scholarly journals Anthrax Toxin Characterization

2002 ◽  
Vol 45 (1) ◽  
pp. 3-5 ◽  
Author(s):  
Jiří Patočka ◽  
Miroslav Špliňo
Keyword(s):  
Blood Cells ◽  
Protective Antigen ◽  
Lethal Factor ◽  
White Blood Cells ◽  
The Body ◽  
Anthrax Toxin ◽  
Signaling System ◽  
Animal Cells ◽  
The Third ◽  
Edema Factor

The anthrax toxin comprises three proteins. When they work together, they can kill humans, especially after spores of the bacteria have been inhaled. One anthrax protein, called protective antigen (PA), chaperones the two other toxins into human or animal cells and shields them from the body’s immune system. The second, lethal factor (LF), destroys the white blood cells that hosts send in defence. The third toxin molecule, edema factor (EF), hijacks the signaling system in the body. This disrupts the energy balance of cells and leads to them accumulating fluid and complete destroy of cells.

Anthrax toxin complexes: heptameric protective antigen can bind lethal factor and edema factor simultaneously

2004 ◽  
Vol 322 (1) ◽  
pp. 258-262 ◽  
Author(s):  
Ruth-Anne L. Pimental ◽  
Kenneth A. Christensen ◽  
Bryan A. Krantz ◽  
R. John Collier
Keyword(s):  
Protective Antigen ◽  
Lethal Factor ◽  
Anthrax Toxin ◽  
Edema Factor

scholarly journals Trapping a translocating protein within the anthrax toxin channel: implications for the secondary structure of permeating proteins

2011 ◽  
Vol 137 (4) ◽  
pp. 343-356 ◽  
Author(s):  
Daniel Basilio ◽  
Laura D. Jennings-Antipov ◽  
Karen S. Jakes ◽  
Alan Finkelstein
Keyword(s):  
Secondary Structure ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Anthrax Toxin ◽  
Bilayer Membranes ◽  
Endosomal Membrane ◽  
Edema Factor ◽  
N Terminus ◽  
Minimum Number ◽  
Pass Through

Anthrax toxin consists of three proteins: lethal factor (LF), edema factor (EF), and protective antigen (PA). This last forms a heptameric channel, (PA63)7, in the host cell’s endosomal membrane, allowing the former two (which are enzymes) to be translocated into the cytosol. (PA63)7 incorporated into planar bilayer membranes forms a channel that translocates LF and EF, with the N terminus leading the way. The channel is mushroom-shaped with a cap containing the binding sites for EF and LF, and an ∼100 Å–long, 15 Å–wide stem. For proteins to pass through the stem they clearly must unfold, but is secondary structure preserved? To answer this question, we developed a method of trapping the polypeptide chain of a translocating protein within the channel and determined the minimum number of residues that could traverse it. We attached a biotin to the N terminus of LFN (the 263-residue N-terminal portion of LF) and a molecular stopper elsewhere. If the distance from the N terminus to the stopper was long enough to traverse the channel, streptavidin added to the trans side bound the N-terminal biotin, trapping the protein within the channel; if this distance was not long enough, streptavidin did not bind the N-terminal biotin and the protein was not trapped. The trapping rate was dependent on the driving force (voltage), the length of time it was applied, and the number of residues between the N terminus and the stopper. By varying the position of the stopper, we determined the minimum number of residues required to span the channel. We conclude that LFN adopts an extended-chain configuration as it translocates; i.e., the channel unfolds the secondary structure of the protein. We also show that the channel not only can translocate LFN in the normal direction but also can, at least partially, translocate LFN in the opposite direction.

scholarly journals Ion selectivity of the anthrax toxin channel and its effect on protein translocation

2015 ◽  
Vol 146 (2) ◽  
pp. 183-192 ◽  
Author(s):  
Aviva Schiffmiller ◽  
Damon Anderson ◽  
Alan Finkelstein
Keyword(s):  
Protective Antigen ◽  
Protein Translocation ◽  
Ion Selectivity ◽  
Lethal Factor ◽  
Phospholipid Bilayer ◽  
Anthrax Toxin ◽  
Terminal Portion ◽  
Bilayer Membranes ◽  
Cation Selectivity ◽  
Edema Factor

