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.

scholarly journals Investigation of the pore-forming mechanism of a cytolytic δ-endotoxin from Bacillus thuringiensis

2003 ◽  
Vol 374 (1) ◽  
pp. 255-259 ◽  
Author(s):  
Boonhiang PROMDONKOY ◽  
David J. ELLAR
Keyword(s):  
Bacillus Thuringiensis ◽  
Blood Cells ◽  
Pore Formation ◽  
Response Curve ◽  
Effect Of Temperature ◽  
Membrane Insertion ◽  
Free Solution ◽  
Forming Mechanism ◽  
Close Proximity ◽  
Membrane Bound

Cyt2Aa1 is a cytolytic protein produced by Bacillus thuringiensis subsp. kyushuensis. Penetration of the toxin into membranes has been studied to learn more about membrane-insertion mechanisms and transmembrane-pore formation. The haemolysis assay of Cyt2Aa1 showed a steep and sigmoidal dose–response curve, indicating that toxin aggregation or oligomerization is required for pore formation. Studies of the effect of temperature on pore formation and fluorimetric studies of acrylodan-labelled toxin suggest that toxin inserts into the membrane before oligomerizing to form a pore. Low temperature neither inhibited membrane binding nor closed pores that have been formed, but markedly inhibited oligomerization of the toxin molecules. When toxin-treated red blood cells at 4 °C were transferred to a toxin-free solution at 37 °C, no significant increase in haemolysis was observed. This result suggests that membrane-bound toxin could not diffuse laterally and interact with other molecules to form a pore. From these results, we propose that Cyt2Aa1 binds and inserts into the membrane as a monomer. Oligomerization occurs when toxin molecules have bound in close proximity to each other and pores are formed from large oligomers.

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 Mutagenic Analysis of a Conserved Region of Domain III in the Cry1Ac Toxin of Bacillus thuringiensis

2002 ◽  
Vol 68 (1) ◽  
pp. 194-200 ◽  
Author(s):  
Luke Masson ◽  
Bruce E. Tabashnik ◽  
Alberto Mazza ◽  
Gabrielle Préfontaine ◽  
Léna Potvin ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Aspartic Acid ◽  
Lipid Bilayers ◽  
Insect Cell Line ◽  
Planar Lipid Bilayers ◽  
Primary Role ◽  
Membrane Pore ◽  
Cry1ac Toxin ◽  
Conserved Region ◽  
Domain Iii

ABSTRACT We used site-directed mutagenesis to probe the function of four alternating arginines located at amino acid positions 525, 527, 529, and 531 in a highly conserved region of domain III in the Cry1Ac toxin of Bacillus thuringiensis. We created 10 mutants: eight single mutants, with each arginine replaced by either glycine (G) or aspartic acid (D), and two double mutants (R525G/R527G and R529G/R531G). In lawn assays of the 10 mutants with a cultured Choristoneura fumiferana insect cell line (Cf1), replacement of a single arginine by either glycine or aspartic acid at position 525 or 529 decreased toxicity 4- to 12-fold relative to native Cry1Ac toxin, whereas replacement at position 527 or 531 decreased toxicity only 3-fold. The reduction in toxicity seen with double mutants was 8-fold for R525G/R527G and 25-fold for R529G/R531G. Five of the mutants (R525G, R525D, R527G, R529D, and R525G/R527G) were tested in bioassays with Plutella xylostella larvae and ion channel formation in planar lipid bilayers. In the bioassays, R525D, R529D, and R525G/R527G showed reduced toxicity. In planar lipid bilayers, the conductance and the selectivity of the mutants were similar to those of native Cry1Ac. Toxins with alteration at position 527 or 529 tended to remain in their subconducting states rather than the maximally conducting state. Our results suggest that the primary role of this conserved region is to maintain both the structural integrity of the native toxin and the full functionality of the formed membrane pore.

