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

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

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

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

Background knowledgeBackground knowledge

McCoy cells – blue (nuclei), green (cytoskeleton)

McCoy cells – blue (nuclei), green (cytoskeleton), red (chlamydial protein transfection - CT223)

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

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

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

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

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

Project #2 - part oneProject #2 - part one

Project #2 - part oneProject #2 - part one

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

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

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

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

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)

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

Thank you for Thank you for your time.your time.

Questions?Questions?