Anthrax Another Reason to Fear Your Mailman By Stefko Waschuk R.C. Liddington, Nature, 415 : (2002)

Outline General Information Pathogenic components Treatment / Management Therapeutic uses

General Information From Bacillus anthracis Two primary forms –cutaneous anthrax (usually curable) –systemic anthrax (usually lethal) Encoded by 2 additional plasmids in genome –pXO1 (184.5 kbp) anthrax toxin  oedema factor (EF), lethal factor (LF), and protective antigen (PA) –pXO2 (95.3 kbp) poly-D-glutamic acid capsule

B. Anthracis cycle M. Mock, A. Fouet, Annu. Rev. Microbiol. 55 : (2001)

Cutaneous Anthrax 95% of all cases Characterized by –tissue swelling (oedema) –skin lesion –impaired neutrophil function Usually self-limiting –80-90% of cases resolve without complication

Cutaneous Anthrax T.C. Dixon. et al. New England Journal of Medicine, 341: (1999)

Systemic Anthrax Mortality rate ~100% Spores germinate within macrophage Toxin released into bloodstream –Toxemia and septicemia Shock and death

So, How Does It Kill Me? M. Mourez et al. Trends Microbiol. 10: (2002)

Requirements for Pathogenesis Anthrax Toxin Receptor Protective Antigen Lethal Factor and/or Oedema Factor

Anthrax Toxin Receptor (ATR) Type I membrane protein Extracellular von Willebrand factor A domain –Directly binds to PA large extracellular domain with 3 N-linked glycosylation sites Highly conserved between different species

Protective Antigen (PA) 83 kDa protein 4 domains Binds ATR Activation requires cleavage Mediates delivery of EF & LF into host cells

Protective Antigen M. Mourez et al. Trends Microbiol. 10: (2002)

Mode of Anthrax Toxin Entry M. Mourez et al. Nature Biotech., 19: (2001)

PA Heptamer M. Mourez et al. Trends Microbiol. 10: (2002)

Lethal Factor (LF) 90 kDa zinc-dependent protease 7 N-terminal residues critical for PA binding Large homology with EF

PA Recognition Site on LF/EF

Lethal Factor Structure A.D. Pannifer et al. Nature, 414: (2001)

Lethal Factor Surgical protease –Cleaves 1 specific bond near N-terminus of six known MAPKKs Removes the docking sequence for MAPK –Lethal effects by unknown mechanism Cleavage of MAPKK inhibits release of pro- inflammatory cytokines

Oedema Factor (EF) 89 kDa adenylate cyclase Contributes to both cutaneous and systemic anthrax Impairs phagocytosis in macrophages Identical 7 PA binding residues as LF Requires activation by calmodulin (CaM)

Oedema Factor Structure (inactive) Active site in interface of C A and C B Catalytic machinery is present, but disordered C.L. Drum et al. Nature, 415: (2002)

Oedema Factor Structure (active) CaM displaces helical domain Switch B becomes ordered –binds ATP –stabilizes EF catalytic residues C.L. Drum et al. Nature, 415: (2002)

More Fun with CaM binding Large binding surface stabilizes structural changes ATP locked into catalytic site by salt bridge Conformational changes to active site do not directly involve catalytic residues –become exposed to solvent in active state

Effects of EF Activation EF-CaM forms an irreversible complex –CaM forced into extended conformation Adenylate cyclase becomes active Conversion of ATP  cAMP Increased [cAMP] perturbs immune effector cell functions –Phagocytosis –Chemotactic response –Cytokine expression

Summary: Anthrax Toxin Action M. Mourez et al. Trends Microbiol. 10: (2002)

Anthrax Toxin Management Vaccinations Antibiotics Other strategies –Polyclonal antibodies –Synthetic inhibitors

Vaccinations Anthrax vaccine adsorbed (AVA) Made from protective antigen

Antibiotics Ciprofloxacin Hydrochloride –C 17 H 18 FN 3 O 3.HCl.H 2 O Cutaneous Anthrax  ~100% effective Systemic Anthrax  before symptomatic

Synthetic Inhibitors EF/LF binding analogues Mutated PA Soluble ATR

EF/LF Binding Analogues M. Mourez et al. Trends Microbiol. 10: (2002)

Domain II Mutant PA M. Mourez et al. Trends Microbiol. 10: (2002)

Domain III Mutant PA J. Mogridge et al. PNAS. 99: (2002)

Soluble ATR M. Mourez et al. Trends Microbiol. 10: (2002)

Therapeutic Uses of Anthrax LF N and EF N can be bound to drugs, imported through ATR & PA Cancer Treatments –Oncogenic proteins (Ras) activate MAPKs –Expression of matrix metalloproteases