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.

Figure 1 A/B: A: deletion and site-directed mutants used in studyB: SDS-PAGE analysis of histone fused tags.

Results:

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

Figure 2 A/B Glucosylation activity of Mutants

A: SDS-PAGE of each mutant and TcdB glucosylation acitivity on RhoA, Rac1 and Cdc 42B: LFnTcdB 1-500 test to see if deletion mutant attenuated modification of substrate

Results …..

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: LFnTcdB1-420

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