1. Characterization of Sporulation-Specific
Kinases in Clostridium perfringens
Bryan Danielson, Mahfuzur Sarker, Ph.D.
http://www.city.hiroshima.jp/shakai/eiken/topics/tp002/baikinman.htm
Dept. of Biomedical Sciences, Bioresource Research
5. Clostridium species
C. difficile
C. botulinum
C. tetani
C. acetobutylicum
http://www.emedicine.com/med/topic3412.htm
http://nabc.ksu.edu/content/factsheets/category/Botulism http://www.accessexcellence.org/LC/SS/ferm_graphics/reactor.html
http://textbookofbacteriology.net/clostridia.html
6. C. perfringens
Reservoir: (ubiquitous)
• Soil, water, intestinal tract of humans and animals
Produces heat-resistant spores
Commonly contaminate foods
Remain viable after cooking
7. C. perfringens
Causes disease in humans and animals through
two routes:
1. Via damaged skin:
Clostridial myonecrosis (gas gangrene)
2. Via gastrointestinal (GI) tract:
1. Food-borne
2. Antibiotic-associated
3. Sporadic
8. Toxins
15 different toxins
Each isolate produces only a subset
Isolates classified by production capabilities of 4
toxins
Toxinotyping
10. Type A Food Poisoning
Third most reported bacterial food poisoning in
the United States
Estimated to cause:
250,000 cases/year
$120 million losses/year
Symptoms:
Appear 8-12 hours post ingestion
Acute abdominal pain, diarrhea
Persist ~24 hours
11. Type A Food Poisoning
Conditions that promote food spoilage:
Slow cooling after cooking and/or
storage of cooked food at warm temperatures
Danger zone: 70° – 120°F (20° - 50°C)
Primary sources for outbreaks:
Banquets, cafeterias
Heating trays
Large quantities of food
Meat, and meat-containing dishes
Generally high-protein foods
12. C. perfringens enterotoxin: CPE
Major virulence factor for type A food poisoning
Damages epithelium of small intestine
Production of CPE is sporulation-specific
Healthy Diseased
13. Type A Food Poisoning
Spore
Contamination
Cooking Germination
Slow cooling
and/or
storage at
moderate
temperature
Rapid proliferation
Ingestion
GI illness
Environment
Disease cycle
14. Type A Food Poisoning
≥107 cells consumed
Cells sporulate in
small intestine
CPE is released
Spores leave through
diarrhea
http://www.drugdevelopment-
technology.com/projects/cilanserton/cilanserton7.html
16. Project Basis
Production of CPE is sporulation-specific
Block sporulation block CPE production
Medical field
Therapeutics
Food industry
Natural inhibitory additives
Safe handling procedures
17. How is Spo0A activated?
Sporulation Pathway
1. Receipt of signal
2. Activation of Spo0A
3. Gene regulation
4. Sporulation
1. 2.
4.
?
3.
Spore
CPE
Spo0A
18. Components in B. subtilis
Phosphorelay in Bacillus subtilis:
Gene sequence similarity:
Signal
kinase Spo0F
Spo0B
Spo0A
6 orthologues Not present (Present)
X
X
~P ~P ~P
P
~
19. Central Hypothesis
One or more kinases bypass intermediate
phosphate messengers to directly activate
Spo0A
Signal Kinase~P Spo0F Spo0B Spo0A
X X
25. Reverse Transcription (RT)-PCR
Purpose:
Transcriptionally active in sporulating conditions
Steps:
Propagate in sporulation-inducing media:
Duncan-Strong (DS)
Isolate total RNA
Reverse transcribe kinase mRNAs to cDNA
Amplify kinase cDNA via polymerase chain reaction
(PCR)
26. Data: RT-PCR
+ +
RT RT
- -
CPE 0213
CPE 1754
Conclusion:
cpe0213 and cpe1754:
1. Are transcriptionally active
2. Are transcribed in sporulating conditions
+ Positive Control
- Negative Control
RT Test
28. Gene Inactivation
Purpose: Evaluate sporulation in kinase-
deficient mutants
Steps:
Construct a mutator plasmid
Transform the plasmid into C. perfringens
Select for putative mutants
29. Gene Inactivation
Mutator plasmid:
1. Kinase gene fragment
2. Chloramphenicol (Cm)
resistance cassette
Kinase ORF
2.
1.
pCR®-XL-TOPO®
(Invitrogen)
400-500 bp
Cm
32. Sporulation Assay
Sporulation induced by 8-hr growth in DS media
Vegetative cells and spores enumerated with a
microscope counting chamber
http://www.hawksley.co.uk
33. Sporulation Assay
Frequency (ν) = [spores] / [spores + cells]
Relative frequency = [mutant ν] / [wild type ν]
Repetition Relative
Frequency
1 0.32
2 0.25
3 0.24
Sporulation in cpe0213 mutant
Average = 0.27
Repetition Relative
Frequency
1 0.25
2 0.50
3 0.30
Sporulation in cpe1754 mutant
Average = 0.33
34. Statistical Analysis
Data for sporulation assays was analyzed with a
two-sample t-test:
Degrees of freedom: 4
p<0.01
Statistical analysis indicates a significant
decrease in sporulation frequency for the kinase
mutants with 99% confidence
36. Complementation
Purpose: to verify that disruption of the target
gene caused the decrease in
sporulation
Steps:
Construct a complementation vector
Transform vector into kinase mutants
Select for transformants
37. Complementation vector:
1. Functional kinase gene
2. Erythromycin resistance (Em)
3. Origin of replication for C.
perfringens
Complementation
2.
1.
Em
3. OriCp
Kinase ORF
pJIR751
2.0 - 2.7 kb
Promoter region
39. Complementation
1. Transformants were selected for by growth in
erythromycin and chloramphenicol
2. Sporulation frequency was evaluated
3. Sporulation frequency was compared to
mutant sporulation frequency
40. Complementation
Result:
No increase in sporulation frequency
Complement wild type:
Severe reduction in sporulation capability
Reliable sporulation assays could not be
performed due to sporulation deficiencies
41. Possible Reasons
The complementation vectors are multicopy. This
may lead to an overproduction of the kinase
A negative feedback system may be triggered to
block all production of the kinase
The overproduced kinase may hinder activity of
kinase(s) involved in sporulation
43. Conclusions
cpe0213 and cpe1754 are transcribed in the
presence of a sporulation signal
cpe0213 and cpe1754 mutants exhibit a
reduced sporulation frequency
cpe0213 and cpe1754 mutants could not be
complemented
44. Future Experiments
Experiments to evaluate the other 4 candidate
kinases
In vitro phosphorylation assays
Overproduction and purification of Spo0A and kinase
45. Future Experiments
Construct stable kinase mutants
Single crossover inactivation technique is reversible
Traditional Double-crossover inactivation
TargeTron™ Gene Knockout System (Sigma-Aldrich)
46. Acknowledgments
The Sarker Lab:
Dr. Mahfuzur Sarker
I-hsiu Huang
Dr. Deepa Raju
Daniel Paredes-Sabja
Nahid Mahfuz
Marcelo Mendez
John Clarke
Dr. Dan Rockey
Bioresource Research:
Wanda Crannell
Dr. Kate Field
Undergraduate Research
Innovation Scholarship and
Creativity (URISC)
Oregon State University
