Anthrax

Another Reason to Fear Your MailmanBy Stefko Waschuk

R.C

. Lid

ding

ton,

Nat

ure,

415

: 373

-374

(20

02)

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. M

ock,

A. F

ouet

, Ann

u. R

ev. M

icro

biol

. 55:

647

-671

(20

01)

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

So, How Does It Kill Me?

M. M

oure

z et

al.

Tre

nds

Mic

robi

ol. 1

0:28

7-29

3 (2

002)

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. M

oure

z et

al.

Tre

nds

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ol. 1

0:28

7-29

3 (2

002)

Mode of Anthrax Toxin Entry

M. M

ou

rez

et a

l. N

atu

re B

iote

ch.,

19:

958-

961

(200

1)

PA Heptamer

M. M

oure

z et

al.

Tre

nds

Mic

robi

ol. 1

0:28

7-29

3 (2

002)

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

. Pan

nife

r et

al.

Nat

ure,

414

: 22

9-23

3 (2

001)

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 CA and CB

• Catalytic machinery is present, but disordered

C.L

. Dru

m e

t al.

Nat

ure,

415

: 39

6-40

2 (2

002)

Oedema Factor Structure (active)

• CaM displaces helical domain• Switch B becomes ordered

– binds ATP– stabilizes EF catalytic residues

C.L

. Dru

m e

t al.

Nat

ure,

415

: 39

6-40

2 (2

002)

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. M

oure

z et

al.

Tre

nds

Mic

robi

ol. 1

0:28

7-29

3 (2

002)

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)

Domain II Mutant PA

M. M

oure

z et

al.

Tre

nds

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robi

ol. 1

0:28

7-29

3 (2

002)

Domain III Mutant PA

J. M

og

rid

ge

et a

l. P

NA

S. 9

9: 7

045-

7048

(2

002)

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