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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

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

Christopher Thompson, PhD

background
Background
  • Chlamydiae are obligate intracellular parasites
  • Vacuolar membrane is protective, but also prohibitive
persistence phenotype
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 phenotype1
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
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
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.
danger of excess iron
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
how chlamydiae acquire iron is unknown
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

putative function of the ytgabcd system
Putative Function of the YtgABCD system

YtgA bound Iron > Mn or Zn in vitro

-Miller et al. (2008)

YtgA

Periplasm

YtgC

YtgD

Cytosol

YtgB

ytgabcd as an iron import system
YtgABCD as an iron-import system

Fe2+

Fe2+

Fe2+

Fe2+

Fe2+

Fe2+

YtgA

Periplasm

YtgC

YtgD

Cytosol

YtgB

ytgabcd as an iron import system1
YtgABCD as an iron-import system

Fe2+

Fe2+

Fe2+

Fe2+

Fe2+

Fe2+

YtgA

Periplasm

YtgC

YtgD

Cytosol

YtgB

ct069 ytgc contains two distinct domains
Ct069 (YtgC) contains two distinct domains

Predicted transmembrane domains

Predicted alpha helical structure

Predicted beta-strand structure

diphtheria toxin repressor superfamily
Diphtheria toxin repressor superfamily
  • DNA binding proteins that repress transcription of specific genes in response to coordination of a metal co-factor
ct069 termed ytgcr contains two distinct domains
Ct069 (termed YtgCR) contains two distinct domains

Fe2+

Fe2+

Fe2+

Fe2+

Fe2+

Fe2+

YtgA

Periplasm

YtgC

YtgD

Cytosol

YtgB

ct069 termed ytgcr contains two distinct domains1
Ct069 (termed YtgCR) contains two distinct domains

Fe2+

Fe2+

Fe2+

Fe2+

Fe2+

Fe2+

YtgA

Periplasm

YtgC

YtgD

Cytosol

YtgB

YtgR

in vitro dna binding assay
In vitro DNA-binding Assay

artificial start site

Biotinylated-DNA sequence

bio layer interferometry assay
Bio-Layer Interferometry Assay

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
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

how does the ytgr domain affect transcription from a membrane anchored localization1
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
YtgCR is cleaved during the course of infection

YtgCR

Lower molecular weight fragment

recombinant ytgcr is heterologously cleaved in e coli
Recombinant YtgCR is heterologously cleaved in E. coli

The lower molecular weight fragments correspond to the C-terminal YtgR domain

(C-terminal epitope)

proposed model for regulation of iron homeostasis
Proposed model for regulation of iron-homeostasis

Fe2+

Fe2+

Fe2+

Fe2+

Fe2+

Fe2+

YtgA

Periplasm

YtgC

YtgD

Cytosol

Iron-starved

YtgB

transcription

YtgR

proposed model for regulation of iron homeostasis1
Proposed model for regulation of iron-homeostasis

Fe2+

Fe2+

Fe2+

Fe2+

Fe2+

Fe2+

YtgA

Periplasm

YtgC

YtgD

Cytosol

Regulated?

Iron-replete

YtgB

YtgR

proposed model for regulation of iron homeostasis2
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

acknowledgements
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)