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