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
Overview
Background
Basis for project
Methods and Results
Conclusions
Future experiments
Background
Clostridium
Ancient genus
Gram +
Rod-shaped
Anaerobic
Spore-formers
Sporulating C. perfringens
Clostridium species
C. difficile
C. tetani
http://www.emedicine.com/med/topic3412.htm
http://textbookofbacteriology.net/clostridia.html
C. botulinum
C. acetobutylicum
http://nabc.ksu.edu/content/factsheets/category/Botulism
http://www.accessexcellence.org/LC/SS/ferm_graphics/reactor.html
C. perfringens
Reservoir: (ubiquitous)
•
Soil, water, intestinal tract of humans and animals
Produces heat-resistant spores
Commonly contaminate foods
Remain viable after cooking
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
Toxins
15 different toxins
Each isolate produces only a subset
Isolates classified by production capabilities of 4
toxins

Toxinotyping
Toxins
C. perfringens toxinotypes
Type
α-toxin β-toxin ε-toxin ι-toxin
A
+
B
+
+
C
+
+
D
+
E
+
+
+
+
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
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
C. perfringens enterotoxin: CPE
Major virulence factor for type A food poisoning
Damages epithelium of small intestine
Healthy
Diseased
Production of CPE is sporulation-specific
Type A Food Poisoning
Spore
Contamination
Cooking
Slow cooling
and/or
storage at
moderate
temperature
Germination
Disease cycle
Environment
GI illness
Ingestion
Rapid proliferation
Type A Food Poisoning
≥107 cells consumed
Cells sporulate in
small intestine
CPE is released
http://www.drugdevelopmenttechnology.com/projects/cilanserton/cilanserton7.html
Spores leave through
diarrhea
Project Basis
Project Basis
Production of CPE is sporulation-specific
Block sporulation  block CPE production
Medical field
Therapeutics
Food industry
Natural inhibitory additives
Safe handling procedures
Sporulation Pathway
How is Spo0A activated?
Spo0A
1. Receipt of signal
2. Activation of Spo0A
1.
?
2.
3.
3. Gene regulation
4. Sporulation
4.
CPE
Spore
Components in B. subtilis
Phosphorelay in Bacillus subtilis:
Gene sequence similarity:
Signal
X
Spo0B
6 orthologues
X
Spo0F ~P
Not present
~
kinase ~P
P
Spo0A ~P
(Present)
Central Hypothesis
One or more kinases bypass intermediate
phosphate messengers to directly activate
Spo0A
Signal
Kinase~P
X
Spo0F
X
Spo0B
Spo0A
Candidate Kinases
6 kinase candidates:






CPE 0986
CPE 1512
CPE 0213
CPE 1754
CPE 1986
CPE 1316
2 selected for project
Objective
Evaluate whether expression of cpe0213 and
cpe1754 is necessary for sporulation to occur
Methods and Results
Project Plan
Assess kinase transcriptional activity
Inactivate each gene to make kinase mutants
Complement mutants with functional kinase
gene
Kinase Transcriptional Activity:
Reverse Transcriptase PCR (RT-PCR)
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)
Data: RT-PCR
+
Positive Control
RT Test
+
RT
-
CPE 1754
+
RT
-
-
Negative Control
CPE 0213
Conclusion:
cpe0213 and cpe1754:
1. Are transcriptionally active
2. Are transcribed in sporulating conditions
Gene Inactivation
Gene Inactivation
Purpose: Evaluate sporulation in kinasedeficient mutants
Steps:



Construct a mutator plasmid
Transform the plasmid into C. perfringens
Select for putative mutants
Gene Inactivation
Mutator plasmid:
1. Kinase gene fragment
pCR®-XL-TOPO®
(Invitrogen)
2. Chloramphenicol (Cm)
resistance cassette
1.
400-500 bp
Kinase ORF
Cm
2.
Gene Inactivation
Transformation:
Electroporation
Cm
Chromosome
Single crossover:
Gene inactivation
Cm
Gene Inactivation
Selection for
chloramphenicol
resistance
Brain-Heart Infusion
Agar plates
http://www.emdchemicals.com
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
Sporulation Assay
Frequency (ν) = [spores] / [spores + cells]
Relative frequency = [mutant ν] / [wild type ν]
Sporulation in cpe0213 mutant
Sporulation in cpe1754 mutant
Repetition Relative
Frequency
Repetition Relative
Frequency
1
0.32
1
0.25
2
0.25
2
0.50
3
0.24
3
0.30
Average = 0.27
Average = 0.33
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
Complementation
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
Complementation
Complementation vector:
3. OriCp
1. Functional kinase gene
2. Erythromycin resistance (Em)
pJIR751
1.
3. Origin of replication for C.
perfringens
2.0 - 2.7 kb
Kinase ORF
Promoter region
2.
Em
Complementation
Transformation: electroporation
Vector
Propagation
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
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
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
Conclusions
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
Future Experiments
Experiments to evaluate the other 4 candidate
kinases
In vitro phosphorylation assays

Overproduction and purification of Spo0A and kinase
Future Experiments
Construct stable kinase mutants



Single crossover inactivation technique is reversible
Traditional Double-crossover inactivation
TargeTron™ Gene Knockout System (Sigma-Aldrich)
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
Questions?