Transcript Mutational Analysis of the Enzymatic Domain of Clostridium difficile

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