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 Single-molecule analysis of the entire perfringolysin O pore formation pathway

2021 ◽  
Author(s):  
Conall Mc Guinness ◽  
James Walsh ◽  
Charles Bayly-Jones ◽  
Michelle Dunstone ◽  
Craig Morton ◽  
...  
Keyword(s):  
Single Molecule ◽  
Pore Formation ◽  
Membrane Insertion ◽  
Formation Pathway ◽  
Perfringolysin O ◽  
Assembly Pathway ◽  
Bacterial Virulence Factor ◽  
Membrane Bound ◽  
Single Molecule Analysis ◽  
Kinetics Of

The cholesterol-dependent cytolysin perfringolysin O (PFO) is secreted by Clostridium perfringens as a bacterial virulence factor able to form giant ring-shaped pores that perforate and ultimately lyse mammalian cell membranes. To resolve the kinetics of all steps in the assembly pathway, we have used single-molecule fluorescence imaging to follow the dynamics of PFO on dye-loaded liposomes that lead to opening of a pore and release of the encapsulated dye. Formation of a long-lived membrane-bound PFO dimer nucleates the growth of an irreversible oligomer. The growing oligomer can insert into the membrane and open a pore at stoichiometries ranging from tetramers to full rings (~35-mers), whereby the rate of insertion increases linearly with the number of subunits. Oligomers that insert before the ring is complete continue to grow by monomer addition post insertion. Overall, our observations suggest that PFO membrane insertion is kinetically controlled.

scholarly journals The Cyt1Aa toxin from Bacillus thuringiensis inserts into target membranes via different mechanisms in insects, red blood cells, and lipid liposomes

2020 ◽  
Vol 295 (28) ◽  
pp. 9606-9617 ◽  
Author(s):  
Janette Onofre ◽  
Sabino Pacheco ◽  
Mary Carmen Torres-Quintero ◽  
Sarjeet S. Gill ◽  
Mario Soberon ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Pore Formation ◽  
Membrane Vesicles ◽  
Membrane System ◽  
Bound State ◽  
Cysteine Residue ◽  
Formation Model ◽  
Small Unilamellar Vesicle ◽  
Membrane Bound ◽  
Detergent Effect

Bacillus thuringiensis subsp. israelensis produces crystal inclusions composed of three-domain Cry proteins and cytolytic Cyt toxins, which are toxic to different mosquito larvae. A key component is the Cyt toxin, which synergizes the activity of the other Cry toxins, thereby resulting in high toxicity. The precise mechanism of action of Cyt toxins is still debated, and two models have been proposed: the pore formation model and the detergent effect. Here, we performed a systematic structural characterization of the Cyt toxin interaction with different membranes, including in Aedes aegypti larval brush border membrane vesicles, small unilamellar vesicle liposomes, and rabbit erythrocytes. We examined Cyt1Aa insertion into these membranes by analyzing fluorescence quenching in solution and in the membrane-bound state. For this purpose, we constructed several Cyt1Aa variants having substitutions with a single cysteine residue in different secondary structures, enabling Cys labeling with Alexa Fluor 488 for quenching analysis using I-soluble quencher in solution and in the membrane-bound state. We identified the Cyt1Aa residues exposed to the solvent upon membrane insertion, predicting a possible topology of the membrane-inserted toxin in the different membranes. Moreover, toxicity assays with these variants revealed that Cyt1Aa exerts its insecticidal activity and hemolysis through different mechanisms. We found that Cyt1Aa exhibits variable interactions with each membrane system, with deeper insertion into mosquito larva membranes, supporting the pore formation model, whereas in the case of erythrocytes and small unilamellar vesicles, Cyt1Aa's insertion was more superficial, supporting the notion that a detergent effect underlies its hemolytic activity.