Anthrax toxin consists of three ∼85-kD proteins: lethal factor (LF), edema factor (EF), and protective antigen (PA). PA63 (the 63-kD, C-terminal portion of PA) forms heptameric channels ((PA63)7) in planar phospholipid bilayer membranes that enable the translocation of LF and EF across the membrane. These mushroom-shaped channels consist of a globular cap domain and a 14-stranded β-barrel stem domain, with six anionic residues lining the interior of the stem to form rings of negative charges. (PA63)7 channels are highly cation selective, and, here, we investigate the effects on both cation selectivity and protein translocation of mutating each of these anionic residues to a serine. We find that although some of these mutations reduce cation selectivity, selectivity alone does not directly predict the rate of protein translocation; local changes in electrostatic forces must be considered as well.

scholarly journals Proton-coupled protein transport through the anthrax toxin channel

2008 ◽  
Vol 364 (1514) ◽  
pp. 209-215 ◽  
Author(s):  
Alan Finkelstein
Keyword(s):  
Protein Transport ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Low Ph ◽  
Phospholipid Bilayers ◽  
Anthrax Toxin ◽  
Large Hole ◽  
The Third ◽  
Folded Proteins ◽  
Acidic Vesicle

Anthrax toxin consists of three proteins (approx. 90 kDa each): lethal factor (LF); oedema factor (OF); and protective antigen (PA). The former two are enzymes that act when they reach the cytosol of a targeted cell. To enter the cytosol, however, which they do after being endocytosed into an acidic vesicle compartment, they require the third component, PA. PA (or rather its proteolytically generated fragment PA 63 ) forms at low pH a heptameric β-barrel channel, (PA 63 ) 7 , through which LF and OF are transported—a phenomenon we have demonstrated in planar phospholipid bilayers. It might appear that (PA 63 ) 7 simply forms a large hole through which LF and OF diffuse. However, LF and OF are folded proteins, much too large to fit through the approximately 15 Å diameter (PA 63 ) 7 β-barrel. This paper discusses how the (PA 63 ) 7 channel both participates in the unfolding of LF and OF and functions in their translocation as a proton–protein symporter.

scholarly journals Toxicity of Anthrax Toxin Is Influenced by Receptor Expression

2008 ◽  
Vol 15 (9) ◽  
pp. 1330-1336 ◽  
Author(s):  
Sarah C. Taft ◽  
Alison A. Weiss
Keyword(s):  
Protective Antigen ◽  
Lethal Factor ◽  
Chinese Hamster ◽  
Receptor Expression ◽  
Anthrax Toxin ◽  
Cellular Receptor ◽  
Wild Type ◽  
Human Form ◽  
Edema Factor ◽  
Surface Pores

ABSTRACT Anthrax toxin protective antigen (PA) binds to its cellular receptor, and seven subunits self-associate to form a heptameric ring that mediates the cytoplasmic entry of lethal factor or edema factor. The influence of receptor type on susceptibility to anthrax toxin components was examined using Chinese hamster ovary (CHO) cells expressing the human form of one of two PA receptors: TEM8 or CMG2. Unexpectedly, PA alone, previously believed to only mediate entry of lethal factor or edema factor, was found to be toxic to CHO-TEM8 cells; cells treated with PA alone displayed reduced cell growth and decreased metabolic activity. PA-treated cells swelled and became permeable to membrane-excluded dye, suggesting that PA formed cell surface pores on CHO-TEM8 cells. While CHO-CMG2 cells were not killed by wild-type PA, they were susceptible to the PA variant, F427A. Receptor expression also conferred differences in susceptibility to edema factor.

scholarly journals Cryo-EM structure of the fully-loaded asymmetric anthrax lethal toxin in its heptameric pre-pore state

2020 ◽  
Author(s):  
Claudia Antoni ◽  
Dennis Quentin ◽  
Alexander E. Lang ◽  
Klaus Aktories ◽  
Christos Gatsogiannis ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Lethal Factor ◽  
Lethal Toxin ◽  
Anthrax Toxin ◽  
Anthrax Lethal Toxin ◽  
Terminal Domain ◽  
Edema Factor ◽  
Major Virulence Factor ◽  
Cell Substrate ◽  
Continuous Chain

