Transcript General Characteristics of Bacillus Anthracis

Dominant-Negative Mutants of a Toxin
Subunit: An Approach to Therapy of Anthrax
Brett R. Sellman, Michael Mourez, R. John Collier
Presented by Michelle Mayer & Young Heinbockel
BIOL475
11/24/03
Question
• What are the three proteins that make up an
Anthrax Toxin?
General Characteristics of Bacillus Anthracis
• First bacterium shown to be the cause of disease
• 1877 Koch grew the organism in pure culture,
demonstrated its ability to form endospores, and produced
experimental anthrax by injecting it into animals
• Very large, Gram +, spore forming rod, 1-1.2 um x 3-5 um
Anthrax
• Spores are found naturally in soil
• In the US, endemic areas include SD, NE, AR,
TX, LA, MS & CA
• Primarily a disease of domesticated and wild
animals
• Humans become infected when brought into
contact with diseased animals (flesh, bones, hides,
hair, excrement)
• In 2001, anthrax spores were used effectively for
the first time in bioterrorist attacks, resulting in 5
deaths
Symptoms of Anthrax in Humans
• In humans, the risk of infection is 1/100,000
• Symptoms usually occur within 7 days
Cutaneous
–95% of anthrax infections
–Bacterium enters a cut or
abrasion on the skin
–20% of untreated cases
result in death
–Resembles insect bite in
the beginning, then develop
into a necrotic ulcer
Symptoms of Anthrax continued
Inhalation (woolsorters’ disease)
–Inhale 8,000 to 50,000 spores
–Initial symptoms may resemble a common cold. After several days, the
symptoms may progress to severe breathing problems and shock
–Usually fatal
Gastrointestinal
–Extremely rare
–Consumption of contaminated meat
–25% ~ 60% of cases result in death
–Initial signs of nausea, loss of appetite, vomiting and fever, followed by
abdominal pain, vomiting of blood and severe diarrhea
Pathogenicity of Bacillus anthracis
• Poly-D-glutamyl capsule
–
–
–
–
All virulent strains form capsule
Nontoxic
Antiphagocytic
Plasmid pX02
• Anthrax toxin
– Powerful toxin of A-B type
– Composed of three factors:
• Protective Antigen (PA): Binding Component
• Active Components: Lethal Factor (LF) & Edema Factor
(EF)
– Plasmid pX01
Anthrax toxin
• Protective antigen (PA): Transports EF & LF to
cytosol
• Edema factor (EF): calmodulin-dependant adenlate
cyclase (causes edema & impairs neutrophil function)
• Lethal factor (LF): Zn 2+ dependant protease (cleaves
MAP kinase kinases, kills macrophages and causes death
to host)
3 nontoxic proteins:
EF + LF is inactive
PA + LF combine to produce lethal activity
PA + EF produce edema
PA + LF + EF produces edema and necrosis
Therapy of Anthrax
• In US, anthrax vaccine for humans is PA from
avirulent, nonencapsulated strain of Bacillus
anthracis
– 3 subcutaneous injections given 2 weeks apart followed
by 3 additional injections at 6, 12 & 18 months
– Annual booster injections
• Treatment of Anthrax – antibiotics
• New approach to treating bacterial infections
– Develop ways to block the action of virulence factors
• Mutant forms of a subunit of anthrax that are
potent inhibitors of toxin action in vitro and in
vivo.
Dominant-Negative Mutants of a Toxin
Subunit: An Approach to Therapy of Anthrax
• Demonstrates that some of translocation
deficient mutants of PA are Dominant
Negative (DN) mutants
• Demonstrates that DN mutants inhibited
translocation activity of WT-PA in vitro
(across endosomal and plasma membranes)
and in vivo.
Model of Anthrax Action
Mutations
Deletion of 2B2-2B3 loop
Point mutations in:
K397
D425
F427
Mutation at these sites would block pore formation
and translocation. But had no effect on its receptor
binding, proteolytic activation or ability to
oligomerize and bind the toxin’s enzymatic
moieties.
