scholarly journals Beticolins, Nonpeptidic, Polycyclic Molecules Produced by the Phytopathogenic Fungus Cercospora beticola, as a New Family of Ion Channel-Forming Toxins

2000 ◽  
Vol 13 (2) ◽  
pp. 203-209 ◽  
Author(s):  
Cyril Goudet ◽  
Marie-Louise Milat ◽  
Hervé Sentenac ◽  
Jean-Baptiste Thibaud
Keyword(s):  
Lipid Bilayers ◽  
Pore Formation ◽  
Phytopathogenic Fungus ◽  
Cercospora Beticola ◽  
Leaf Spot Disease ◽  
Channel Formation ◽  
Channel Conductance ◽  
New Family ◽  
Conductance States ◽  
Multiple Conductance States

Beticolins are toxins produced by Cercospora beticola, a phytopathogenic fungus responsible for the leaf spot disease of sugar beet. They form a family of 20 nonpeptidic compounds (named B0 to B19) that share the same polycyclic skeleton but differ by isomeric configuration (ortho- or para-) and by a variable residue R (bridging two carbons in one of the six cycles). It has been previously shown that B0 assembles itself into a multimeric structure and forms ion channels into planar lipid bilayers (C. Goudet, A.-A. Véry, M.-L. Milat, M. Ildefonse, J.-B. Thibaud, H.Sentenac, and J.-P. Blein, Plant J. 14:359-364, 1998). In the present work, we investigate pore formation by three ortho-beticolins, B0, B2, and B4, and their related (i.e., same R) para-isomers, B13, B1, and B3, respectively, using planarlipid bilayers. All beticolins were able to form ionchannels with multiple conductance states, although the type of cyclization (ortho- or para-) and residue (R) result in variations of channel conductance and ionic permeability, respectively. Channel formation by beticolins is likely to be involved in the biological activity of these toxins.

scholarly journals Structural and Functional Analysis of the Pore-Forming Toxin NetB from Clostridium perfringens

mBio ◽  
2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Xu-Xia Yan ◽  
Corrine J. Porter ◽  
Simon P. Hardy ◽  
David Steer ◽  
A. Ian Smith ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Clostridium Perfringens ◽  
Pore Formation ◽  
Random Mutagenesis ◽  
Planar Lipid Bilayers ◽  
Necrotic Enteritis ◽  
Channel Conductance ◽  
Content Type ◽  
Alpha Hemolysin ◽  
Insight Into

ABSTRACT Clostridium perfringens is an anaerobic bacterium that causes numerous important human and animal diseases, primarily as a result of its ability to produce many different protein toxins. In chickens, C. perfringens causes necrotic enteritis, a disease of economic importance to the worldwide poultry industry. The secreted pore-forming toxin NetB is a key virulence factor in the pathogenesis of avian necrotic enteritis and is similar to alpha-hemolysin, a β-barrel pore-forming toxin from Staphylococcus aureus. To address the molecular mechanisms underlying NetB-mediated tissue damage, we determined the crystal structure of the monomeric form of NetB to 1.8 Å. Structural comparisons with other members of the alpha-hemolysin family revealed significant differences in the conformation of the membrane binding domain. These data suggested that NetB may recognize different membrane receptors or use a different mechanism for membrane-protein interactions. Consistent with this idea, electrophysiological experiments with planar lipid bilayers revealed that NetB formed pores with much larger single-channel conductance than alpha-hemolysin. Channel conductance varied with phospholipid net charge. Furthermore, NetB differed in its ion selectivity, preferring cations over anions. Using hemolysis as a screen, we carried out a random-mutagenesis study that identified several residues that are critical for NetB-induced cell lysis. Mapping of these residues onto the crystal structure revealed that they were clustered in regions predicted to be required for oligomerization or membrane binding. Together these data provide an insight into the mechanism of NetB-mediated pore formation and will contribute to our understanding of the mode of action of this important toxin. IMPORTANCE Necrotic enteritis is an economically important disease of the worldwide poultry industry and is mediated by Clostridium perfringens strains that produce NetB, a β-pore-forming toxin. We carried out structural and functional studies of NetB to provide a mechanistic insight into its mode of action and to assist in the development of a necrotic enteritis vaccine. We determined the structure of the monomeric form of NetB to 1.8 Å, used both site-directed and random mutagenesis to identify key residues that are required for its biological activity, and analyzed pore formation by NetB and its substitution-containing derivatives in planar lipid bilayers.

scholarly journals The Mode of Action of the Bacillus thuringiensis Vegetative Insecticidal Protein Vip3A Differs from That of Cry1Ab δ-Endotoxin

2003 ◽  
Vol 69 (8) ◽  
pp. 4648-4657 ◽  
Author(s):  
Mi Kyong Lee ◽  
Frederick S. Walters ◽  
Hope Hart ◽  
Narendra Palekar ◽  
Jeng-Shong Chen
Keyword(s):  
Lipid Bilayers ◽  
Manduca Sexta ◽  
Mode Of Action ◽  
Pore Formation ◽  
Brush Border Membrane ◽  
Danaus Plexippus ◽  
Membrane Vesicles ◽  
Monarch Butterfly ◽  
New Family ◽  
Voltage Clamping

