ReviewMembrane translocation by anthrax toxin
Introduction
For many disease-causing bacteria a key strategy for survival within mammalian hosts is to deliver selected enzymes (effector proteins) into the cytosol of host cells, primarily with the aim of killing or disabling key cellular elements of the immune system. Delivery of an effector protein requires specialized machinery to enable it to cross a membrane at some level within the host cell, and much attention has been devoted to defining such machinery and understanding how it functions. Some of the simplest and most extensively studied systems for protein translocation are found in intracellularly acting bacterial toxins. Our current understanding of translocation by anthrax toxin, which has proven to be one of the most tractable toxins for studying this process, is summarized here. Readers may also wish to consult other recent reviews relevant to this topic (Collier and Young, 2003, Finkelstein, 2009, Puhar and Montecucco, 2007, Young and Collier, 2007).
Anthrax toxin is an ensemble of three large, multidomain proteins, which are secreted from Bacillus anthracis as monomers and self-assemble on receptor-bearing cells into a series of toxic, hetero-oligomeric complexes (Pimental et al., 2004, Smith, 2000). Two of the proteins are enzymic intracellular effectors: Lethal Factor (LF, 90 kDa), a Zn++-dependent protease (Duesbery et al., 1998, Vitale et al., 1998), and Edema Factor (EF, 89 kDa), a Ca++- and calmodulin-dependent adenylyl cyclase (Leppla, 1982). The third is a receptor binding and pore-forming protein, called Protective Antigen (PA, 83 kDa), which transports EF and LF from the extracellular milieu to the cytosolic compartment of mammalian cells. EF and LF can be transported to the cytosol by PA and act independently of one other, a fact that has given rise to the terms Edema Toxin, EdTx, and Lethal Toxin, LeTx, for the binary combinations, EF + PA and LF + PA, respectively (Ezzell et al., 1984, Friedlander, 1986). However, all three components of the toxin are produced during B. anthracis infections, and can combine to form ternary complexes as well as binary complexes during self-assembly at the cell surface (Pimental et al., 2004). In addition, any given host cell that is affected by EF is almost certainly affected by LF, and vice versa; and there is evidence of synergy between these two effector proteins (Cui et al., 2007, Rossi Paccani et al., 2007, Tournier et al., 2005). Thus, while the terms Edema Toxin and Lethal Toxin are useful in analyzing and describing experimental findings, it is also appropriate to think of the ensemble of PA, EF, and LF as a single system.
Section snippets
Activation, acidic pH, and pore formation
Leppla and co-workers showed that PA must be proteolytically activated in order to interact with LF and EF (Singh et al., 1989). The activation involves cleavage of the native protein into N-terminal 20-kDa and C-terminal 63-kDa fragments (PA20 and PA63, respectively) and may be effected in vivo by cell-associated furin-family proteases (Klimpel et al., 1992) or by proteases in the blood of animals (Ezzell and Abshire, 1992, Moayeri et al., 2007). For research purposes trypsin is commonly used
Systems for study
A cell-based assay developed by Olsnes and co-workers for probing the translocation of diphtheria toxin across the plasma membrane was adapted to anthrax toxin (Falnes et al., 1994, Wesche et al., 1998). In that assay, radiolabeled translocation ligands were bound to proteolytically activated PA at the surface of CHO or L6 cells, and translocation was induced by lowering the pH of the medium. The cells were then treated with Pronase E to degrade exposed label at the cell surface, and
Acknowledgements
Work in the author’s laboratory on anthrax toxin has been supported by NIH Grant AI022021. Some of the proteins used were produced by the Biomolecule Production Core under the New England Regional Center of Excellence under Grant AI057159. The author holds equity in PharmAthene, Inc.
References (74)
- et al.
Nucleotide sequence and analysis of the lethal factor gene (lef) from Bacillus anthracis
Gene
(1989) - et al.
Inhibition of membrane translocation of diphtheria toxin A-fragment by internal disulfide bridges
J. Biol. Chem.
(1994) Macrophages are sensitive to anthrax lethal toxin through an acid-dependent process
J. Biol. Chem.
(1986)- et al.
Exchange characteristics of calcium ions bound to anthrax protective antigen
Biochem. Biophys. Res. Commun.
(2003) - et al.
Protein translocation through the anthrax toxin transmembrane pore is driven by a proton gradient
J. Mol. Biol.
(2006) - et al.
Acid-induced unfolding of the amino-terminal domains of the lethal and edema factors of anthrax toxin
J. Mol. Biol.
(2004) - et al.
Structural determinants for the binding of anthrax lethal factor to oligomeric protective antigen
J. Biol. Chem.
(2006) - et al.
Anthrax protective antigen forms oligomers during intoxication of mammalian cells
J. Biol. Chem.
(1994) - et al.
Anthrax toxin protective antigen: inhibition of channel function by chloroquine and related compounds and study of binding kinetics using the current noise analysis
Biophys. J.
(2005) - et al.
Anthrax toxin complexes: heptameric protective antigen can bind lethal factor and edema factor simultaneously
Biochem. Biophys. Res. Commun.
(2004)
Where and how do anthrax toxins exit endosomes to intoxicate host cells?
