. anguillarum have been shown to contribute to virulence. While two hemolysin gene clusters, vah1-plp and rtxACHBDE, have been previously identified and described, the activities of the protein encoded by the plp gene were not known. Here we describe the biochemical activities of the plp-encoded protein and its role in pathogenesis.ResultsThe plp gene, one of the components in vah1 cluster, encodes a 416-amino-acid protein (Plp), which has homology to lipolytic enzymes containing the catalytic site amino acid signature SGNH. Hemolytic activity of the plp mutant increased 2-3-fold on sheep blood agar indicating that plp represses vah1; however, hemolytic activity of the plp mutant decreased by 2-3-fold on fish blood agar suggesting that Plp has different effects against erythrocytes from different species. His6-tagged recombinant Plp protein (rPlp) was over-expressed in E. coli. Purified and re-folded active rPlp exhibited phospholipase A2 activity against phosphatidylcholine and no activity against phosphatidylserine, phosphatidylethanolamine, or sphingomyelin. Characterization of rPlp revealed broad optimal activities at pH 5-9 and at temperatures of 30-64°C. Divalent cations and metal chelators did not affect activity of rPlp. We also demonstrated that Plp was secreted using thin layer chromatography and immunoblot analysis. Additionally, rPlp had strong hemolytic activity towards rainbow trout erythrocytes, but not to sheep erythrocytes suggesting that rPlp is optimized for lysis of phosphatidylcholine-rich fish erythrocytes. Further, only the loss of the plp gene had a significant effect on hemolytic activity of culture supernatant on fish erythrocytes, while the loss of rtxA and/or vah1 had little effect. However, V. anguillarum strains with mutations in plp or in plp and vah1 exhibited no significant reduction in virulence compared to the wild type strain when used to infect rainbow trout.ConclusionThe plp gene of V. anguillarum encoding a phospholipase with A2 activity is specific for phosphatidylcholine and, therefore, able to lyse fish erythrocytes, but not sheep erythrocytes. Mutation of plp does not affect the virulence of V. anguillarum in rainbow trout.

Dataset Information

Quantifying transient interactions between Bacillus phosphatidylinositol-specific phospholipase-C and phosphatidylcholine-rich vesicles.

Publications

Quantifying transient interactions between Bacillus phosphatidylinositol-specific phospholipase-C and phosphatidylcholine-rich vesicles.

Similar Datasets

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