Identification of iron-responsive proteins expressed by Chlamydia trachomatis reticulate bodies during intracellular growth

Abstract

The obligate intracellular bacterium Chlamydia trachomatis serovar E is the most prevalent cause of bacterial sexually transmitted disease. With an established requirement for iron, the developmental cycle arrests at the intracellular reticulate body stage during iron restriction, resulting in a phenomenon termed persistence. Persistence has implications in natural infections for altered expression of virulence factors and antigens, in addition to a potential role in producing chronic infection. In this study, chlamydial proteins in iron-restricted, infected HEC-1B cells were radiolabelled during mid-developmental cycle growth, harvested, and separated using twodimensional polyacrylamide gel electrophoresis (2D-PAGE). Of~250 radiolabelled protein species visualized, densitometric analysis revealed 25 proteins that increased in expression under iron restriction compared to iron-sufficient control samples; ten protein species identified by mass spectrometry are involved in the oxidative damage response (alkyl hydroperoxide reductase, 6-phosphogluconolactonase and acyl carrier protein synthase), transcription (RNA polymerase subunit alpha and transcription anti-termination factors NusA and NusG), protein modification (peptide deformylase and trigger factor), and virulence (Chlamydia protein associating with death domains, CADD). Transcript-level expression patterns of ahpC, devB, cadd, fabF and ct538 were measured by quantitative RT-PCR throughout the developmental cycle, and each gene examined demonstrated a significant but small mid-cycle increase in transcript level in iron-restricted cultures compared to iron-replete controls. Taken together, these data suggest that the primary response of chlamydiae to reduced iron availability is to increase expression of proteins involved in protection against oxidative damage via iron-catalysed generation of reactive oxygen species and adaptation to stress by increasing expression of transcriptional machinery and other stressresponsive proteins.

Cotranslational Proteolysis Dominates Glutathione Homeostasis to Support Proper Growth and Development

“…The inhibition of NME causes cell cycle arrest in tumor cells (Yeh et al, 2000;Zhang et al, 2000), developmental defects (Boxem et al, 2004;Yeh et al, 2006), and early senescence in plants (Ross et al, 2005). Accordingly, recent data have also suggested that the expression of NME enzymes is tightly regulated throughout development, in response to abiotic stress and tumorigenesis (Selvakumar et al, 2006;Dill et al, 2009), suggesting that the fraction of proteins undergoing NME may vary over space and time. Despite the demonstration that this process is essential in all organisms and its identification as a valuable target for the design of new antibacterial and anticancer agents, the molecular mechanism underlying the physiological requirement for NME function in living cells remains largely unknown.…”

Chlamydia trachomatis YtgA is an iron-binding periplasmic protein induced by iron restriction

“…Bacteria avoid the detrimental effects of iron by limiting its availability, restricting uptake, and sensing adequate iron levels (Clarke et al, 2001). In addition, new findings report that C. trachomatis responds to limited iron availability by increasing expression of proteins involved in protection against oxidative damage (Dill et al, 2009). C. pneumoniae protein levels under iron-limited conditions have also been examined, showing that iron stress induced observable changes in expression in at least six proteins (Wehrl et al, 2004).…”

“…Desferal was previously documented to reduce the epithelial cell intracellular ferritin level significantly (Dill et al, 2009). Protein samples from both cell culture growth conditions were removed throughout the chlamydial developmental cycle (0, 24, 36, 48, 60 and 72 hp.i.…”

Discovery and Validation of New Regulatory RNA Elements in Chlamydia trachomatis

“…The endonucleolytic reaction performed by the RNAP catalytic center is slow, but is greatly stimulated by transcript cleavage factors including GreA (Fish and Kane, 2002;Sergei et al, 2005;Stepanova et al, 2007). A recent study of iron induced persistence in C. trachomatis E showed that both NusA and NusG were regulated (Dill et al, 2009). This study is intriguing as it shows that Nus factors are regulated during persistence and might explain why CTIG643 shows such discrepancy in termination under normal and stress conditions.…”