Transcript ytgA
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 Chlamydia undergo a unique biphasic development 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 Persistence Phenotype Chronic infection has been linked to the serious sequelae of Chlamydia: • Blindness (trachoma) • Infertility • Ectopic pregnancy • Pelvic Inflammatory Disease 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 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 • Corynebacterium diphtheriae Chlamydia enters the persistent growth mode upon low-iron availability. Danger of Excess Iron Excess Fe2+ can catalyze the generation of toxic freeradicals Most organisms employ regulatory mechanisms to maintain iron-homeostasis o Prokaryotes iron-dependent DNA-binding transcriptional repressors 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 Putative Function of the YtgABCD system YtgA bound Iron > Mn or Zn in vitro YtgA -Miller et al. (2008) Periplasm YtgC YtgD Cytosol YtgB YtgABCD as an iron-import system Fe2+ Fe2+ Fe2+ Fe2+ Periplasm Fe2+ Fe2+ YtgC YtgA YtgD Cytosol YtgB YtgABCD as an iron-import system Fe2+ Fe2+ Fe2+ Fe2+ Fe2+ YtgA Periplasm YtgC YtgD Cytosol YtgB Fe2+ Is transcription of the ytgABCD operon regulated in response to fluctuation in available iron? Transcription of ytgA is responsive to iron-starvation control iron-starved How does this iron-dependent transcriptional regulation occur? Ct069 (YtgC) contains two distinct domains DtxR CT069 (YtgCR) Predicted transmembrane domains Predicted alpha helical structure Predicted beta-strand structure Diphtheria toxin repressor superfamily DNA binding proteins that repress transcription of specific genes in response to coordination of a metal co-factor The metal coordinating residues of DtxR are conserved in the YtgR domain ‘YtgR’ MntR TroR IdeR DtxR 1 2 Ct069 (termed YtgCR) contains two distinct domains Fe2+ Fe2+ Fe2+ Fe2+ Fe2+ YtgA Periplasm YtgC YtgD Cytosol YtgB Fe2+ Ct069 (termed YtgCR) contains two distinct domains Fe2+ Fe2+ Fe2+ Fe2+ Fe2+ YtgA Periplasm YtgC YtgD Cytosol YtgB YtgR Fe2+ Is the ‘YtgR’ domain a functional DNAbinding protein? In vitro DNA-binding Assay artificial start site DtxR CT069 (YtgCR) Biotinylated-DNA sequence Bio-Layer Interferometry Assay YtgR apo-YtgR apo-YtgR + Fe 2+ apo-YtgR + Mn 2+ apo-YtgR + Zn 2+ apo-YtgR + Cu 2+ apo-YtgR + Co 2+ Negative controls- purified YtgR +/- cofactor removal step All metals supplemented at 150 µM KD = 3.4x10-8 M How does the YtgR domain affect transcription from a membrane-anchored localization? YtgA Periplasm YtgC Fe2+ Cytosol Fe2+ Fe2+ Fe2+ Fe2+ Fe2+ YtgD YtgB YtgR 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 YtgCR is cleaved during the course of infection UI EBs Hours post-infection 1 6 24 50 - YtgCR 37 - Lower molecular weight fragment 25 20 α-CT069 Recombinant YtgCR is heterologously cleaved in E. coli EV D-glucose L-arabinose + _ pBAD-YtgCR + _ _ _ _ + 50 - pBAD-YtgCR D-glucose + L-arabinose _ _ + 50 - 37 - 37 - 25 - 25 - The lower molecular weight fragments correspond to the C-terminal YtgR domain 20 - 20 - α-CT069 α-V5 (C-terminal epitope) A model for maintenance of ironhomeostasis in Chlamydia Proposed model for regulation of iron-homeostasis Fe2+ Fe2+ Fe2+ Fe2+ Fe2+ YtgA Periplasm YtgC YtgD Cytosol Iron-starved YtgB transcription YtgR Fe2+ Proposed model for regulation of iron-homeostasis Fe2+ YtgA Periplasm YtgC Cytosol Iron-replete Fe2+ Fe2+ Fe2+ YtgD Regulated? Fe2+ YtgB YtgR Fe2+ Proposed model for regulation of iron-homeostasis Fe2+ YtgA Periplasm YtgC Cytosol Fe2+ YtgD Fe2+ Fe2+ Iron-replete YtgB YtgR Fe2+ Fe2+ Fe2+ system repressed Acknowledgements Prof. Myra McClure Dr. Rey Carabeo Jefferiss Research Trust PDRA Fellowship Dr. Guaming Zhong Dr. Scott Grieshaber Sophie Nicod Denise Malcolm 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 irondependent transcriptional repressor. PNAS (2012)