ABSTRACT The Vip3A protein, secreted by Bacillus spp. during the vegetative stage of growth, represents a new family of insecticidal proteins. In our investigation of the mode of action of Vip3A, the 88-kDa Vip3A full-length toxin (Vip3A-F) was proteolytically activated to an approximately 62-kDa core toxin either by trypsin (Vip3A-T) or lepidopteran gut juice extracts (Vip3A-G). Biotinylated Vip3A-G demonstrated competitive binding to lepidopteran midgut brush border membrane vesicles (BBMV). Furthermore, in ligand blotting experiments with BBMV from the tobacco hornworm, Manduca sexta (Linnaeus), activated Cry1Ab bound to 120-kDa aminopeptidase N (APN)-like and 250-kDa cadherin-like molecules, whereas Vip3A-G bound to 80-kDa and 100-kDa molecules which are distinct from the known Cry1Ab receptors. In addition, separate blotting experiments with Vip3A-G did not show binding to isolated Cry1A receptors, such as M. sexta APN protein, or a cadherin Cry1Ab ecto-binding domain. In voltage clamping assays with dissected midgut from the susceptible insect, M. sexta, Vip3A-G clearly formed pores, whereas Vip3A-F was incapable of pore formation. In the same assay, Vip3A-G was incapable of forming pores with larvae of the nonsusceptible insect, monarch butterfly, Danaus plexippus (Linnaeus). In planar lipid bilayers, both Vip3A-G and Vip3A-T formed stable ion channels in the absence of any receptors, supporting pore formation as an inherent property of Vip3A. Both Cry1Ab and Vip3A channels were voltage independent and highly cation selective; however, they differed considerably in their principal conductance state and cation specificity. The mode of action of Vip3A supports its use as a novel insecticidal agent.

Effect of Sterol Side Chain on Ion Channel Formation by Amphotericin B in Lipid Bilayers

Biochemistry ◽  
2014 ◽  
Vol 53 (19) ◽  
pp. 3088-3094 ◽  
Author(s):  
Yasuo Nakagawa ◽  
Yuichi Umegawa ◽  
Tetsuro Takano ◽  
Hiroshi Tsuchikawa ◽  
Nobuaki Matsumori ◽  
...  
Keyword(s):  
Ion Channel ◽  
Amphotericin B ◽  
Lipid Bilayers ◽  
Side Chain ◽  
Channel Formation

scholarly journals Daptomycin Pore Formation and Stoichiometry Depend on Membrane Potential of Target Membrane

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Gabriela Seydlová ◽  
Albert Sokol ◽  
Petra Lišková ◽  
Ivo Konopásek ◽  
Radovan Fišer
Keyword(s):  
Membrane Potential ◽  
Lipid Bilayers ◽  
Pore Formation ◽  
Content Type ◽  
Calcium Dependent ◽  
Screening Experiment ◽  
Serious Infections ◽  
Fluorescence Methods ◽  
Initial Membrane ◽  
Target Membrane

ABSTRACT Daptomycin is a calcium-dependent lipodepsipeptide antibiotic clinically used to treat serious infections caused by Gram-positive pathogens. Its precise mode of action is somewhat controversial; the biggest issue is daptomycin pore formation, which we directly investigated here. We first performed a screening experiment using propidium iodide (PI) entry to Bacillus subtilis cells and chose the optimum and therapeutically relevant conditions (10 µg/ml daptomycin and 1.25 mM CaCl2) for the subsequent analyses. Using conductance measurements on planar lipid bilayers, we show that daptomycin forms nonuniform oligomeric pores with conductance ranging from 120 pS to 14 nS. The smallest conductance unit is probably a dimer; however, tetramers and pentamers occur in the membrane most frequently. Moreover, daptomycin pore-forming activity is exponentially dependent on the applied membrane voltage. We further analyzed the membrane-permeabilizing activity in B. subtilis cells using fluorescence methods [PI and DiSC3(5)]. Daptomycin most rapidly permeabilizes cells with high initial membrane potential and dissipates it within a few minutes. Low initial membrane potential hinders daptomycin pore formation.

Comparative Structural Study of Leaf Spot Disease of Safflower and Sugar Beet by Cercospora beticola

2007 ◽  
Vol 6 (1) ◽  
pp. 37-43 ◽  
Author(s):  
Robert T. Lartey ◽  
Andrew W. Lenssen ◽  
Robert G. Evans ◽  
Soumitra Ghoshroy
Keyword(s):  
Sugar Beet ◽  
Structural Study ◽  
Leaf Spot ◽  
Cercospora Beticola ◽  
Leaf Spot Disease ◽  
Spot Disease

scholarly journals Exchange of Gramicidin between Lipid Bilayers: Implications for the Mechanism of Channel Formation and Gating

2011 ◽  
Vol 100 (3) ◽  
pp. 501a ◽  
Author(s):  
Kevin Lum ◽  
Helgi I. Ingólfsson ◽  
Roger E. Koeppe ◽  
Olaf S. Andersen
Keyword(s):  
Lipid Bilayers ◽  
Channel Formation
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