AbstractAnthrax toxin is the major virulence factor secreted by Bacillus anthracis, causing high mortality in humans and other mammals. It consists of a membrane translocase, known as protective antigen (PA), that catalyzes the unfolding of its cytotoxic substrates lethal factor (LF) and edema factor (EF), followed by translocation into the host cell. Substrate recruitment to the heptameric PA pre-pore and subsequent translocation, however, are not well understood. Here, we report three high-resolution cryo-EM structures of the fully-loaded anthrax lethal toxin in its heptameric pre-pore state, which differ in the position and conformation of LFs. The structures reveal that three LFs interact with the heptameric PA and upon binding change their conformation to form a continuous chain of head-to-tail interactions. As a result of the underlying symmetry mismatch, one LF binding site in PA remains unoccupied. Whereas one LF directly interacts with a part of PA called α-clamp, the others do not interact with this region, indicating an intermediate state between toxin assembly and translocation. Interestingly, the interaction of the N-terminal domain with the α-clamp correlates with a higher flexibility in the C-terminal domain of the protein. Based on our data, we propose a model for toxin assembly, in which the order of LF binding determines which factor is translocated first.

scholarly journals Peptide- and proton-driven allosteric clamps catalyze anthrax toxin translocation across membranes

2016 ◽  
Vol 113 (34) ◽  
pp. 9611-9616 ◽  
Author(s):  
Debasis Das ◽  
Bryan A. Krantz
Keyword(s):  
Active Sites ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Anthrax Toxin ◽  
Sufficient Information ◽  
Binding Affinities ◽  
Transmembrane Channel ◽  
Edema Factor ◽  
Substrate Peptide ◽  
Catalytic Active Sites

Anthrax toxin is an intracellularly acting toxin in which sufficient information is available regarding the structure of its transmembrane channel, allowing for detailed investigation of models of translocation. Anthrax toxin, comprising three proteins—protective antigen (PA), lethal factor (LF), and edema factor—translocates large proteins across membranes. Here we show that the PA translocase channel has a transport function in which its catalytic active sites operate allosterically. We find that the phenylalanine clamp (ϕ-clamp), the known conductance bottleneck in the PA translocase, gates as either a more closed state or a more dilated state. Thermodynamically, the two channel states have >300-fold different binding affinities for an LF-derived peptide. The change in clamp thermodynamics requires distant α-clamp and ϕ-clamp sites. Clamp allostery and translocation are more optimal for LF peptides with uniform stereochemistry, where the least allosteric and least efficiently translocated peptide had a mixed stereochemistry. Overall, the kinetic results are in less agreement with an extended-chain Brownian ratchet model but, instead, are more consistent with an allosteric helix-compression model that is dependent also on substrate peptide coil-to-helix/helix-to-coil cooperativity.

scholarly journals Direct imaging of anthrax intoxication in animals reveals shared and individual functions of CMG-2 and TEM-8 in cellular toxin entry

2021 ◽  
Author(s):  
Carly Merritt ◽  
Elizabeth M. Chun ◽  
Rasem J. Fattah ◽  
Mahtab Moayeri ◽  
Dennis Paliga ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Fluorescent Protein ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Chimeric Protein ◽  
Anthrax Toxin ◽  
Specific Cell ◽  
Anthrax Lethal Toxin ◽  
Surface Receptors ◽  
Edema Factor

SUMMARYThe virulence of Bacillus anthracis is linked to the secretion of anthrax lethal toxin and anthrax edema toxin. These binary toxins consist of a common cell-binding moiety, protective antigen (PA), and the enzymatic moieties, lethal factor (LF) and edema factor (EF). PA binds either of two specific cell surface receptors, capillary morphogenesis protein-2 (CMG-2) or tumor endothelial marker-8 (TEM-8), which triggers the binding, endocytosis, and cytoplasmic translocation of LF and EF. The cellular distribution of functional TEM-8 and CMG-2 receptors during anthrax toxin intoxication in animals is not fully elucidated. Herein, we describe a novel assay to image anthrax toxin intoxication in live animals, and we use the assay to visualize TEM-8- and CMG-2-dependent intoxication. Specifically, we generated a chimeric protein consisting of the N-terminal domain of LF fused to a nuclear localization signal-tagged Cre recombinase (LFn-NLS-Cre). When PA and LFn-NLS-Cre were co-administered to transgenic mice that ubiquitously express a red fluorescent protein in the absence of Cre activity and a green fluorescent protein in the presence of Cre activity, anthrax toxin intoxication could be visualized at single-cell resolution by confocal microscopy. By using this assay, we show that CMG-2 is critical for intoxication in the liver and heart, whereas TEM-8 is required for full intoxication in the kidney and spleen. Other tissues examined were largely unaffected by single deficiences in either receptor, suggesting extensive overlap in TEM-8 and CMG-2 expression. The novel assay will be useful for basic and clinical/translational studies of Bacillus anthracis infection and for identifying on- and off-targets for reengineered toxin variants in the clinical development of cancer treatments.BackgroundAssays for imaging of anthrax toxin intoxication in animals are not available.ResultsAnthrax toxin-Cre fusions combined with fluorescent Cre reporter mice enabled imaging of anthrax toxin intoxication in animals.ConclusionShared and distinct functions of toxin receptors in cellular entry were uncovered. Significance. A simple and versatile assay for anthrax toxin intoxication is described.