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 In vivo nanoscale analysis of the dynamic synergistic interaction of Bacillus thuringiensis Cry11Aa and Cyt1Aa toxins in Aedes aegypti

2021 ◽  
Vol 17 (1) ◽  
pp. e1009199
Author(s):  
Samira López-Molina ◽  
Nathaly Alexandre do Nascimento ◽  
Maria Helena Neves Lobo Silva-Filha ◽  
Adán Guerrero ◽  
Jorge Sánchez ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Pore Formation ◽  
Lower Layer ◽  
Larval Mortality ◽  
Membrane Insertion ◽  
Cell Cytoplasm ◽  
Mutant Proteins ◽  
Posterior Midgut ◽  
High Larval Mortality

The insecticidal Cry11Aa and Cyt1Aa proteins are produced by Bacillus thuringiensis as crystal inclusions. They work synergistically inducing high toxicity against mosquito larvae. It was proposed that these crystal inclusions are rapidly solubilized and activated in the gut lumen, followed by pore formation in midgut cells killing the larvae. In addition, Cyt1Aa functions as a Cry11Aa binding receptor, inducing Cry11Aa oligomerization and membrane insertion. Here, we used fluorescent labeled crystals, protoxins or activated toxins for in vivo localization at nano-scale resolution. We show that after larvae were fed solubilized proteins, these proteins were not accumulated inside the gut and larvae were not killed. In contrast, if larvae were fed soluble non-toxic mutant proteins, these proteins were found inside the gut bound to gut-microvilli. Only feeding with crystal inclusions resulted in high larval mortality, suggesting that they have a role for an optimal intoxication process. At the macroscopic level, Cry11Aa completely degraded the gastric caeca structure and, in the presence of Cyt1Aa, this effect was observed at lower toxin-concentrations and at shorter periods. The labeled Cry11Aa crystal protein, after midgut processing, binds to the gastric caeca and posterior midgut regions, and also to anterior and medium regions where it is internalized in ordered “net like” structures, leading finally to cell break down. During synergism both Cry11Aa and Cyt1Aa toxins showed a dynamic layered array at the surface of apical microvilli, where Cry11Aa is localized in the lower layer closer to the cell cytoplasm, and Cyt1Aa is layered over Cry11Aa. This array depends on the pore formation activity of Cry11Aa, since the non-toxic mutant Cry11Aa-E97A, which is unable to oligomerize, inverted this array. Internalization of Cry11Aa was also observed during synergism. These data indicate that the mechanism of action of Cry11Aa is more complex than previously anticipated, and may involve additional steps besides pore-formation activity.

scholarly journals Notch receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

2019 ◽  
Vol 2019 (4) ◽  
Author(s):  
Thiruma V. Arumugam ◽  
Christopher Sobey
Keyword(s):  
Cell Fate ◽  
Single Agent ◽  
Notch Signalling ◽  
Type I ◽  
Notch Receptors ◽  
Anti Cancer ◽  
Close Proximity ◽  
Ligand Interactions ◽  
Transcriptional Modulation ◽  
Membrane Bound

The canonical Notch signalling pathway has four type I transmembrane Notch receptors (Notch1-4) and five ligands (DLL1, 2 and 3, and Jagged 1-2). Each member of this highly conserved receptor family plays a unique role in cell-fate determination during embryogenesis, differentiation, tissue patterning, proliferation and cell death [2]. As the Notch ligands are also membrane bound, cells have to be in close proximity for receptor-ligand interactions to occur. Cleavage of the intracellular domain (ICD) of activated Notch receptors by γ-secretase is required for downstream signalling and Notch-induced transcriptional modulation [15, 3, 11, 22]. This is why γ-secretase inhibitors can be used to downregulate Notch signalling and explains their anti-cancer action. One such small molecule is RO4929097 [8], although development of this compound has been terminated following an unsuccessful Phase II single agent clinical trial in metastatic colorectal cancer [19].Aberrant Notch signalling is implicated in a number of human cancers [12, 20, 6, 16], with demcizumab and tarextumab identified as antibody inhibitors of ligand:receptor binding [13].

