Review
The anthrax lethal factor and its MAPK kinase-specific metalloprotease activity

https://doi.org/10.1016/j.mam.2009.07.006Get rights and content

Abstract

The anthrax lethal factor is a multi-domain protein toxin released by Bacillus anthracis which enters cells in a process mediated by the protective antigen and specific cell receptors. In the cytosol, the lethal factor cleaves the N-terminal tail of many MAPK kinases, thus deranging a major cell signaling pathway. The structural features at the basis of these activities of LF are reviewed here with particular attention to the proteolytic activity and to the identification of specific inhibitors. A significant similarity between the metalloprotease domain of the lethal factor and of that of the clostridial neurotoxins has been noted and is discussed.

Introduction

The presence of exotoxins in culture filtrates of Bacillus anthracis was demonstrated long ago, but it was only in the fifties that Harry Smith and his colleagues isolated three proteins, termed protective antigen (PA, 83 kDa), oedema factor (EF, 89 kDa) and lethal factor (LF, 90 kDa). These names derive from a series of clever experiments showing that each of these factors were innocuous, whilst the combination of PA and LF caused death in intravenously-injected animals and PA plus EF caused oedema upon subcutaneous injection (Smith, 2002).

The genes encoding for PA, EF and LF are contained within a virulence plasmid termed pXO1 and their expression is regulated by host-specific factors such as elevated temperature (⩾37 °C) and carbon dioxide concentration (⩾5%), and by the presence of serum components, conditions similar to those met by the bacterium inside the mammalian host (Hudson et al., 2008). The expression system is under the regulation of the transcriptional activator AtxA, whose activity appears to be affected by the host-specific factors mentioned above (see review by Koehler in this volume).

These bacterial toxins have been studied much less intensively than other protein toxins, and the identification of their enzymatic activity followed accordingly. EF was shown to be an intracellular calmodulin-activated adenylate cyclase (Leppla, 1982) following the work done in previous years on cholera and pertussis toxins (Gill and Meren, 1978, Moss and Vaughan, 1979, Katada and Ui, 1979) and LF was demonstrated to be a zinc-metalloprotease specific for the N-terminus of most isoforms of the MAPK kinase (Duesbery et al., 1998, Vitale et al., 1998, Vitale et al., 2000) following the discovery of the metalloprotease activity of tetanus and botulinum neurotoxins (Schiavo et al., 1992a, Schiavo et al., 1992b).

The determination of their three-dimensional structures and ensuing mutational studies have revealed the structural basis of the multiple activities of EF and LF (Pannifer et al., 2001; review of Tang in this volume). In fact, as nearly all other bacterial protein toxins with intracellular targets do, EF and LF enter cells via a four-steps mechanism (Montecucco et al., 1994): (a) binding, (b) internalization, (c) membrane translocation and d) cytosolic action. To perform all these activities, these toxins are multi-domain proteins which have evolved by events of gene fusion, duplication and mutation (Ascenzi et al., 2002, Baldari et al., 2006). To trace the origin of these genetic events has been so far impossible, and sequence similarities are so little as to have given no clues, apart from providing the first hint that LF was a metalloprotease (Klimpel et al., 1994, Kochi et al., 1994). Only the determination of the LF crystallographic structure provided evolutionary indications still awaiting to be properly followed.

Section snippets

The crystallographic structure of LF

The 776-residues LF is an elongated protein (>10 nm) consisting of four domains (Pannifer et al., 2001). Domain 1 (residues 1–262) comprises a four-stranded and a two-stranded β-sheets (segment 78–138) packed together with a bundle of 9-helices, as shown in Fig. 1A (blue trace).1 This domain has a high structural similarity with domain 1 of EF (see overlap in Fig. 1B) indicating its function, which

