1. 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

2. Question What are the three proteins that make up an Anthrax Toxin?

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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

9. 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.

10. 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.

11. Model of Anthrax Action

12. Mutations Deletion of 2B 2 -2B 3 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.

13. 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

14. 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 LF n -DTA (baseline) –WT-PA & LF n -DTA (control) –6 mutants + WT-PA & LF n -DTA Removed medium & replaced with Leu-free HAM F-12 supplemented with 3 H- Leu Incubated (1, 4, 18 hrs) and washed with PBS, followed by 10% TCA Quantity of 3 H-Leu incorporated into TCA-precipitable material was measured and expressed as % of that incorporated in the absence of PA Translocation-deficient mutant Experimental procedure (figure 2)

15. Translocation-deficient mutants Double mutant: K397D, D425K 2Bs-2B3 loop deletion F427A D425K SSSR K397D

16. Hybrid Complex Formation Experimentation (figure 3) Does the inhibition by DN mutants depend the on formation of WT-PA 63 + DN hybrid complexes? Tested inhibition of protein synthesis by LFn-DTA (protein inhibitor) in CHO-K1 cells: Homo-heptamers of WT PA 63 (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.

17. Hybrid Complex Formation

18. 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 [ 35 S]LF n Lysed cells and measured radiolabel Study 1A CHO-K1 cells incubated with trypsin activated PA & various mutants Cells washed and incubated with [ 35 S]LF n Incubated 1 min @ 37C with pH 5.0 buffer Digested cell surface [ 35 S]LF n 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 Across Plasma Membrane (figure 4)

19. Translocation Assay

20. 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

21. Quantity of Protein(µg) WTDeletionDoubleF427ASSSR 40 90 ± 11 min 40 Survived 40 Survived 40 Survived 40 Survived 40 40--Survived 40 40 Survived 40 40100 ± 3 min 4010 Survived 40 10 Survived 40 10 Survived TTM Inhibition of Toxin Action in Rats

22. 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