The role of proteolytic cleavage in the onset of the Chlamydia trachomatis persistence phenotypeChristopher 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 free-radicals

Most organisms employ regulatory mechanisms to maintain iron-homeostasis

oProkaryotes 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

Periplasm

Cytosol

YtgC YtgD

YtgB

YtgA bound Iron > Mn or Zn in vitro-Miller et al. (2008)

YtgABCD as an iron-import system

Fe2+

Fe2+

Fe2+

Fe2+

Fe2+

YtgAPeriplasm

Cytosol

YtgC YtgD

YtgB

Fe2+

YtgABCD as an iron-import system

Fe2+

Fe2+

Fe2+

Fe2+

Fe2+

YtgAPeriplasm

Cytosol

YtgC YtgD

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

Predicted transmembrane domainsPredicted alpha helical structurePredicted 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

Ct069 (termed YtgCR) contains two distinct domains

Fe2+

Fe2+

Fe2+

Fe2+

Fe2+

YtgAPeriplasm

Cytosol

YtgC YtgD

YtgB

Fe2+

Ct069 (termed YtgCR) contains two distinct domains

Fe2+

Fe2+

Fe2+

Fe2+

Fe2+

YtgAPeriplasm

Cytosol

YtgC YtgD

YtgB

Fe2+YtgR

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

In vitro DNA-binding Assay

artificial start site

Biotinylated-DNA sequence

Bio-Layer Interferometry Assay

KD = 3.4x10-8 M

Negative controls- purified YtgR +/- cofactor removal step

All metals supplemented at 150 µM

How does the YtgR domain affect transcription from a membrane-anchored localization?

Fe2+

Fe2+

Fe2+

Fe2+

Fe2+

YtgAPeriplasm

Cytosol

YtgC YtgD

YtgB

Fe2+

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

YtgCR

Lower molecular weight fragment

Recombinant YtgCR is heterologously cleaved in E. coli

(C-terminal epitope)

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

A model for maintenance of iron-homeostasis in Chlamydia

Proposed model for regulation of iron-homeostasis

Fe2+

Fe2+

Fe2+Fe2+

Fe2+

YtgAPeriplasm

Cytosol

YtgC YtgD

YtgB

Fe2+YtgR

transcription

Iron-starved

Proposed model for regulation of iron-homeostasis

Fe2+

Fe2+ Fe2+

Fe2+

Fe2+

YtgAPeriplasm

Cytosol

YtgC YtgD

YtgB

Fe2+YtgR

Iron-repleteRegulated?

Proposed model for regulation of iron-homeostasis

Fe2+

Fe2+

Fe2+

Fe2+

YtgAPeriplasm

Cytosol

YtgC YtgD

YtgB

Fe2+YtgR

system repressed

Iron-replete

Fe2+ Fe2+

Acknowledgements

Prof. Myra McClureDr. Rey Carabeo

Dr. Guaming ZhongDr. Scott Grieshaber

Sophie NicodDenise 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)