Enzymatic activity

LF is an endopeptidase with highly restricted specificity. The only known protein substrates are mitogen-activated protein kinase kinases (MAPKKs), which are cleaved within their N-terminus (Vitale et al., 1998, Vitale et al., 2000, Liang et al., 2004). MAPKKs are recognized via multiple interactions which includes the cleaved N-terminal tails and other regions. This was first suggested by the fact that a clone encoding for a MAPKK lacking the N-terminus was found as prey in a yeast two hybrid

Conclusions and perspectives

The recent scientific history of the study of the anthrax lethal factor is almost incredible since its structure, function, mode of binding, routing into the cytosol and immunosuppressive properties were determined in less than ten years. It is a perfect example of how far the focused effort of state-of the-art science can go. Even so, much remains to be done. On the applied side, we expect that specific inhibitors, to be developed as therapeutics, will be found. On the more basic side, we

References (76)

  • S.L. Johnson et al.

    A high-throughput screening approach to anthrax lethal factor inhibition

    Bioorg. Chem.

    (2007)
  • S.L. Johnson et al.

    Structure–activity relationship studies of a novel series of anthrax lethal factor inhibitors

    Bioorg. Med. Chem.

    (2009)
  • S.K. Kochi et al.

    Zinc content of the Bacillus anthracis lethal factor

    FEMS Microbiol. Lett.

    (1994)
  • D.B. Lacy et al.

    Mapping the anthrax protective antigen binding site on the lethal and edema factors

    J. Biol. Chem.

    (2002)
  • X. Liang et al.

    Involvement of domain II in toxicity of anthrax lethal factor

    J. Biol. Chem.

    (2004)
  • C. Montecucco et al.

    Bacterial protein toxins penetrate cells via a four-step mechanism

    FEBS Lett.

    (1994)
  • C. Montecucco et al.

    Stop the killer: how to inhibit the anthrax lethal factor metalloprotease

    Trends Biochem. Sci.

    (2004)
  • T. Neumeyer et al.

    Anthrax edema factor, voltage-dependent binding to the protective antigen ion channel and comparison to LF binding

    J. Biol. Chem.

    (2006)
  • J. Peinado et al.

    Cross-inhibition between furin and lethal factor inhibitors

    Biochem. Biophys. Res. Commun.

    (2004)
  • M. Rigoni et al.

    Site-directed mutagenesis identifies active-site residues of the light chain of botulinum neurotoxin type A

    Biochem. Biophys. Res. Commun.

    (2001)
  • O. Rossetto et al.

    Active-site mutagenesis of tetanus neurotoxin implicates TYR-375 and GLU-271 in metalloproteolytic activity

    Toxicon

    (2001)
  • O. Rossetto et al.

    Tetanus and botulinum neurotoxins: turning bad guys into good by research

    Toxicon

    (2001)
  • G. Schiavo et al.

    Botulinum neurotoxins A, B and E are zinc proteins

    J. Biol. Chem.

    (1992)
  • F. Tonello et al.

    The metalloproteolytic activity of the anthrax lethal factor is substrate-inhibited

    J. Biol. Chem.

    (2003)
  • F. Tonello et al.

    Tyrosine-728 and glutamic acid-735 are essential for the metalloproteolytic activity of the lethal factor of Bacillus anthracis

    Biochem. Biophys. Res. Commun.

    (2004)
  • 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)
  • M. Zakharova et al.

    Substrate recognition of anthrax lethal factor examined by combinatorial and pre-steady-state kinetic approaches

    J. Biol. Chem.

    (2009)
  • Y. Xiong et al.

    The discovery of a potent and selective lethal factor inhibitor for adjunct therapy of anthrax infection

    Bioorg. Med. Chem. Lett.

    (2006)
  • A. Agrawal et al.

    Thioamide hydroxypyrothiones supersede amide hydroxypyrothiones in potency against anthrax lethal factor

    J. Med. Chem.

    (2009)
  • N. Arora et al.

    Fusions of anthrax toxin lethal factor with shiga toxin and diphtheria toxin enzymatic domains are toxic to mammalian cells

    Infect. Immun.

    (1994)
  • J. Ballard et al.

