Review
Membrane translocation by anthrax toxin

https://doi.org/10.1016/j.mam.2009.06.003Get rights and content

Abstract

Much attention has been focused on anthrax toxin recently, both because of its central role in the pathogenesis of Bacillus anthracis and because it has proven to be one of the most tractable toxins for studying how enzymic moieties of intracellularly acting toxins traverse membranes. The Protective Antigen (PA) moiety of the toxin, after being proteolytically activated at the cell surface, self-associates to form a heptameric pore precursor (prepore). The prepore binds up to three molecules of Edema Factor (EF), Lethal Factor (LF), or both, forming a series of complexes that are then endocytosed. Under the influence of acidic pH within the endosome, the prepore undergoes a conformational transition to a mushroom-shaped pore, with a globular cap and 100 Å-long stem that spans the membrane. Electrophysiological studies in planar bilayers indicate that EF and LF translocate through the pore in unfolded form and in the N- to C-terminal direction. The pore serves as an active transporter, which translocates its proteinaceous cargo across the endosomal membrane in response to ΔpH and perhaps, to a degree, Δψ. A ring of seven Phe residues (Phe427) in the lumen of the pore forms a seal around the translocating polypeptide and blocks the passage of ions, presumably preserving the pH gradient. A charge state-dependent Brownian ratchet mechanism has been proposed to explain how the pore translocates EF and LF. This transport mechanism of the pore may function in concert with molecular chaperonins to effect delivery of effector proteins in catalytically active form to the cytosolic compartment of host cells.

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)

  • A. Puhar et al.

    Where and how do anthrax toxins exit endosomes to intoxicate host cells?

    Trend. Microbiol.

    (2007)
  • C.P. Quinn et al.

    Functional mapping of anthrax toxin lethal factor by in-frame insertion mutagenesis

    J. Biol. Chem.

    (1991)
  • S.L. Rybak et al.

    Theoretical considerations on the role of membrane potential in the regulation of endosomal pH

    Biophys. J.

    (1997)
  • G.E. Schulz

    Bacterial porins: structure and function

    Curr. Opin. Cell. Biol.

    (1993)
  • B.R. Sellman et al.

    Point mutations in anthrax protective antigen that block translocation

    J. Biol. Chem.

    (2001)
  • Y. Singh et al.

    A deleted variant of Bacillus anthracis protective antigen is non-toxic and blocks anthrax toxin action in vivo

    J. Biol. Chem.

    (1989)
  • Y. Singh et al.

    A dominant negative mutant of Bacillus anthracis protective antigen inhibits anthrax toxin action in vivo

    J. Biol. Chem.

    (2001)
  • Y. Singh et al.

    The carboxyl-terminal end of protective antigen is required for receptor binding and anthrax toxin activity

    J. Biol. Chem.

    (1991)
  • H. Smith

    Discovery of the anthrax toxin: the beginning of in vivo studies on pathogenic bacteria

    Trend. Microbiol.

    (2000)
  • N.D. Sonawane et al.

    Chloride concentration in endosomes measured using a ratioable fluorescent Cl-indicator: evidence for chloride accumulation during acidification

    J. Biol. Chem.

    (2002)
  • J. Sun et al.

    Insertion of anthrax protective antigen into liposomal membranes: effects of a receptor

    J. Biol. Chem.

    (2007)
  • G. Vitale et al.

    Anthrax lethal factor cleaves the N-terminus of MAPKKs and induces tyrosine/threonine phosphorylation of MAPKs in cultured macrophages

    Biochem. Biophys. Res. Commun.

    (1998)
  • D.J. Wigelsworth et al.

    Binding stoichiometry and kinetics of the interaction of a human anthrax toxin receptor, CMG2, with protective antigen

    J. Biol. Chem.

    (2004)
  • S. Zhang et al.

    Protein translocation through anthrax toxin channels formed in planar lipid bilayers

    Biophys. J.

    (2004)
  • J. Zhao et al.

    Effect of anthrax toxin’s lethal factor on ion channels formed by the protective antigen

    J. Biol. Chem.

    (1995)
  • D. Basilio et al.

    Evidence for a proton–protein symport mechanism in the anthrax toxin channel

    J. Gen. Physiol.

    (2009)
  • E.L. Benson et al.

    Identification of residues lining the anthrax protective antigen channel

    Biochemistry

    (1998)
  • S.R. Blanke et al.

    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

    (1996)
  • R.O. Blaustein et al.

    Voltage-dependent block of anthrax toxin channels in planar phospholipid bilayer membranes by symmetric tetraalkylammonium ions. Effects on macroscopic conductance

    J. Gen. Physiol.

    (1990)
  • R.O. Blaustein et al.

    Anthrax toxin: channel-forming activity of protective antigen in planar phospholipid bilayers

    Proc. Natl. Acad. Sci. USA

    (1989)
  • K.A. Bradley et al.

    Identification of the cellular receptor for anthrax toxin

    Nature

    (2001)
  • R.J. Collier et al.

    Anthrax toxin

    Annu. Rev. Cell. Dev. Biol.

    (2003)
  • X. Cui et al.

    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.

    (2007)
  • K. Cunningham et al.

    Mapping the lethal factor and edema factor binding sites on oligomeric anthrax protective antigen

    Proc. Natl. Acad. Sci. USA

    (2002)
  • C.L. Drum et al.

    Structural basis for the activation of anthrax adenylyl cyclase exotoxin by calmodulin

    Nature

    (2002)
  • N.S. Duesbery et al.

    Proteolytic inactivation of MAP-kinase–kinase by anthrax lethal factor

    Science

    (1998)
  • J.L. Elliott et al.

    A quantitative study of the interactions of Bacillus anthracis edema factor and lethal factor with activated protective antigen

    Biochemistry

    (2000)
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