Production of plasmid-encoded chlamydial proteins in host cells Jeffrey Burnett Dr. Dan Rockey Lab

Chlamydia trachomatis ▫ most common STI ▫ 2.8 million cases annually ▫ causes: ▫ Pelvic Inflammatory Disease ▫ Ectopic pregnancy ▫ Infertility ▫ Preventable blindness ▫ asymptomatic

C. trachomatis ▫ Gram (-) bacterium ▫ Obligate intracellular aerobe ▫ Cannot make its own ATP ▫ Inclusion ▫ Biphasic lifecycle ▫ Non-infectious metabolically active form ▫ Infectious “spore” form

Background knowledge Horoschak and Moulder Horoschak and Moulder Decrease in amount of cells Decrease in amount of cells Bose and Liebhaber Bose and Liebhaber No decrease in DNA synthesis No decrease in DNA synthesis Greene and Zhong Greene and Zhong Interactions between chlamydia and host cell cycle Interactions between chlamydia and host cell cycle

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

Background information Rockey et al Rockey et al Inclusion membrane proteins (inc) Inclusion membrane proteins (inc) CT119 - incA CT119 - incA Identification of CT 223 as inc gene Identification of CT 223 as inc gene

My Project Two projects: Two projects: Find active domain of CT 223 Find active domain of CT 223 Double transfection with genes in inc operon Double transfection with genes in inc operon

Project #1

Project #1 - conclusion Active site of CT223 is c-terminus 56 AA sequence Active site of CT223 is c-terminus 56 AA sequence To do: To do: What is the relevance? What is the relevance? Note: Chlamydia blocks cytokinesis Note: Chlamydia blocks cytokinesis Find binding partners Find binding partners

Project #2 - part one Make expression vectors Transformation Send for sequencing gene # / restriction siteSequence CT223 C. trachomatis / Eco RIagcaGAATTCttgagatctagaaaaatagaagc CT223 C. trachomatis / KpnIagcaGGTACCaatggtgagtttagcattagg CT224 C. trachomatis / EcoRIagcaGAATTCatgagttttgttggagatagt CT224 C. trachomatis / XhoIagcaCTCGAGctaatcattgggaaaaattga CT225 C. trachomatis / EcoRIagcaGAATTCatggtggctaacaactcctttatt CT225 C. trachomatis / EcoRVagcaGATATCttaatcccacccatgaaattt CT226 C. trachomatis / EcoRIagcaGAATTCatgttggccttttttttgcga CT226 C. trachomatis / EcoRVagcaGATATCttatatcagactttcttccaa CT227 C. trachomatis / EcoRIagcaGAATTCatgtcttatcttttttgttcc CT227 C. trachomatis / EcoRVagcaGATATCtcatgagacacttatagtcac

Project #2 - part one

Project #2 - part two Goal of part two: Goal of part two: 1. Attain double transfection of same host mammalian cell 2. Conclude whether or not there is an effect on polynuclear cells

Project #2 - part two

Results No effect on polynuclear morphology between single transfection CT223 and double transfections No effect on polynuclear morphology between single transfection CT223 and double transfections To do: To do: Transfections with CT225, and CT226 to see if same conclusion can be drawn Transfections with CT225, and CT226 to see if same conclusion can be drawn

Summary Project #1 Project #1 Found active domain of CT223 - last 56 aa Found active domain of CT223 - last 56 aa To Do: relevance / active partner in host To Do: relevance / active partner in host Project #2 Project #2 Found no increase in rate of polynuclear cells in double- transfected cells Found no increase in rate of polynuclear cells in double- transfected cells To Do: look at other genes (CT225, CT226) To Do: look at other genes (CT225, CT226)

Acknowledgements Howard Hughes Medical Institute Howard Hughes Medical Institute Dr. Kevin Ahern Dr. Kevin Ahern Dr. Dan Rockey and his Lab Dr. Dan Rockey and his Lab Damir Alzhanov Damir Alzhanov Department of Biomedical Sciences in the college of Veterinary Medicine Department of Biomedical Sciences in the college of Veterinary Medicine

Thank you for your time. Questions?