Trend. Microbiol.
Functional mapping of anthrax toxin lethal factor by in-frame insertion mutagenesis
J. Biol. Chem.
Theoretical considerations on the role of membrane potential in the regulation of endosomal pH
Biophys. J.
Bacterial porins: structure and function
Curr. Opin. Cell. Biol.
Point mutations in anthrax protective antigen that block translocation
J. Biol. Chem.
A deleted variant of Bacillus anthracis protective antigen is non-toxic and blocks anthrax toxin action in vivo
J. Biol. Chem.
A dominant negative mutant of Bacillus anthracis protective antigen inhibits anthrax toxin action in vivo
J. Biol. Chem.
The carboxyl-terminal end of protective antigen is required for receptor binding and anthrax toxin activity
J. Biol. Chem.
Discovery of the anthrax toxin: the beginning of in vivo studies on pathogenic bacteria
Trend. Microbiol.
Chloride concentration in endosomes measured using a ratioable fluorescent Cl-indicator: evidence for chloride accumulation during acidification
J. Biol. Chem.
Insertion of anthrax protective antigen into liposomal membranes: effects of a receptor
J. Biol. Chem.
Anthrax lethal factor cleaves the N-terminus of MAPKKs and induces tyrosine/threonine phosphorylation of MAPKs in cultured macrophages
Biochem. Biophys. Res. Commun.
Binding stoichiometry and kinetics of the interaction of a human anthrax toxin receptor, CMG2, with protective antigen
J. Biol. Chem.
Protein translocation through anthrax toxin channels formed in planar lipid bilayers
Biophys. J.
Effect of anthrax toxin’s lethal factor on ion channels formed by the protective antigen
J. Biol. Chem.
Evidence for a proton–protein symport mechanism in the anthrax toxin channel
J. Gen. Physiol.
Identification of residues lining the anthrax protective antigen channel
Biochemistry
Fused polycationic peptide mediates delivery of diphtheria toxin A chain to the cytosol in the presence of anthrax protective antigen
Proc. Natl. Acad. Sci. USA
Voltage-dependent block of anthrax toxin channels in planar phospholipid bilayer membranes by symmetric tetraalkylammonium ions. Effects on macroscopic conductance
J. Gen. Physiol.
Anthrax toxin: channel-forming activity of protective antigen in planar phospholipid bilayers
Proc. Natl. Acad. Sci. USA
Identification of the cellular receptor for anthrax toxin
Nature
Anthrax toxin
Annu. Rev. Cell. Dev. Biol.
Bacillus anthracis edema and lethal toxin have different hemodynamic effects but function together to worsen shock and outcome in a rat model
J. Infect. Dis.
Mapping the lethal factor and edema factor binding sites on oligomeric anthrax protective antigen
Proc. Natl. Acad. Sci. USA
Structural basis for the activation of anthrax adenylyl cyclase exotoxin by calmodulin
Nature
Proteolytic inactivation of MAP-kinase–kinase by anthrax lethal factor
Science
A quantitative study of the interactions of Bacillus anthracis edema factor and lethal factor with activated protective antigen
Biochemistry
Cited by (127)
Anthrax Toxin: Model System for Studying Protein Translocation
2024, Journal of Molecular BiologyBacterial AB toxins and host–microbe interactions
2022, Advances in Microbial PhysiologyCitation Excerpt :PA63 bound to its receptor is self-assembled into a heptameric pre-pore structure, which further induces the seven bound receptors to cluster in lipid raft-microdomains, followed by toxin endocytosis. The PA63 heptamer binds up to three LF or EF subunits through direct interactions between the PA63 and each of up to three LF or EF (Collier, 2009) (Fig. 2). This holotoxin formation activates src-like kinases to initiate toxin endocytosis, induce a conformational change in the PA63 heptamer that is favorable for the translocation of LF or EF from the vesicle to the cytoplasm (Collier, 2009; Young & Collier, 2007).
Anthrax toxin channel: What we know based on over 30 years of research
2021, Biochimica et Biophysica Acta - BiomembranesABC toxins: Self-assembling nanomachines for the targeted cellular delivery of bioactive proteins
2019, Comprehensive Nanoscience and NanotechnologyEffect of late endosomal DOBMP lipid and traditional model lipids of electrophysiology on the anthrax toxin channel activity
2018, Biochimica et Biophysica Acta - BiomembranesCitation Excerpt :To avoid confusion with the bone morphogenetic protein, which is a popular growth factor also abbreviated as BMP, here we will refer to bis(monoacylglycero)phosphate as BMP/LBPA. The previously reported model bilayer membrane anthrax toxin translocation experiments (reviewed in refs. [10, 38, 39]), in all their elegance and sophistication, were exclusively performed in painted solvent-containing diphytanoylphosphatidylcholine (DPhPC) bilayers. This synthetic branched-chain lamellar lipid, due to its unique properties, such as high chemical and physical stability and low water and ion permeability, is commonly used in model bilayer lipid experiments to investigate ion channel behavior [40–43].
Direct Detection of Membrane-Inserting Fragments Defines the Translocation Pores of a Family of Pathogenic Toxins
2018, Journal of Molecular Biology