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 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 Edema Toxin Sensitizes DBA/2J Mice to Lethal Toxin

2007 ◽  
Vol 75 (5) ◽  
pp. 2120-2125 ◽  
Author(s):  
Aaron M. Firoved ◽  
Mahtab Moayeri ◽  
Jason F. Wiggins ◽  
Yuequan Shen ◽  
Wei-Jen Tang ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Lethal Factor ◽  
Endocrine System ◽  
Lethal Toxin ◽  
Anthrax Toxin ◽  
Edema Factor ◽  
Edema Toxin ◽  
Separate Protein ◽  
Protein Components ◽  
Higher Doses

ABSTRACT Anthrax toxin is made up of three separate protein components: the receptor-binding protective antigen (PA), the adenylyl cyclase edema factor (EF), and the metalloproteinase lethal factor (LF). EF and PA constitute edema toxin (ET), which causes edema when injected subcutaneously. At higher doses, ET causes severe pathologies and death in BALB/cJ mice (A. M. Firoved et al., Am. J. Pathol. 167:1309-1320, 2005). A striking effect of ET at lethal doses is adrenal necrosis. Here we show that low doses of ET (10 μg) that produce no overt signs of illness in mice still cause substantial adrenal lesions. These lesions are not associated with reduced corticosterone production; instead, ET-treated mice have increased corticosterone production. Because the resistance of mice to the other component of anthrax toxin, lethal toxin (LT; LF plus PA), has been shown to be overcome by the perturbation of the endocrine system, we hypothesized that sublethal doses of ET might sensitize LT-resistant DBA/2J mice to LT-mediated lethality. We report that a low dose of ET (5 μg) is sufficient to sensitize DBA/2J mice when given concurrently with LT. Higher doses of ET (e.g., 15 μg) given to male and female DBA/2J mice 18 h prior to LT challenge also sensitize them to LT. This study using highly purified ET and LT demonstrates how the components of anthrax toxin can work together to increase lethality.

scholarly journals Cross-Linked Forms of the Isolated N-Terminal Domain of the Lethal Factor Are Potent Inhibitors of Anthrax Toxin

2007 ◽  
Vol 75 (10) ◽  
pp. 5052-5058 ◽  
Author(s):  
Stephen J. Juris ◽  
Roman A. Melnyk ◽  
Robert E. Bolcome ◽  
Joanne Chan ◽  
R. John Collier
Keyword(s):  
Mammalian Cells ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Anthrax Toxin ◽  
Zebrafish Model ◽  
Ion Conductance ◽  
New Approach ◽  
Terminal Domain ◽  
Edema Factor ◽  
C Terminus

ABSTRACT The proteins that comprise anthrax toxin self-assemble at the mammalian cell surface into a series of toxic complexes, each containing a heptameric form of protective antigen (PA) plus up to a total of three molecules of the enzymatic moieties of the toxin (lethal factor [LF] and edema factor [EF]). These complexes are trafficked to the endosome, where the PA heptamer forms a pore in the membrane under the influence of low pH, and bound LF and EF unfold and translocate through the pore to the cytosol. To explore the hypothesis that the PA pore can translocate multiple, cross-linked polypeptides simultaneously, we cross-linked LFN, the N-terminal domain of LF, via an introduced cysteine at its N or C terminus and characterized the products. Both dimers and trimers of LFN retained the ability to bind to PA pores and block ion conductance, but they were unable to translocate across the membrane, even at high voltages or with a transmembrane pH gradient. The multimers were remarkably potent inhibitors of toxin action in mammalian cells (20- to 50-fold more potent than monomeric LFN) and in a zebrafish model system. These findings show that the PA pore cannot translocate multimeric, cross-linked polypeptides and demonstrate a new approach to generating potent inhibitors of anthrax toxin.

scholarly journals Inactivation of Rho GTPases by Statins Attenuates Anthrax Lethal Toxin Activity

