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The role of proteolytic cleavage in the onset of the Chlamydia trachomatis persistence phenotype

The role of proteolytic cleavage in the onset of the Chlamydia trachomatis persistence phenotype. Christopher Thompson, PhD. Background. Chlamydiae are obligate intracellular parasites Vacuolar membrane is protective, but also prohibitive.

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The role of proteolytic cleavage in the onset of the Chlamydia trachomatis persistence phenotype

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  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 • Fe2+ <-> Fe3+ • Able to accept and donate single electrons • Essential for conserved cellular processes • e.g. electron transport, nucleotide biosynthesis • Often a limiting nutrient for pathogens • Corynebacteriumdiphtheriae • Chlamydia enters the persistent growth mode upon low-iron availability.

  8. Danger of Excess Iron • Excess Fe2+ can catalyze the generation of toxic free-radicals • Most organisms employ regulatory mechanisms to maintain iron-homeostasis • Prokaryotes  iron-dependent DNA-binding transcriptional repressors

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

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

  11. YtgABCD as an iron-import system Fe2+ Fe2+ Fe2+ Fe2+ Fe2+ Fe2+ YtgA Periplasm YtgC YtgD Cytosol YtgB

  12. YtgABCD as an iron-import system Fe2+ Fe2+ Fe2+ Fe2+ Fe2+ Fe2+ YtgA Periplasm YtgC YtgD Cytosol YtgB

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

  14. Transcription of ytgA is responsive to iron-starvation control iron-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 Fe2+ Fe2+ Fe2+ Fe2+ Fe2+ Fe2+ YtgA Periplasm YtgC YtgD Cytosol YtgB

  20. Ct069 (termed YtgCR) contains two distinct domains Fe2+ Fe2+ Fe2+ Fe2+ Fe2+ Fe2+ YtgA Periplasm YtgC YtgD Cytosol YtgB 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 Negative controls- purified YtgR +/- cofactor removal step All metals supplemented at 150 µM KD = 3.4x10-8 M

  24. How does the YtgR domain affect transcription from a membrane-anchored localization? Fe2+ Fe2+ Fe2+ Fe2+ Fe2+ Fe2+ YtgA Periplasm YtgC YtgD Cytosol YtgB 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 The lower molecular weight fragments correspond to the C-terminal YtgR domain (C-terminal epitope)

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

  29. Proposed model for regulation of iron-homeostasis Fe2+ Fe2+ Fe2+ Fe2+ Fe2+ Fe2+ YtgA Periplasm YtgC YtgD Cytosol Iron-starved YtgB transcription YtgR

  30. Proposed model for regulation of iron-homeostasis Fe2+ Fe2+ Fe2+ Fe2+ Fe2+ Fe2+ YtgA Periplasm YtgC YtgD Cytosol Regulated? Iron-replete YtgB YtgR

  31. Proposed model for regulation of iron-homeostasis Fe2+ Fe2+ Fe2+ Fe2+ Fe2+ Fe2+ Fe2+ YtgA Periplasm YtgC YtgD Cytosol Iron-replete YtgB YtgR system repressed

  32. Acknowledgements • Prof. Myra McClure • Dr. Rey Carabeo • Dr. GuamingZhong • 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)

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