Mutational Analysis of the Enzymatic Domain of Clostridium difficile Toxin B Reveals Novel Inhibitor

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 QaDan, 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
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.
11
Table 1Glucosylhydrolase activity using
UDP-glucose as a substrate to determine if
defective hydrolase activity was the reason for
inability for target modification
12
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
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 LFnTcdB1-420 Open LFnTcdB
w102A Dotted LFnTcdB c365w Checkered LFnTcdB
33-556 Hatched LFn TcdB1-500
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 TcdB1-556
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
• -dont 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 Tcna

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