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Characterization of Sporulation-Specific Characterization of Sporulation-Specific Kinases in Kinases in Clostridium perfringensClostridium perfringens
Bryan Danielson, Mahfuzur Sarker, Ph.D.Bryan Danielson, Mahfuzur Sarker, Ph.D.
http://www.city.hiroshima.jp/shakai/eiken/topics/tp002/baikinman.htm
Dept. of Biomedical Sciences, Bioresource Research
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OverviewOverview
• BackgroundBackground• Basis for projectBasis for project• Methods and ResultsMethods and Results• ConclusionsConclusions• Future experimentsFuture experiments
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BackgroundBackground
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ClostridiumClostridium
• Ancient genusAncient genus• Gram +Gram +• Rod-shapedRod-shaped• AnaerobicAnaerobic• Spore-formersSpore-formers
Sporulating Sporulating C. perfringensC. perfringens
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Clostridium speciesClostridium speciesC. difficileC. difficile
C. botulinumC. botulinum
C. tetaniC. tetani
C. acetobutylicumC. 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
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C. perfringensC. perfringens
• Reservoir: (ubiquitous)Reservoir: (ubiquitous)• Soil, water, intestinal tract of humans and animalsSoil, water, intestinal tract of humans and animals
• Produces heat-resistant sporesProduces heat-resistant sporesCommonly contaminate foodsCommonly contaminate foods
Remain viable after cookingRemain viable after cooking
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C. perfringensC. perfringens
Causes disease in humans and animals through Causes disease in humans and animals through two routes:two routes:1. Via damaged skin:1. Via damaged skin:
Clostridial myonecrosis (gas gangrene)Clostridial myonecrosis (gas gangrene)
2. Via gastrointestinal (GI) tract:2. Via gastrointestinal (GI) tract:1. Food-borne1. Food-borne
2. Antibiotic-associated2. Antibiotic-associated
3. Sporadic3. Sporadic
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ToxinsToxins
15 different toxins15 different toxins
Each isolate produces only a subsetEach isolate produces only a subset
Isolates classified by production capabilities of 4 Isolates classified by production capabilities of 4 toxinstoxins ToxinotypingToxinotyping
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ToxinsToxins
TypeType αα-toxin-toxin ββ-toxin-toxin εε-toxin-toxin ιι-toxin-toxin
AA ++
BB ++ ++ ++
CC ++ ++
DD ++ ++
EE ++ ++
C. perfringensC. perfringens toxinotypes toxinotypes
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Type A Food PoisoningType A Food Poisoning
Third most reported bacterial food poisoning in Third most reported bacterial food poisoning in the United Statesthe United States
Estimated to cause:Estimated to cause:250,000 cases/year250,000 cases/year$120 million losses/year$120 million losses/year
Symptoms:Symptoms:Appear 8-12 hours post ingestionAppear 8-12 hours post ingestionAcute abdominal pain, diarrheaAcute abdominal pain, diarrheaPersist ~24 hoursPersist ~24 hours
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Type A Food PoisoningType A Food Poisoning
Conditions that promote food spoilage:Conditions that promote food spoilage: Slow cooling after cooking and/orSlow cooling after cooking and/or storage of cooked food at warm temperaturesstorage of cooked food at warm temperatures
Danger zone: 70Danger zone: 70°° – 120 – 120°°F (20° - 50°C)F (20° - 50°C)
Primary sources for outbreaks:Primary sources for outbreaks: Banquets, cafeteriasBanquets, cafeterias
Heating traysHeating traysLarge quantities of foodLarge quantities of food
Meat, and meat-containing dishesMeat, and meat-containing dishesGenerally high-protein foodsGenerally high-protein foods
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C. perfringens C. perfringens enterotoxin: CPEenterotoxin: CPE
Major virulence factor for type A food poisoningMajor virulence factor for type A food poisoning
Damages epithelium of small intestineDamages epithelium of small intestine
Production of CPE is sporulation-specificProduction of CPE is sporulation-specific
HealthyHealthy DiseasedDiseased
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Type A Food PoisoningType A Food Poisoning
Spore Contamination
Cooking GerminationSlow cooling and/or storage at moderate temperature
Rapid proliferationIngestionGI illnessEnvironment
Disease cycle
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Type A Food PoisoningType A Food Poisoning
≥≥101077 cells consumed cells consumed
Cells sporulate in Cells sporulate in small intestinesmall intestine
CPE is releasedCPE is released
Spores leave through Spores leave through diarrheadiarrheahttp://www.drugdevelopment-technology.com/projects/
cilanserton/cilanserton7.html
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Project BasisProject Basis
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Project BasisProject Basis
Production of CPE is sporulation-specificProduction of CPE is sporulation-specific
Block sporulation Block sporulation block CPE production block CPE production
Medical fieldMedical fieldTherapeuticsTherapeutics
Food industryFood industryNatural inhibitory additivesNatural inhibitory additives
Safe handling proceduresSafe handling procedures
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How is Spo0A activated?How is Spo0A activated?