    Anthrax toxin-mediated delivery of a cytotoxic T-cell epitope in vivo

    Proc. Natl. Acad. Sci. USA

    (1996)
  • A. Bardwell et al.

    A conserved docking site in MEKs mediates high-affinity binding to MAP kinases and operates with a scaffold protein to enhance signal transmission

    J. Biol. Chem.

    (2001)
  • A. Bardwell et al.

    Anthrax lethal factor-cleavage products of MAPK (mitogen-activated protein kinase) kinases exhibit reduced binding to their cognate MAPKs

    Biochem. J.

    (2004)
  • F. Beall et al.

    Rapid lethal effect in rats of a third component found upon fractionating the toxin of Bacillus anthracis

    J. Bacteriol.

    (1962)
  • V. Chauhan et al.

    Identification of amino acid residues of anthrax protective antigen involved in binding with lethal factor

    Infect. Immun.

    (2002)
  • R.T. Cummings et al.

    A peptide-based fluorescence resonance energy transfer assay for Bacillus anthracis lethal factor protease

    Proc. Natl. Acad. Sci. USA

    (2002)
  • K. Cunningham et al.

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

    Proc. Natl. Acad. Sci. USA

    (2002)
  • F. Dal Molin et al.

    Cell entry and cAMP imaging of anthrax edema toxin

    EMBO J.

    (2006)
  • Cited by (67)

    • Bacillus anthracis and other Bacillus species

      2023, Molecular Medical Microbiology, Third Edition
    • Preparation of Clostridium perfringens binary iota-toxin pore complex for structural analysis using cryo-EM

      2021, Methods in Enzymology
      Citation Excerpt :

      On the other hand, Bacillus anthracis also produces an A-B binary toxin called anthrax toxin (Collier & Young, 2003), which is composed of either edema factor (EF) or lethal factor (LF) as the A component and protective antigen (PA) as the B component; thus, the functional binary toxin is either EF-PA or LF-PA. EF is a calmodulin-dependent adenyl cyclase, while LF is a Zn2 +-dependent protease that cleaves mitogen-activated-protein kinase (Tang & Guo, 2009; Tonello & Montecucco, 2009). As described in the clostridial binary toxin section, EF and LF are translocated into the target cell via the pore formed by PA and causes damage to the target cells.

    • Anthrax lethal toxin rapidly reduces c-Jun levels by inhibiting c-Jun gene transcription and promoting c-Jun protein degradation

      2017, Journal of Biological Chemistry
      Citation Excerpt :

      Extracellular stimuli such as growth factors or cytokines initiate activation of MKKKs that phosphorylate MKKs, which, in turn, phosphorylate MAPKs. LF cleavage of MKKs at their docking sites (D sites) disrupts the activation of MAPKs, including Erk1/2, which are activated by MKK1 and MKK2; p38 MAPKs, which are activated by MKK3 and MKK6; and JNKs, which are activated by MKK4 and MKK7 (7, 12–14). Studies from our laboratory and others have revealed that LT treatment induces cell cycle arrest and inhibition of cell proliferation (15–20).

    • High metal substitution tolerance of anthrax lethal factor and characterization of its active copper-substituted analogue

      2014, Journal of Inorganic Biochemistry
      Citation Excerpt :

      This amino acid residue in LF (Tyr728) has been implicated mainly in the protonation of the amine leaving group following peptide bond cleavage [13,52,53], although recent theoretical calculations suggest that it might be involved in stabilizing the tetrahedral intermediate (as has been postulated for thermolysin) [54]. Nonetheless, the observation of LF and TeNT being highly activated by Mn2 + has served as the basis for postulating a closer structural relationship of LF to clostridial neurotoxins than to thermolysin [52]. Furthermore, a notable metal substitution tolerance has also been observed with astacin and serralysin (see Table 3), both of which harbour a Tyr residue (Tyr149 and Tyr216 in astacin and serralysin, respectively) serving as a coordinating ligand in the active sites of these enzymes.

    View all citing articles on Scopus
    View full text