production of plasmid-encoded chlamydial proteins in host cells jeffrey burnett dr. dan rockey lab
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Production of plasmid-Production of plasmid-encoded chlamydial encoded chlamydial proteins in host cellsproteins in host cells
Jeffrey BurnettJeffrey Burnett
Dr. Dan Rockey LabDr. Dan Rockey Lab
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Chlamydia trachomatisChlamydia trachomatis
▫ most common STI most common STI ▫ 2.8 million cases annually2.8 million cases annually
▫ causes:causes:▫ Pelvic Inflammatory Pelvic Inflammatory
DiseaseDisease▫ Ectopic pregnancyEctopic pregnancy▫ InfertilityInfertility▫ Preventable blindnessPreventable blindness
▫ asymptomaticasymptomatic
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C. trachomatisC. trachomatis
▫ Gram (-) bacteriumGram (-) bacterium▫ Obligate Obligate
intracellular aerobeintracellular aerobe▫ Cannot make its own Cannot make its own
ATP ATP ▫ InclusionInclusion▫ Biphasic lifecycleBiphasic lifecycle
▫ Non-infectious Non-infectious metabolically active metabolically active formform
▫ Infectious “spore” Infectious “spore” formform
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Background knowledgeBackground knowledge
Horoschak and MoulderHoroschak and Moulder Decrease in amount of cellsDecrease in amount of cells
Bose and LiebhaberBose and Liebhaber No decrease in DNA synthesisNo decrease in DNA synthesis
Greene and ZhongGreene and Zhong Interactions between chlamydia and Interactions between chlamydia and
host cell cyclehost cell cycle
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Background knowledgeBackground knowledge
McCoy cells – blue (nuclei), green (cytoskeleton)
McCoy cells – blue (nuclei), green (cytoskeleton), red (chlamydial protein transfection - CT223)
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Background informationBackground information
Rockey et alRockey et al Inclusion membrane proteins (inc)Inclusion membrane proteins (inc) CT119 - CT119 - incAincA
Identification of CT 223 as Identification of CT 223 as incinc gene gene
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My ProjectMy Project
Two projects:Two projects: Find active domain of CT 223Find active domain of CT 223 Double transfection with genes in Double transfection with genes in
inc operoninc operon
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Project #1Project #1
Construct Structure of construct PN Oligos
p23f
p23c
p23cL1/2
p23cR1/2
p23f2/3
Hydrophobic domain Hydrophilic regions
23% DA97/DA116
25% DA71/DA116
8% DA71/DA121
15% DA122/DA116
7% DA97/DA119
1 270
96 270
96 158
159 270
1 214
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Project #1 - conclusionProject #1 - conclusion
Active site of CT223 is c-terminus 56 Active site of CT223 is c-terminus 56 AA sequenceAA sequence
To do:To do: What is the relevance?What is the relevance?
Note: Chlamydia blocks cytokinesisNote: Chlamydia blocks cytokinesis Find binding partnersFind binding partners
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Project #2 - part oneProject #2 - part one
Make expression Make expression vectorsvectors
TransformationTransformation
Send for Send for sequencingsequencing
gene # / restriction site Sequence
CT223 C. trachomatis / Eco RI agcaGAATTCttgagatctagaaaaatagaagc
CT223 C. trachomatis / KpnI agcaGGTACCaatggtgagtttagcattagg
CT224 C. trachomatis / EcoRI agcaGAATTCatgagttttgttggagatagt
CT224 C. trachomatis / XhoI agcaCTCGAGctaatcattgggaaaaattga
CT225 C. trachomatis / EcoRI agcaGAATTCatggtggctaacaactcctttatt
CT225 C. trachomatis / EcoRV agcaGATATCttaatcccacccatgaaattt
CT226 C. trachomatis / EcoRI agcaGAATTCatgttggccttttttttgcga
CT226 C. trachomatis / EcoRV agcaGATATCttatatcagactttcttccaa
CT227 C. trachomatis / EcoRI agcaGAATTCatgtcttatcttttttgttcc
CT227 C. trachomatis / EcoRV agcaGATATCtcatgagacacttatagtcac
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Project #2 - part oneProject #2 - part one
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Project #2 - part oneProject #2 - part one
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Project #2 - part twoProject #2 - part two
Goal of part two:Goal of part two:
1.1. Attain double transfection of same host Attain double transfection of same host mammalian cellmammalian cell
2.2. Conclude whether or not there is an effect on Conclude whether or not there is an effect on polynuclear cellspolynuclear cells
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Project #2 - part twoProject #2 - part twoTransfected Polynuclear Average % of total Total
224/223 25 6 2425 6 2825 7 2025 5 20
23 100/24
227/223 25 5 2025 4 1625 7 2825 4 16
20 100/20
233 only 25 6 2425 6 2425 3 1225 5 20
20 100/20
RFP/GFP 25 3 1225 2 825 1 425 0 0
6 100/6
Mock 25 2 825 1 425 0 025 1 4
4 100/4
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Project #2 - part twoProject #2 - part two
0
5
10
15
20
25
30
Relative number of polynuclear cells (%)
223/224
227/223
223
RFP/GFP
Mock
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ResultsResults
No effect on polynuclear morphology No effect on polynuclear morphology between single transfection CT223 between single transfection CT223 and double transfectionsand double transfections
To do:To do: Transfections with CT225, and CT226 Transfections with CT225, and CT226
to see if same conclusion can be drawnto see if same conclusion can be drawn
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SummarySummary
Project #1Project #1 Found active domain of CT223 - last 56 aaFound active domain of CT223 - last 56 aa To Do: relevance / active partner in hostTo Do: relevance / active partner in host
Project #2Project #2 Found no increase in rate of polynuclear cells Found no increase in rate of polynuclear cells
in double-transfected cellsin double-transfected cells To Do: look at other genes (CT225, CT226)To Do: look at other genes (CT225, CT226)
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AcknowledgementsAcknowledgements
Howard Hughes Medical Howard Hughes Medical InstituteInstitute Dr. Kevin AhernDr. Kevin Ahern
Dr. Dan Rockey and his LabDr. Dan Rockey and his Lab Damir AlzhanovDamir Alzhanov
Department of Biomedical Department of Biomedical Sciences in the college of Sciences in the college of Veterinary MedicineVeterinary Medicine
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Thank you for Thank you for your time.your time.
Questions?Questions?