Sporulation PathwaySporulation Pathway
1. Receipt of signal
2. Activation of Spo0A
3. Gene regulation
4. Sporulation
1. 2.
4.
?3.
SporeCPE
Spo0A
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Components in Components in B. subtilisB. subtilis
Phosphorelay in Phosphorelay in Bacillus subtilisBacillus subtilis::
Gene sequence similarity:Gene sequence similarity:
Signal
kinase Spo0F
Spo0B
Spo0A
6 orthologues Not present (Present)
XX
~P ~P ~PP~
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Central HypothesisCentral Hypothesis
One or more kinases bypass intermediate One or more kinases bypass intermediate phosphate messengers to directly activate phosphate messengers to directly activate Spo0ASpo0A
Signal Kinase~P Spo0F Spo0B Spo0AX X
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Candidate KinasesCandidate Kinases
6 kinase candidates:6 kinase candidates: CPE 0986CPE 0986 CPE 1512CPE 1512 CPE 0213CPE 0213 CPE 1754CPE 1754 CPE 1986CPE 1986 CPE 1316CPE 1316
2 selected for project
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ObjectiveObjective
Evaluate whether expression of Evaluate whether expression of cpe0213cpe0213 and and cpe1754cpe1754 is necessary for sporulation to occur is necessary for sporulation to occur
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Methods and ResultsMethods and Results
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Project PlanProject PlanAssess kinase transcriptional activityAssess kinase transcriptional activity
Inactivate each gene to make kinase mutantsInactivate each gene to make kinase mutants
Complement mutants with functional kinase Complement mutants with functional kinase genegene
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Kinase Transcriptional Activity:Kinase Transcriptional Activity:Reverse Transcriptase PCR (RT-PCR)Reverse Transcriptase PCR (RT-PCR)
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Reverse Transcription (RT)-PCRReverse Transcription (RT)-PCR
Purpose:Purpose: Transcriptionally active in sporulating conditionsTranscriptionally active in sporulating conditions
Steps:Steps: Propagate in sporulation-inducing media:Propagate in sporulation-inducing media:
Duncan-Strong (DS)Duncan-Strong (DS) Isolate total RNAIsolate total RNA Reverse transcribe kinase mRNAs to cDNAReverse transcribe kinase mRNAs to cDNA Amplify kinase cDNA via polymerase chain reaction Amplify kinase cDNA via polymerase chain reaction
(PCR)(PCR)
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Data: RT-PCRData: RT-PCR
+ +RT RT- -CPE 0213CPE 1754
Conclusion:
cpe0213 and cpe1754:
1. Are transcriptionally active
2. Are transcribed in sporulating conditions
+ Positive Control
- Negative Control
RT Test
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Gene InactivationGene Inactivation
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Gene InactivationGene Inactivation
Purpose: Evaluate sporulation in kinase-Purpose: Evaluate sporulation in kinase- deficient mutantsdeficient mutants
Steps:Steps: Construct a mutator plasmidConstruct a mutator plasmid Transform the plasmid into Transform the plasmid into C. perfringensC. perfringens Select for putative mutantsSelect for putative mutants
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Gene InactivationGene Inactivation
Mutator plasmid:Mutator plasmid:1.1. Kinase gene fragmentKinase gene fragment
2.2. Chloramphenicol (Cm) Chloramphenicol (Cm) resistance cassetteresistance cassette
Kinase ORF
2.1.