scholarly journals A cytolytic δ-endotoxin from Bacillus thuringiensis var. israelensis forms cation-selective channels in planar lipid bilayers

FEBS Letters ◽  
1989 ◽  
Vol 244 (2) ◽  
pp. 259-262 ◽  
Author(s):  
Barbara H. Knowles ◽  
Michael R. Blatt ◽  
Mark Tester ◽  
Jane M. Horsnell ◽  
Joe Carroll ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Lipid Bilayers ◽  
Planar Lipid Bilayers

scholarly journals Estimation of the radius of the pores formed by the Bacillus thuringiensis Cry1C δ-endotoxin in planar lipid bilayers

2002 ◽  
Vol 1567 ◽  
pp. 113-122 ◽  
Author(s):  
Olivier Peyronnet ◽  
Brian Nieman ◽  
Francis Généreux ◽  
Vincent Vachon ◽  
Raynald Laprade ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Lipid Bilayers ◽  
Planar Lipid Bilayers

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

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

Lepidopteran-specific crystal toxins from Bacillus thuringiensis form cation- and anion-selective channels in planar lipid bilayers

1993 ◽  
Vol 132 (1) ◽  
Author(s):  
Jean-Louis Schwartz ◽  
Line Garneau ◽  
Diane Savaria ◽  
Luke Masson ◽  
Roland Brousseau ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Lipid Bilayers ◽  
Planar Lipid Bilayers ◽  
Crystal Toxins

scholarly journals Acetylcholine receptor in planar lipid bilayers. Characterization of the channel properties of the purified nicotinic acetylcholine receptor from Torpedo californica reconstituted in planar lipid bilayers.

1984 ◽  
Vol 83 (4) ◽  
pp. 473-496 ◽  
Author(s):  
P Labarca ◽  
J Lindstrom ◽  
M Montal
Keyword(s):  
Steady State ◽  
Acetylcholine Receptor ◽  
Lipid Bilayers ◽  
Applied Voltage ◽  
Time Course ◽  
Single Channel ◽  
Electric Organ ◽  
Membrane Conductance ◽  
Planar Lipid Bilayers ◽  
Agonist Concentration

The properties of the channel of the purified acetylcholine receptor (AChR) were investigated after reconstitution in planar lipid bilayers. The time course of the agonist-induced conductance exhibits a transient peak that relaxes to a steady state value. The macroscopic steady state membrane conductance increases with agonist concentration, reaching saturation at 10(-5) M for carbamylcholine (CCh). The agonist-induced membrane conductance was inhibited by d-tubocurarine (50% inhibition, IC50, at approximately 10(-6) M) and hexamethonium (IC50 approximately 10(-5) M). The single channel conductance, gamma, is ohmic and independent of the agonist. At 0.3 M monovalent salt concentrations, gamma = 28 pS for Na+, 30 pS for Rb+, 38 pS for Cs+, and 50 pS for NH+4. The distribution of channel open times was fit by a sum of two exponentials, reflecting the existence of two distinct open states. tau o1 and tau o2, the fast and slow components of the distribution of open times, are independent of the agonist concentration: for CCh this was verified in the range of 10(-6) M less than C less than 10(-3)M. tau 01 and tau o2 are approximately three times longer for suberyldicholine ( SubCh ) than for CCh. tau o1 and tau o2 are moderately voltage dependent, increasing as the applied voltage in the compartment containing agonist is made more positive with respect to the other. At desensitizing concentrations of agonist, the AChR channel openings occurred in a characteristic pattern of sudden paroxysms of channel activity followed by quiescent periods. A local anesthetic derivative of lidocaine ( QX -222) reduced both tau o1 and tau o2. This effect was dependent on both the concentration of QX -222 and the applied voltage. Thus, the AChR purified from Torpedo electric organ and reconstituted in planar lipid bilayers exhibits ion conduction and kinetic and pharmacological properties similar to AChR in intact muscle postsynaptic membranes.

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