dominant-negative mutants of a toxin subunit: an approach to therapy of anthrax brett r. sellman,...
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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.
Polypeptide products of recessive and dominant mutations
Phenotypes of heterozygotes carrying a wild-type allele and different types of mutant alleles
Recessive Loss-of-Function Mutations & Dominant Gain-of-Function Mutations
Translocation-deficient mutantExperimental 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 mutantExperimental procedure (figure 2)
Translocation-deficient mutants
•Double mutant: K397D, D425K•2Bs-2B3 loop deletion•F427A•D425K
SSSR
K397D
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
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 1ACHO-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 lysedMeasured radiolabel
Note: data presented as % of cell associated label that became Pronase-resistant in cells treated with low pH
Translocation Across Plasma Membrane (figure 4)
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
Quantity of Protein(µg)
WT Deletion Double F427A SSSR40 90 ± 11 min 40 Survived 40 Survived 40 Survived40 40 Survived40 40 - - Survived40 40 Survived40 40 100 ± 3 min40 10 Survived40 10 Survived40 10 Survived
TTM
Inhibition of Toxin Action in Rats
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