pCR®-XL-TOPO®
(Invitrogen)
400-500 bp
Cm
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Gene InactivationGene Inactivation
Transformation:Transformation:
ElectroporationElectroporation
Single crossover:Single crossover:
Gene inactivationGene inactivation
Cm
Cm
Chromosome
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Gene InactivationGene Inactivation
Selection for Selection for chloramphenicol chloramphenicol resistanceresistance
Brain-Heart Infusion Brain-Heart Infusion Agar platesAgar plates
http://www.emdchemicals.com
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Sporulation AssaySporulation Assay
Sporulation induced by 8-hr growth in DS mediaSporulation induced by 8-hr growth in DS media
Vegetative cells and spores enumerated with a Vegetative cells and spores enumerated with a microscope counting chambermicroscope counting chamber
http://www.hawksley.co.uk
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Sporulation AssaySporulation Assay
Frequency (Frequency (νν) = [spores] / [spores + cells]) = [spores] / [spores + cells]
Relative frequency = [mutant Relative frequency = [mutant νν] / [wild type ] / [wild type νν]]
RepetitionRepetition Relative Relative FrequencyFrequency
11 0.320.32
22 0.250.25
33 0.240.24
Sporulation in cpe0213 mutant
Average = 0.27
RepetitionRepetition Relative Relative FrequencyFrequency
11 0.250.25
22 0.500.50
33 0.300.30
Sporulation in cpe1754 mutant
Average = 0.33
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Statistical AnalysisStatistical Analysis
Data for sporulation assays was analyzed with a Data for sporulation assays was analyzed with a two-sample t-test:two-sample t-test: Degrees of freedom: 4Degrees of freedom: 4 p<0.01p<0.01
Statistical analysis indicates a significant Statistical analysis indicates a significant decrease in sporulation frequency for the kinase decrease in sporulation frequency for the kinase mutants with 99% confidencemutants with 99% confidence
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ComplementationComplementation
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ComplementationComplementation
Purpose: to verify that disruption of the target Purpose: to verify that disruption of the target gene caused the decrease in gene caused the decrease in sporulation sporulation
Steps:Steps: Construct a complementation vectorConstruct a complementation vector Transform vector into kinase mutantsTransform vector into kinase mutants Select for transformantsSelect for transformants
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Complementation vector:Complementation vector:1. Functional kinase gene1. Functional kinase gene
2. Erythromycin resistance (Em)2. Erythromycin resistance (Em)
3. Origin of replication for 3. Origin of replication for C. C. perfringensperfringens
ComplementationComplementation
2.
1.Em
3. OriCp
Kinase ORF
pJIR751
2.0 - 2.7 kb
Promoter region
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ComplementationComplementation
Transformation: electroporationTransformation: electroporation
Propagation
Vector
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ComplementationComplementation
1.1. Transformants were selected for by growth in Transformants were selected for by growth in erythromycin erythromycin andand chloramphenicol chloramphenicol
2.2. Sporulation frequency was evaluatedSporulation frequency was evaluated
3.3. Sporulation frequency was compared to Sporulation frequency was compared to mutant sporulation frequencymutant sporulation frequency
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ComplementationComplementationResult:Result: No increase in sporulation frequencyNo increase in sporulation frequency
Complement wild type:Complement wild type: Severe reduction in sporulation capabilitySevere reduction in sporulation capability
Reliable sporulation assays could not be Reliable sporulation assays could not be performed due to sporulation deficienciesperformed due to sporulation deficiencies
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Possible ReasonsPossible Reasons
The complementation vectors are multicopy. This The complementation vectors are multicopy. This may lead to an overproduction of the kinasemay lead to an overproduction of the kinase
A negative feedback system may be triggered to A negative feedback system may be triggered to block all production of the kinaseblock all production of the kinase
The overproduced kinase may hinder activity of The overproduced kinase may hinder activity of kinase(s) involved in sporulationkinase(s) involved in sporulation
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ConclusionsConclusions
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ConclusionsConclusions
cpe0213cpe0213 and and cpe1754cpe1754 are transcribed in the are transcribed in the presence of a sporulation signalpresence of a sporulation signal
cpe0213cpe0213 and and cpe1754cpe1754 mutants exhibit a reduced mutants exhibit a reduced sporulation frequencysporulation frequency
cpe0213cpe0213 and and cpe1754cpe1754 mutants could not be mutants could not be complementedcomplemented
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Future ExperimentsFuture Experiments
Experiments to evaluate the other 4 candidate Experiments to evaluate the other 4 candidate kinaseskinases
In vitroIn vitro phosphorylation assays phosphorylation assays Overproduction and purification of Spo0A and kinaseOverproduction and purification of Spo0A and kinase
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Future ExperimentsFuture Experiments
Construct stable kinase mutantsConstruct stable kinase mutants Single crossover inactivation technique is reversibleSingle crossover inactivation technique is reversible Traditional Double-crossover inactivationTraditional Double-crossover inactivation TargeTron™ Gene Knockout System (Sigma-Aldrich)TargeTron™ Gene Knockout System (Sigma-Aldrich)
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AcknowledgmentsAcknowledgments
The Sarker Lab:The Sarker Lab:Dr. Mahfuzur SarkerDr. Mahfuzur Sarker
I-hsiu HuangI-hsiu Huang
Dr. Deepa RajuDr. Deepa Raju
Daniel Paredes-SabjaDaniel Paredes-Sabja
Nahid MahfuzNahid Mahfuz
Marcelo MendezMarcelo Mendez
John ClarkeJohn Clarke
Dr. Dan RockeyDr. Dan Rockey
Bioresource Research:Bioresource Research:Wanda CrannellWanda Crannell
Dr. Kate FieldDr. Kate Field
Undergraduate Research Undergraduate Research Innovation Scholarship and Innovation Scholarship and Creativity (URISC)Creativity (URISC)
Oregon State UniversityOregon State University
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Questions?Questions?