EFFECT OF TEMPERATURE ON THE PATHOGENESIS OF BACILLUS THURINGIENSIS BERLINER IN LARVAE OF THE SPRUCE BUDWORM, CHORISTONEURA FUMIFERANA CLEM. (LEPIDOPTERA: TORTRICIDAE)

1994 ◽  
Vol 126 (4) ◽  
pp. 1061-1065 ◽  
Author(s):  
Kees van Frankenhuyzen
Keyword(s):  
Bacillus Thuringiensis ◽  
Bacterial Growth ◽  
Choristoneura Fumiferana ◽  
Larval Mortality ◽  
Lethal Dose ◽  
Spruce Budworm ◽  
Effect Of Temperature ◽  
Vegetative Cells ◽  
Time Required ◽  
Peak Abundance

AbstractThe relationship between temperature and pathogenesis of Bacillus thuringiensis Berliner var. kurstaki in infected larvae of the eastern spruce budworm, Choristoneura fumiferana Clem., was investigated to determine if more rapid death of larvae with an increase in temperature could be accounted for by enhanced bacterial growth. Cumulative mortality of larvae force-fed with a lethal dose of HD-1-S-1980 peaked within 2 days at 25 °C, 3 days at 19 °C, 7 days at 16 °C, and 21 days at 13 °C. The progress of bacterial growth in the larvae was followed from spore germination to cell lysis, and was completed within 4 days at 25 °C, 6 days at 22 °C, 12 days at 19 °C, 14 days at 16 °C, and > 28 days at 13 °C. Peak abundance of vegetative cells in the larvae was observed after 1 day at 25 °C, 2 days at 22 °C, 3 days at 19 °C, 7 days at 16 °C, and 21 days at 13 °C, and thus coincided almost exactly with the time required for maximum larval mortality. This correlation suggests that the observed effect of temperature on progression of larval mortality was due to its effect on the proliferation of vegetative cells in the infected larvae, and that bacterial septicemia makes an important contribution to death.

scholarly journals Polymorphism of amyloid β peptide in different environments: implications for membrane insertion and pore formation

Soft Matter ◽  
2011 ◽  
Vol 7 (11) ◽  
pp. 5267 ◽  
Author(s):  
Fernando Terán Arce ◽  
Hyunbum Jang ◽  
Srinivasan Ramachandran ◽  
Preston B. Landon ◽  
Ruth Nussinov ◽  
...  
Keyword(s):  
Pore Formation ◽  
Amyloid Β ◽  
Membrane Insertion ◽  
Amyloid Β Peptide

Studies of the endoplasmic reticulum and plasma membrane-bound ribosomes in erythropoietic cells

1978 ◽  
Vol 31 (1) ◽  
pp. 165-178
Author(s):  
J.A. Grasso ◽  
A.L. Sullivan ◽  
S.C. Chan
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Exact Role ◽  
Whole Cells ◽  
Cytoplasmic Face ◽  
Close Proximity ◽  
Haem Biosynthesis ◽  
Hypotonic Buffer ◽  
Membrane Bound ◽  
Hypotonic Lysis

Erythropoietic cells of 5 species, including man, contain endoplasmic reticulum present as individual cisternae or tubules scattered throughout the cytoplasm of all stages except mature RBCs. The endoplasmic reticulum is mainly agranular but occurs frequently as a variant of granular ER which is characterized by an asymmetrical and irregular distribution of ribosomes along one cytoplasmic face. In most cells, the endoplasmic reticulum occurs in close proximity to mitochondria or the plasma membrane, suggesting that the organelle may be involved in functions related to these structures, e.g. haem biosynthesis. Endoplasmic reticulum is more abundant in early than in late erythroid cells. Its exact role in RBC development is unclear. Since endoplasmic reticulum could account for ‘plasma membrane-bound ribosomes’ reported in lysed reticulocytes, studies were performed which ruled out this possibility and which suggested that such ribosomes were an artifact of the lysing conditions. Hypotonic lysis in less than 20 vol. of magnesium-containing buffers yielded ghosts variably contaminated by ribosomes and other structures. Lysis of reticulocytes in 20–30 vol. of magnesium-free buffer or homogenization of whole cells or crude membrane fractions in hypotonic buffer removed virtually all contaminating ribosomes from the purified membrane fraction.

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