1. Mutational Analysis of the Enzymatic Domain of Clostridium difficile Toxin B Reveals Novel Inhibitors of the Wild-Type Toxin Authors: Lea M. Spyres, Jeremy Daniel, Amy Hensley, Maen Qa’Dan, William Ortiz-Leduc, and Jimmy D, Ballard Presented by: S. Camphor

2. Background Clostridium difficile (C.difficile) -gram positive -anaerobic -spore former -major cause of hospital acquired diarrhea & Pseudomembranous colitis -some cases colitis is life-threatening

3. Background …. What is pseudomembranous colitis? -severe irritation of the colon -caused by C.difficile when the normal flora of the gut is wiped out due to antibiotic use -illness characteristics 1. diarrhea 2. fever 3. abdominal cramping/pain

4. Background continued….. Statistics (U.S.) C. difficile Found that: -20% of hospitalized patients suffer (about 3 million cases/year) -30% of these develop diarrhea Asymptomatic: -2%-3% of healthy adults -70% of healthy infants and youth -low mortality/morbidity rate (10%-30% of seriously ill will die)

5. Introduction…. Current treatment: -Antibiotics ( could this perpetuate the problem?) -supportive therapy New treatments: -Tx with Saccharomyeces boulardii Future treatments: -therapeutics that target major virulent factors to prevent major cell specific cytoxic activities.

6. Intro continued……. Toxins A & B -LCTs (Large clostridial toxins) -involved in development of colitis -toxin A enterotoxin Toxin B (cytotoxin) -glucosylates isoforms of Rho, Rac, and Cdc 42. -structures:- enzymatic - translocation - receptor binding domains -triggers caspase-dependent / independent apoptosis

7. Intro continued….. Tcd B enzymatic domain focus: -activity requires all 546 amino terminal amino acids -if deletion in amino or carboxy terminal a reduction in activity is seen

8. Toxin B as a target for drug therapy? Toxin B - possible drug therapy through activity inhibition - Paper: investigates use of mutants to block CPEs (cytopathic effects)

9. Materials and Methods… Created fusion proteins using lfn ( encodes Ag binding region of anthrax toxin lethal factor) - 4 with deletions - 3 site-directed mutations (mutations and deletions in enzymatic domain) Fusion proteins used in various assays to determine inhibition capabilities of Tcd B.

10. Figure 1 A/B: A: deletion and site-directed mutants used in study B: SDS-PAGE analysis of histone fused tags. Results: Lfn- used for mutants to gain entry into the cell.

11. Table 1:Glucosylhydrolase activity using UDP-glucose as a substrate to determine if defective hydrolase activity was the reason for inability for target modification

12. Figure 2 A/B Glucosylation activity of Mutants A: SDS-PAGE of each mutant and TcdB glucosylation acitivity on RhoA, Rac1 and Cdc 42 B: LFnTcdB 1-500 test to see if deletion mutant attenuated modification of substrate Results …..

13. Figure 3A: actin condensation and cell rounding in the inhibitor assay.

14. Figure 3B: Summary of inhibitors capable of blocking TcdB CPEs. - Antagonistic impact on Toxin B intoxication - Inhibition decrease over time Legend: Solid: LFnTcdB 1- 420 Open: LFnTcdB w102A Dotted: LFnTcdB c365w Checkered: LFnTcdB 33-556 Hatched: LFn TcdB 1-500

15. Figure 4: Monitoring of CPEs. Are the inhibitory effects really limited? - more than 50% of cells show no sign of CPEs.

16. Figure 5: Is inhibition occurring in the cytosol? Use CHO cell line that induce expression of TcdB1-556 Eventually presence of CPEs because of cells continuous production of TcdB 1-556

17. Figure 6: Is inhibition due to competition for substrate or co-substrate? -TcdB1-500 added to see if protection from TcsL -Both TcsL and TcdB share Rac as a common substrate. almost 50% block of TcsL

18. Discussion Mutants -don’t modify substrate -have cytosolic functions that allow inhibition Question: -Is the inhibition occurring because of prevented access of UDP-glucose to toxin B?

19. Discussion… TcsL assay -show inhibition at cosubstrate level b/c only Rac in common with Toxin B -effects would be less effective on TcsL if Rho, Rac and Cdc42 involved -inhibition at the co-substrate level because no effect seen with Tcnα

20. Future use… This study provides possibility of useful therapeutic treatments in the future, targeting toxin B. Cell surface studies to better understand the surface interacting regions More studies on inactive mutants in other viruses