Transcript The Anthrax Toxin - University of Guelph

R.C. Liddington, Nature, 415: 373-374 (2002)
Anthrax
Another Reason to Fear Your Mailman
By Stefko Waschuk
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
M. Mock, A. Fouet, Annu. Rev. Microbiol. 55: 647-671 (2001)
B. Anthracis cycle
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: 815-826 (1999)
Systemic Anthrax
• Mortality rate ~100%
• Spores germinate within macrophage
• Toxin released into bloodstream
– Toxemia and septicemia
• Shock and death
M. Mourez et al. Trends Microbiol. 10:287-293 (2002)
So, How Does It Kill Me?
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
M. Mourez et al. Trends Microbiol. 10:287-293 (2002)
Protective Antigen
M. Mourez et al. Nature Biotech., 19: 958-961 (2001)
Mode of Anthrax Toxin Entry
M. Mourez et al. Trends Microbiol. 10:287-293 (2002)
PA Heptamer
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
A.D. Pannifer et al. Nature, 414: 229-233 (2001)
Lethal Factor Structure
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 proinflammatory 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)
C.L. Drum et al. Nature, 415: 396-402 (2002)
Oedema Factor Structure (inactive)
• Active site in interface of CA and CB
• Catalytic machinery is present, but
disordered
C.L. Drum et al. Nature, 415: 396-402 (2002)
Oedema Factor Structure (active)
• CaM displaces helical domain
• Switch B becomes ordered
– binds ATP
– stabilizes EF catalytic residues
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
M. Mourez et al. Trends Microbiol. 10:287-293 (2002)
Summary: Anthrax Toxin Action
Anthrax Toxin Management
• Vaccinations
• Antibiotics
• Other strategies
– Polyclonal antibodies
– Synthetic inhibitors
Vaccinations
• Anthrax vaccine adsorbed (AVA)
• Made from protective antigen
Antibiotics
• Ciprofloxacin Hydrochloride
– C17H18FN3O3.HCl.H2O
• 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. 10:287-293 (2002)
M. Mourez et al. Trends Microbiol. 10:287-293 (2002)
Domain II Mutant PA
J. Mogridge et al. PNAS. 99: 7045-7048 (2002)
Domain III Mutant PA
Soluble ATR
M. Mourez et al. Trends Microbiol. 10:287-293 (2002)
Therapeutic Uses of Anthrax
• LFN and EFN can be bound to drugs, imported
through ATR & PA
• Cancer Treatments
– Oncogenic proteins (Ras) activate MAPKs
– Expression of matrix metalloproteases