1. The role of proteolytic cleavage in the onset of the Chlamydia trachomatis persistence phenotype Christopher Thompson, PhD

2. Background Chlamydiae are obligate intracellular parasites Vacuolar membrane is protective, but also prohibitive

3. Chlamydia undergo a unique biphasic development

4. Persistence Phenotype Viable but non-cultivatable Aberrant morphology, lack of cytokinesis Induced by certain stress conditions, though normal development can be reactivated stress Removal of stress

5. Persistence Phenotype Chronic infection has been linked to the serious sequelae of Chlamydia: Blindness (trachoma) Infertility Ectopic pregnancy Pelvic Inflammatory Disease

6. Mediators of persistence, in vitro IFN-gamma treatment Penicillin treatment Iron-restriction Amino acid starvation Glucose deficiency Growth within monocytes Co-infection with certain intracellular parasites Culture with adenosine Heat shock Chlamydiophage infection Inhibition of Type three secretion

7. The Importance of Iron Stable in multiple oxidation states Fe 2+ Fe 3+ Able to accept and donate single electrons Essential for conserved cellular processes e.g. electron transport, nucleotide biosynthesis Often a limiting nutrient for pathogens Corynebacterium diphtheriae Chlamydia enters the persistent growth mode upon low-iron availability.

8. Danger of Excess Iron Excess Fe 2+ can catalyze the generation of toxic free- radicals Most organisms employ regulatory mechanisms to maintain iron-homeostasis o Prokaryotes  iron-dependent DNA-binding transcriptional repressors

9. How Chlamydiae acquire iron is unknown No siderophore secretion/adsorption systems No recognized receptors for host iron-proteins ATP-binding cassette (ABC) transport system? Homology to divalent metal transport systems in other bacteria

10. Putative Function of the YtgABCD system YtgA Periplasm Cytosol YtgC YtgD YtgB YtgA bound Iron > Mn or Zn in vitro -Miller et al. (2008)

11. YtgABCD as an iron-import system Fe 2+ YtgA Periplasm Cytosol YtgC YtgD YtgB Fe 2+

12. YtgABCD as an iron-import system Fe 2+ YtgA Periplasm Cytosol YtgC YtgD YtgB Fe 2+

13. Is transcription of the ytgABCD operon regulated in response to fluctuation in available iron?

14. Transcription of ytgA is responsive to iron-starvation controliron-starved

15. How does this iron-dependent transcriptional regulation occur?

16. Ct069 (YtgC) contains two distinct domains Predicted transmembrane domains Predicted alpha helical structure Predicted beta-strand structure

17. Diphtheria toxin repressor superfamily DNA binding proteins that repress transcription of specific genes in response to coordination of a metal co-factor

18. The metal coordinating residues of DtxR are conserved in the YtgR domain

19. Ct069 (termed YtgCR) contains two distinct domains Fe 2+ YtgA Periplasm Cytosol YtgC YtgD YtgB Fe 2+

20. Ct069 (termed YtgCR) contains two distinct domains Fe 2+ YtgA Periplasm Cytosol YtgC YtgD YtgB Fe 2+ YtgR

21. Is the ‘YtgR’ domain a functional DNA- binding protein?

22. In vitro DNA-binding Assay artificial start site Biotinylated-DNA sequence

23. Bio-Layer Interferometry Assay K D = 3.4x10 -8 M Negative controls- purified YtgR +/- cofactor removal step All metals supplemented at 150 µM

24. How does the YtgR domain affect transcription from a membrane-anchored localization? Fe 2+ YtgA Periplasm Cytosol YtgC YtgD YtgB Fe 2+ YtgR

25. How does the YtgR domain affect transcription from a membrane-anchored localization? Proteolytic liberation from membrane sequestration is a common mechanism for the regulation of transcription in both prokaryotes and eukaryotes

26. YtgCR is cleaved during the course of infection YtgCR Lower molecular weight fragment

27. Recombinant YtgCR is heterologously cleaved in E. coli (C-terminal epitope) The lower molecular weight fragments correspond to the C-terminal YtgR domain

28. A model for maintenance of iron- homeostasis in Chlamydia

29. Proposed model for regulation of iron-homeostasis Fe 2+ YtgA Periplasm Cytosol YtgC YtgD YtgB Fe 2+ YtgR transcription Iron-starved

30. Proposed model for regulation of iron-homeostasis Fe 2+ YtgA Periplasm Cytosol YtgC YtgD YtgB Fe 2+ YtgR Iron-replete Regulated?

31. Proposed model for regulation of iron-homeostasis Fe 2+ YtgA Periplasm Cytosol YtgC YtgD YtgB Fe 2+ YtgR system repressed Iron-replete Fe 2+

32. Acknowledgements Prof. Myra McClure Dr. Rey Carabeo Dr. Guaming Zhong Dr. Scott Grieshaber Sophie Nicod Denise Malcolm Jefferiss Research Trust PDRA Fellowship An optimal method of iron starvation of the obligate intracellular pathogen, Chlamydia trachomatis. Frontiers in Microbiology (2011) Cleavage of a putative metal permease in Chlamydia trachomatis yields an iron- dependent transcriptional repressor. PNAS (2012)