Recessive Loss-of-Function Mutations &
Dominant Gain-of-Function Mutations
Polypeptide products of recessive
and dominant mutations
Phenotypes of heterozygotes
carrying a wild-type allele and
different types of mutant alleles
Translocation-deficient mutant
Experimental procedure (figure 2)
Do translocation-deficient PA mutants inhibit toxin action?
• Tested inhibition of protein synthesis in CHO-K1cells:
– Without PA or LFn-DTA (baseline)
– WT-PA & LFn-DTA (control)
– 6 mutants + WT-PA & LFn-DTA
• Removed medium & replaced with Leu-free HAM F-12
supplemented with 3H- Leu
• Incubated (1, 4, 18 hrs) and washed with PBS, followed by
10% TCA
• Quantity of 3H-Leu incorporated into TCA-precipitable
material was measured and expressed as % of that
incorporated in the absence of PA
Translocation-deficient mutants
•Double mutant: K397D, D425K
•2Bs-2B3 loop deletion
•F427A
•D425K
K397D
SSSR
Hybrid Complex Formation
Experimentation (figure 3)
Does the inhibition by DN mutants depend the on formation of
WT-PA63 + DN hybrid complexes?
Tested inhibition of protein synthesis by LFn-DTA (protein
inhibitor) in CHO-K1 cells:
•Homo-heptamers of WT PA63 (control)
•Homo-heptamers of five translocation-deficient PA mutants
•Hetero heptamers were prepared by mixing each mutant PA
1:1 with WT-PA
Note: Used the same protein inhibition protocol as in Translocation-deficient
Mutant Experimentation.
Hybrid Complex Formation
Translocation Across Plasma
Membrane (figure 4)
Will Dominant-negative PA mutants inhibit translocation
across the plasma membrane?
• Study 1
(baseline)
CHO-K1 cells incubated with trypsin activated PA & various mutants
Cells washed and incubated with [35S]LFn
Lysed cells and measured radiolabel
• Study 1A
CHO-K1 cells incubated with trypsin activated PA & various mutants
Cells washed and incubated with [35S]LFn
Incubated 1 min @ 37C with pH 5.0 buffer
Digested cell surface [35S]LFn with Pronase, cells washed and lysed
Measured radiolabel
Note: data presented as % of cell associated label that became Pronase-resistant in cells treated with low
pH
Translocation Assay
Toxin Inhibition In Vivo
Experimentation (table 1)
Do the DN mutants inhibit toxin action in vivo?
Injected rats with:
• LF (8 ug) and WT-PA 40 ug (~10x minimal lethal dosage)
• LF & DN-PA
– Deletion, Double & F427A
• DN-PA + WT-PA/LF mixture at a 1:1 ratio (40 ug:40 ug)
– Deletion, Double, F427A & SSSR
• DN-PA + WT-PA/LF mixture at a 0.25:1 ratio (10 ug:40 ug)
– Deletion, Double & F427A
Inhibition of Toxin Action in Rats
Quantity of Protein(µg)
WT
Deletion
Double
40
40
40
TTM
F427A
SSSR
40
40
40
40
40
40
40
40
40
40
40
40
10
10
10
90 ± 11 min
Survived
Survived
Survived
Survived
Survived
Survived
100 ± 3 min
Survived
Survived
Survived
Comments
This article is a culmination of work started back in mid
1990’s. The research is being continued. The latest article
is dated to September 2003 by John Collier.
Constructive Criticism
• Abrupt transition:
– Figures 1, 2 & 3 study translocation across endosomal membrane.
– Figure 4 studies translocation across plasma membrane.
• Figure 4 lacking baseline graph
Things to Come
• DN-PA therapy?
• Dual action anthrax vaccine targeting both toxin and capsule