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

OverviewOverview

• BackgroundBackground• Basis for projectBasis for project• Methods and ResultsMethods and Results• ConclusionsConclusions• Future experimentsFuture experiments

BackgroundBackground

ClostridiumClostridium

• Ancient genusAncient genus• Gram +Gram +• Rod-shapedRod-shaped• AnaerobicAnaerobic• Spore-formersSpore-formers

Sporulating Sporulating C. perfringensC. perfringens

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

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

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

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

ToxinsToxins

TypeType αα-toxin-toxin ββ-toxin-toxin εε-toxin-toxin ιι-toxin-toxin

AA ++

BB ++ ++ ++

CC ++ ++

DD ++ ++

EE ++ ++

C. perfringensC. perfringens toxinotypes toxinotypes

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

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

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

Type A Food PoisoningType A Food Poisoning

Spore Contamination

Cooking GerminationSlow cooling and/or storage at moderate temperature

Rapid proliferationIngestionGI illnessEnvironment

Disease cycle

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

Project BasisProject Basis

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

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

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~

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

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

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

Methods and ResultsMethods and Results

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

Kinase Transcriptional Activity:Kinase Transcriptional Activity:Reverse Transcriptase PCR (RT-PCR)Reverse Transcriptase PCR (RT-PCR)

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)

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

Gene InactivationGene Inactivation

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

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

Gene InactivationGene Inactivation

Transformation:Transformation:

ElectroporationElectroporation

Single crossover:Single crossover:

Gene inactivationGene inactivation

Cm

Cm

Chromosome

Gene InactivationGene Inactivation

Selection for Selection for chloramphenicol chloramphenicol resistanceresistance

Brain-Heart Infusion Brain-Heart Infusion Agar platesAgar plates

http://www.emdchemicals.com

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

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

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

ComplementationComplementation

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

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

ComplementationComplementation

Transformation: electroporationTransformation: electroporation

Propagation

Vector

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

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

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

ConclusionsConclusions

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

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

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)

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

Questions?Questions?