scholarly journals Anthrax toxin blocks priming of neutrophils by lipopolysaccharide and by muramyl dipeptide.

1986 ◽  
Vol 164 (5) ◽  
pp. 1700-1709 ◽  
Author(s):  
G G Wright ◽  
G L Mandell
Keyword(s):  
Phorbol Myristate Acetate ◽  
Superoxide Anion ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Muramyl Dipeptide ◽  
Polymorphonuclear Neutrophils ◽  
Anthrax Toxin ◽  
Chemotactic Peptide ◽  
Myristate Acetate ◽  
Edema Factor

We studied the pretreatment of human polymorphonuclear neutrophils (PMN) with purified preparations of the anthrax toxin components--protective antigen (PA), edema factor (EF), and lethal factor (LF)--and their effects on release of superoxide anion (O-2) after stimulation with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). PMN isolated in the absence of lipopolysaccharide (LPS) (less than 0.1 ng/ml) released only small amounts of O-2 after FMLP stimulation; pretreatment with anthrax toxin had little effect. The release of O-2 was increased fivefold by prior treatment with 3 ng/ml LPS for 1 h at 37 degrees C, an effect referred to as priming. PMN were primed to an equivalent extent by treatment with 100 ng/ml N-acetyl-muramyl-L-alanyl-D-isoglutamine (muramyl dipeptide [MDP]). Pretreatment of PMN with anthrax toxin components PA plus EF or PA plus LF inhibited priming by LPS or MDP, as shown by the reduction in the release of O-2 up to 90% relative to controls not treated with toxin; single toxin components were inactive. The inhibition was markedly reduced when priming with LPS or MDP was carried out before exposure to toxin. O-2 release after stimulation by phorbol myristate acetate was not increased by priming, and pretreatment with toxin did not inhibit O-2 release after this stimulus. Evidently, anthrax toxin inhibits the priming that is normally induced in PMN by bacterial products and is necessary for the full expression of antimicrobial effects.

scholarly journals Anthrax toxin lethal factor domain 3 is highly mobile and responsive to ligand binding

2014 ◽  
Vol 70 (11) ◽  
pp. 2813-2822 ◽  
Author(s):  
Kimberly M. Maize ◽  
Elbek K. Kurbanov ◽  
Teresa De La Mora-Rey ◽  
Todd W. Geders ◽  
Dong-Jin Hwang ◽  
...  
Keyword(s):  
Small Molecule ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Anthrax Toxin ◽  
Mitogen Activated Protein Kinase ◽  
Host Immune System ◽  
Peptide Substrates ◽  
Edema Factor ◽  
Mitogen Activated Protein ◽  
Domain 3

The secreted anthrax toxin consists of three components: the protective antigen (PA), edema factor (EF) and lethal factor (LF). LF, a zinc metalloproteinase, compromises the host immune system primarily by targeting mitogen-activated protein kinase kinases in macrophages. Peptide substrates and small-molecule inhibitors bind LF in the space between domains 3 and 4 of the hydrolase. Domain 3 is attached on a hinge to domain 2viaresidues Ile300 and Pro385, and can move through an angular arc of greater than 35° in response to the binding of different ligands. Here, multiple LF structures including five new complexes with co-crystallized inhibitors are compared and three frequently populated LF conformational states termed `bioactive', `open' and `tight' are identified. The bioactive position is observed with large substrate peptides and leaves all peptide-recognition subsites open and accessible. The tight state is seen in unliganded and small-molecule complex structures. In this state, domain 3 is clamped over certain substrate subsites, blocking access. The open position appears to be an intermediate state between these extremes and is observed owing to steric constraints imposed by specific bound ligands. The tight conformation may be the lowest-energy conformation among the reported structures, as it is the position observed with no bound ligand, while the open and bioactive conformations are likely to be ligand-induced.

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