2008 ◽  
Vol 77 (1) ◽  
pp. 348-359 ◽  
Author(s):  
Aimee M. deCathelineau ◽  
Gary M. Bokoch
Keyword(s):  
Rho Gtpases ◽  
Rho Gtpase ◽  
Protective Antigen ◽  
Biological Effects ◽  
Lethal Factor ◽  
Lethal Toxin ◽  
Mitogen Activated Protein Kinase ◽  
Gtpase Activity ◽  
Anthrax Lethal Toxin ◽  
Reductase Inhibitors

ABSTRACT Anthrax lethal factor (LF), secreted by Bacillus anthracis, interacts with protective antigen to form a bipartite toxin (lethal toxin [LT]) that exerts pleiotropic biological effects resulting in subversion of the innate immune response. Although the mitogen-activated protein kinase kinases (MKKs) are the major intracellular protein targets of LF, the pathology induced by LT is not well understood. The statin family of HMG-coenzyme A reductase inhibitors have potent anti-inflammatory effects independent of their cholesterol-lowering properties, which have been attributed to modulation of Rho family GTPase activity. The Rho GTPases regulate vesicular trafficking, cytoskeletal dynamics, and cell survival and proliferation. We hypothesized that disruption of Rho GTPase function by statins might alter LT action. We show here that statins delay LT-induced death and MKK cleavage in RAW macrophages and that statin-mediated effects on LT action are attributable to disruption of Rho GTPases. The Rho GTPase-inactivating toxin, toxin B, did not significantly affect LT binding or internalization, suggesting that the Rho GTPases regulate trafficking and/or localization of LT once internalized. The use of drugs capable of inhibiting Rho GTPase activity, such as statins, may provide a means to attenuate intoxication during B. anthracis infection.

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 Plant-Based Vaccine: Mice Immunized with Chloroplast-Derived Anthrax Protective Antigen Survive Anthrax Lethal Toxin Challenge

2005 ◽  
Vol 73 (12) ◽  
pp. 8266-8274 ◽  
Author(s):  
Vijay Koya ◽  
Mahtab Moayeri ◽  
Stephen H. Leppla ◽  
Henry Daniell
Keyword(s):  
Protective Antigen ◽  
Expression System ◽  
Lethal Factor ◽  
Vaccine Antigen ◽  
Total Soluble Protein ◽  
Continuous Illumination ◽  
Anthrax Lethal Toxin ◽  
Anthrax Vaccine ◽  
Edema Factor ◽  
Subcutaneous Immunization

ABSTRACT The currently available human vaccine for anthrax, derived from the culture supernatant of Bacillus anthracis, contains the protective antigen (PA) and traces of the lethal and edema factors, which may contribute to adverse side effects associated with this vaccine. Therefore, an effective expression system that can provide a clean, safe, and efficacious vaccine is required. In an effort to produce anthrax vaccine in large quantities and free of extraneous bacterial contaminants, PA was expressed in transgenic tobacco chloroplasts by inserting the pagA gene into the chloroplast genome. Chloroplast integration of the pagA gene was confirmed by PCR and Southern analysis. Mature leaves grown under continuous illumination contained PA as up to 14.2% of the total soluble protein. Cytotoxicity measurements in macrophage lysis assays showed that chloroplast-derived PA was equal in potency to PA produced in B. anthracis. Subcutaneous immunization of mice with partially purified chloroplast-derived or B. anthracis-derived PA with adjuvant yielded immunoglobulin G titers up to 1:320,000, and both groups of mice survived (100%) challenge with lethal doses of toxin. An average yield of about 150 mg of PA per plant should produce 360 million doses of a purified vaccine free of bacterial toxins edema factor and lethal factor from 1 acre of land. Such high expression levels without using fermenters and the immunoprotection offered by the chloroplast-derived PA should facilitate development of a cleaner and safer anthrax vaccine at a lower production cost. These results demonstrate the immunogenic and immunoprotective properties of plant-derived anthrax vaccine antigen.

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.

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