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 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 Oligomerization of Anthrax Toxin Protective Antigen and Binding of Lethal Factor during Endocytic Uptake into Mammalian Cells

1999 ◽  
Vol 67 (4) ◽  
pp. 1853-1859 ◽  
Author(s):  
Yogendra Singh ◽  
Kurt R. Klimpel ◽  
Seema Goel ◽  
Prabodha K. Swain ◽  
Stephen H. Leppla
Keyword(s):  
Gel Electrophoresis ◽  
Mammalian Cells ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Anthrax Toxin ◽  
Proteolytic Activation ◽  
Edema Factor ◽  
Plastic Surface ◽  
Kinetics Of

ABSTRACT The protective antigen (PA) protein of anthrax toxin binds to a cellular receptor and is cleaved by cell surface furin to produce a 63-kDa fragment (PA63). The receptor-bound PA63 oligomerizes to a heptamer and acts to translocate the catalytic moieties of the toxin, lethal factor (LF) and edema factor (EF), from endosomes to the cytosol. In this report, we used nondenaturing gel electrophoresis to show that each PA63 subunit in the heptamer can bind one LF molecule. Studies using PA immobilized on a plastic surface showed that monomeric PA63 is also able to bind LF. The internalization of PA and LF by cells was studied with radiolabeled and biotinylated proteins. Uptake was relatively slow, with a half-time of 30 min. The number of moles of LF internalized was nearly equal to the number of moles of PA subunit internalized. The essential role of PA oligomerization in LF translocation was shown with PA protein cleaved at residues 313-314. The oligomers formed by these proteins during uptake into cells were not as stable when subjected to heat and detergent as were those formed by native PA. The results show that the structure of the toxin proteins and the kinetics of proteolytic activation, LF binding, and internalization are balanced in a way that allows each PA63 subunit to internalize an LF molecule. This set of proteins has evolved to achieve highly efficient internalization and membrane translocation of the catalytic components, LF and EF.

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.

Oligomerization of Anthrax Toxin Protective Antigen and Binding of Lethal Factor during Endocytic Uptake into Mammalian Cells

1999 ◽  
Vol 67 (4) ◽  
pp. 1853-1859
Author(s):  
Yogendra Singh ◽  
Kurt R. Klimpel ◽  
Seema Goel ◽  
Prabodha K. Swain ◽  
Stephen H. Leppla
Keyword(s):  
Mammalian Cells ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Anthrax Toxin

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 Cytosolic Delivery and Characterization of the TcdB Glucosylating Domain by Using a Heterologous Protein Fusion

2001 ◽  
Vol 69 (1) ◽  
pp. 599-601 ◽  
Author(s):  
Lea M. Spyres ◽  
Maen Qa'Dan ◽  
Amy Meader ◽  
James J. Tomasek ◽  
Eric W. Howard ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Small Gtpases ◽  
Anthrax Toxin ◽  
Coding Region ◽  
Protein Fusion ◽  
Actin Reorganization ◽  
Amino Terminal ◽  
Cell Extracts

ABSTRACT TcdB from Clostridium difficile glucosylates small GTPases (Rho, Rac, and Cdc42) and is an important virulence factor in the human disease pseudomembranous colitis. In these experiments, in-frame genetic fusions between the genes for the 255 amino-terminal residues of anthrax toxin lethal factor (LFn) and the TcdB1-556 coding region were constructed, expressed, and purified from Escherichia coli. LFnTcdB1-556was enzymatically active and glucosylated recombinant RhoA, Rac, Cdc42, and substrates from cell extracts. LFnTcdB1-556 plus anthrax toxin protective antigen intoxicated cultured mammalian cells and caused actin reorganization and mouse lethality, all similar to those caused by wild-type TcdB.

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 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 Cytotoxic T-Lymphocyte Epitopes Fused to Anthrax Toxin Induce Protective Antiviral Immunity

1999 ◽  
Vol 67 (7) ◽  
pp. 3290-3296 ◽  
Author(s):  
Amy M. Doling ◽  
Jimmy D. Ballard ◽  
Hao Shen ◽  
Kaja Murali Krishna ◽  
Rafi Ahmed ◽  
...  
Keyword(s):  
Fusion Protein ◽  
T Lymphocyte ◽  
Mammalian Cells ◽  
Protective Antigen ◽  
Cytotoxic T Lymphocyte ◽  
Lethal Factor ◽  
Antiviral Immunity ◽  
Anthrax Toxin ◽  
Listeriolysin O

ABSTRACT We have investigated the use of the protective antigen (PA) and lethal factor (LF) components of anthrax toxin as a system for in vivo delivery of cytotoxic T-lymphocyte (CTL) epitopes. During intoxication, PA directs the translocation of LF into the cytoplasm of mammalian cells. Here we demonstrate that antiviral immunity can be induced in BALB/c mice immunized with PA plus a fusion protein containing the N-terminal 255 amino acids of LF (LFn) and an epitope from the nucleoprotein (NP) of lymphocytic choriomeningitis virus. We also demonstrate that BALB/c mice immunized with a single LFn fusion protein containing NP and listeriolysin O protein epitopes in tandem mount a CTL response against both pathogens. Furthermore, we show that NP-specific CTL are primed in both BALB/c and C57BL/6 mice when the mice are immunized with a single fusion containing two epitopes, one presented by Ld and one presented by Db. The data presented here demonstrate the versatility of the anthrax toxin delivery system and indicate that this system may be used as a general approach to vaccinate outbred populations against a variety of pathogens.

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