Identification of Chlamydial Iron-Responsive Proteins during Intracellular Growth.

Chlamydia trachomatis is an obligate intracellular bacterium and the most prevalent cause of bacterial sexually transmitted disease. Genital chlamydial infections, marked by chronic, intense inflammation, can lead to genital tissue scarring and infertility and is a contributing factor to development of pelvic inflammatory disease and ectopic pregnancy. Iron is required as a cofactor for numerous highly conserved pathways, and nearly all studied organisms rely on iron for growth. In response to iron restriction, the chlamydial developmental cycle arrests at the intracellular reticulate body stage, resulting in a phenomenon termed persistence. Persistence likely plays a role in chlamydial pathogenesis through the expression of virulence factors and antigens in addition to sustaining chronic infection; however, little is known concerning how chlamydiae respond to iron limitation at the molecular level, and no systems for iron acquisition have been identified in Chlamydia. This dissertation presents an investigation into the chlamydial response to iron restriction. Chlamydial heat shock protein 60 (cHsp60) has been implicated in development of the more severe disease sequelae and has been found to increase in expression following iron restriction; however, three cHsp60 homologues were identified following the sequencing of the chlamydial genome. Here, iron restriction is shown to increase expression of cHsp60-2 but not the two other homologs, cHsp60-1 or -3. Next, in order to investigate an alternate model for restricting iron availability to chlamydiae, a cell line with inducible expression of recombinant ferroportin, a eukaryotic iron efflux protein, was examined. Lastly, 10 chlamydial proteins differentially expressed during growth in iron-restricted host cells were identified by proteomic analysis of radiolabeled proteins followed by mass spectrometry analysis; transcripts encoding 5 iron responsive proteins were examined across a timecourse of infection and revealed increased transcript levels at 18 and/or 24 hours post infection. Together, these studies have examined the molecular response of chlamydiae to reduced iron availability and have underlined the importance for pathways involved in protection against oxidative damage and adaptation to stress