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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 Background • Clostridium difficile (C.difficile) -gram positive -anaerobic -spore former -major cause of hospital acquired diarrhea & Pseudomembranous colitis -some cases colitis is life-threatening 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 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) 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. 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 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 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) 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. Results: Figure 1 A/B: A: deletion and site-directed mutants used in study B: SDS-PAGE analysis of histone fused tags. Lfn- used for mutants to gain entry into the cell. Table 1:Glucosylhydrolase activity using UDP-glucose as a substrate to determine if defective hydrolase activity was the reason for inability for target modification Results ….. 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 Figure 3A: actin condensation and cell rounding in the inhibitor assay. Figure 3B: Summary of inhibitors capable of blocking TcdB CPEs. - Antagonistic impact on Toxin B intoxication - Inhibition decrease over time Legend: Solid: LFnTcdB1420 Open: LFnTcdB w102A Dotted: LFnTcdB c365w Checkered: LFnTcdB 33-556 Hatched: LFn TcdB1-500 Figure 4: Monitoring of CPEs. Are the inhibitory effects really limited? - more than 50% of cells show no sign of CPEs. 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 TcdB1-556 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 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? 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 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