Pore Formation by S. aureus α-toxin in Liposomes and Planar Lipid Bilayers: Effects of Nonelectrolytes

1996 ◽  
Vol 150 (1) ◽  
pp. 37-45 ◽  
Author(s):  
C.L. Bashford ◽  
G.M. Alder ◽  
L.G. Fulford ◽  
Y.E. Korchev ◽  
E. Kovacs ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Pore Formation ◽  
Planar Lipid Bilayers

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 Rapid topology probing using fluorescence spectroscopy in planar lipid bilayer: the pore-forming mechanism of the toxin Cry1Aa of Bacillus thuringiensis

2010 ◽  
Vol 136 (5) ◽  
pp. 497-513 ◽  
Author(s):  
Nicolas Groulx ◽  
Marc Juteau ◽  
Rikard Blunck
Keyword(s):  
Bacillus Thuringiensis ◽  
Lipid Bilayers ◽  
Pore Formation ◽  
Time Course ◽  
Structural Information ◽  
Dynamic Change ◽  
Fluorescence Emission ◽  
Aqueous Environment ◽  
Planar Lipid Bilayers ◽  
Forming Mechanism

Pore-forming toxins, many of which are pathogenic to humans, are highly dynamic proteins that adopt a different conformation in aqueous solution than in the lipid environment of the host membrane. Consequently, their crystal structures obtained in aqueous environment do not reflect the active conformation in the membrane, making it difficult to deduce the molecular determinants responsible for pore formation. To obtain structural information directly in the membrane, we introduce a fluorescence technique to probe the native topology of pore-forming toxins in planar lipid bilayers and follow their movement during pore formation. Using a Förster resonance energy transfer (FRET) approach between site-directedly labeled proteins and an absorbing compound (dipicrylamine) in the membrane, we simultaneously recorded the electrical current and fluorescence emission in horizontal planar lipid bilayers formed in plastic chips. With this system, we mapped the topology of the pore-forming domain of Cry1Aa, a biological pesticide from Bacillus thuringiensis, by determining the location of the loops between its seven α helices. We found that the majority of the toxins initially traverse from the cis to the trans leaflet of the membrane. Comparing the topologies of Cry1Aa in the active and inactive state in order to identify the pore-forming mechanism, we established that only the α3–α4 hairpin translocates through the membrane from the trans to the cis leaflet, whereas all other positions remained constant. As toxins are highly dynamic proteins, populations that differ in conformation might be present simultaneously. To test the presence of different populations, we designed double-FRET experiments, where a single donor interacts with two acceptors with very different kinetics (dipicrylamine and oxonol). Due to the nonlinear response of FRET and the dynamic change of the acceptor distribution, we can deduce the distribution of the acceptors in the membrane from the time course of the donor fluorescence. We found that Cry1Aa is present on both membrane leaflets.

Molecular Mechanism of Action of β-Hairpin Antimicrobial Peptide Arenicin: Oligomeric Structure in Dodecylphosphocholine Micelles and Pore Formation in Planar Lipid Bilayers

Biochemistry ◽  
2011 ◽  
Vol 50 (28) ◽  
pp. 6255-6265 ◽  
Author(s):  
Zakhar O. Shenkarev ◽  
Sergey V. Balandin ◽  
Kirill I. Trunov ◽  
Alexander S. Paramonov ◽  
Stanislav V. Sukhanov ◽  
...  
Keyword(s):  
Antimicrobial Peptide ◽  
Molecular Mechanism ◽  
Lipid Bilayers ◽  
Mechanism Of Action ◽  
Pore Formation ◽  
Planar Lipid Bilayers ◽  
Oligomeric Structure ◽  
Molecular Mechanism Of Action

Membrane pore formation by pentraxin proteins from Limulus, the American horseshoe crab

2008 ◽  
Vol 413 (2) ◽  
pp. 305-313 ◽  
Author(s):  
John M. Harrington ◽  
Hui-Ting Chou ◽  
Thomas Gutsmann ◽  
Christoph Gelhaus ◽  
Henning Stahlberg ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Pore Formation ◽  
Horseshoe Crab ◽  
Planar Lipid Bilayers ◽  
C Reactive Protein ◽  
Membrane Pore ◽  
Reactive Protein ◽  
Independent Form ◽  
Transmembrane Pores ◽  
Amyloid P

The pentraxins are a family of highly conserved plasma proteins of metazoans known to function in immune defence. The canonical members, C-reactive protein and serum amyloid P component, have been identified in arthropods and humans. Mammalian pentraxins are known to bind lipid bilayers, and a pentraxin representative from the American horseshoe crab, Limulus polyphemus, binds and permeabilizes mammalian erythrocytes. Both activities are Ca2+-dependent. Utilizing model liposomes and planar lipid bilayers, in the present study we have investigated the membrane-active properties of the three pentraxin representatives from Limulus and show that all of the Limulus pentraxins permeabilize lipid bilayers. Mechanistically, Limulus C-reactive protein forms transmembrane pores in asymmetric planar lipid bilayers that mimic the outer membrane of Gram-negative bacteria and exhibits a Ca2+-independent form of membrane binding that may be sufficient for pore formation.

scholarly journals Localization in diphtheria toxin fragment B of a region that induces pore formation in planar lipid bilayers at low pH

FEBS Letters ◽  
1983 ◽  
Vol 160 (1-2) ◽  
pp. 82-86 ◽  
Author(s):  
Michel Deleers ◽  
Nadine Beugnier ◽  
Paul Falmagne ◽  
Véronique Cabiaux ◽  
Jean-Marie Ruysschaert
Keyword(s):  
Lipid Bilayers ◽  
Diphtheria Toxin ◽  
Pore Formation ◽  
Low Ph ◽  
Planar Lipid Bilayers

scholarly journals Saturation behavior of single, amiloride-sensitive Na+ channels in planar lipid bilayers

1984 ◽  
Vol 46 (6) ◽  
pp. 831-835 ◽  
Author(s):  
L. Olans ◽  
S. Sariban-Sohraby ◽  
D.J. Benos
Keyword(s):  
Lipid Bilayers ◽  
Planar Lipid Bilayers ◽  
Na Channels

scholarly journals Stimulation‐dependent gating of TRPM3 channel in planar lipid bilayers

2015 ◽  
Vol 30 (3) ◽  
pp. 1306-1316 ◽  
Author(s):  
Kunitoshi Uchida ◽  
Lusine Demirkhanyan ◽  
Swapna Asuthkar ◽  
Alejandro Cohen ◽  
Makoto Tominaga ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Planar Lipid Bilayers
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