Transcriptional analysis of Chlamydial persistence

Field: Immunology and Microbiology (Life Sciences)

Chlamydial infections have been associated with several chronic human diseases, including trachoma, pelvic inflammatory disease, chronic obstructive pulmonary disease and atherosclerotic cardiovascular disease. In Chlamydia-associated disease, the organisms are believed to exist in an atypical, persistent phase that is not well understood at the genetic level. The research presented in this thesis investigated chlamydial gene expression in in vitro cell culture models of persistence.

The first set of studies analysed a continuous-infection model of persistence that has been recently developed for two C. pneumoniae isolates (TW-183 and CM-1). The spontaneous establishment and unique cyclical nature of continuous infections could be particularly relevant to in vivo events. An initial analysis using a semi-quantitative reverse transcriptase PCR (sqRT-PCR) approach provided evidence of differential gene expression in C. pneumoniae TW-183 continuous infections relative to acute control infections. Using a subsequently established fully quantitative real-time reverse transcriptase PCR (rtRT-PCR) assay, up-regulated expression profiles were confirmed for five genes (CPn0483, nlpD, ompA, pmp1 and porB) in the continuous C. pneumoniae TW-183 infections. The omcB, pmp1 and porB genes, all of which encode membrane proteins, showed similar patterns of expression over both the acute and continuous time courses tested. Gene expression data for a second C. pneumoniae isolate, CM-1, revealed similar overall expression trends to those seen for C. pneumoniae TW-183 but also supported previous observations of different growth characteristics between the two isolates in the continuous-infection model.

The rtRT-PCR assay was further optimised for use in gene expression studies of the gamma interferon (IFN-γ)-mediated model of C. pneumoniae A-03 persistence, in which altered growth and morphological traits typical of chlamydial persistence have been well characterised. Meanwhile, chlamydial genes such as euo, ftsK and hctB were emerging from the literature as reliable genetic markers of persistence. Therefore, a preliminary rtRT-PCR analysis of marker gene expression was used to assess the likely extent of persistence in individual IFN-γ-treated C. pneumoniae A-03 infections from a series of experiments that had been prepared for this persistence model. In this way, an appropriate pair of duplicate experiments was selected for further studies based on strong genetic evidence of persistence in IFN-γ-treated samples at 48 h post-infection (PI) in those experiments.

Using rtRT-PCR, 14 genes of interest from the related peptidoglycan, aminosugars and lipopolysaccharide (LPS) biosynthetic pathways were analysed in the validated experiments of the IFN-γ-mediated C. pneumoniae A-03 persistence model. Selective up- and down-regulated expression trends were associated with IFN-γ-treatment at 48 h PI for genes encoding products that are located at specific enzymatic points in these pathways. Most strikingly, the expression of glmU, the product of which controls the amount of an essential precursor metabolite that enters both peptidoglycan and LPS biosynthesis, was strongly and reproducibly down-regulated in the 48-h PI IFN-γ-treated samples. This expression profile may contribute to a reduced rate of peptidoglycan biosynthesis in this persistence model and may therefore be related to the inhibited cell division and RB-to-EB differentiation that characterise chlamydial persistence. While most other genes in these pathways showed unchanged expression associated with IFN-γ treatment, murA and kdsB (from peptidoglycan and LPS biosynthesis, respectively) were selectively up-regulated in the 48-h PI IFN-γ-treated samples. Taken together, these data supported the concept of a persistence stimulon in C. pneumoniae that is regulated at key points in various metabolic pathways.

In addition to the analysis of biosynthetic genes, the up-regulated gene set from continuous C. pneumoniae TW-183 infections was also analysed in the validated IFN-γ-mediated C. pneumoniae A-03 persistence experiments. The data revealed similarities and differences in gene expression patterns between these two in vitro persistence models. Furthermore, the profiles obtained for genes such as pmp1 and porB provided insights into the widely predicted phenomenon of late developmental gene shut-down during chlamydial persistence.

A final investigation into an analogous IFN-γ-mediated persistence system for C. trachomatis serovar L2 focussed on one up-regulated (murA) and one down-regulated (glmU) gene from the validated IFN-γ-mediated persistent C. pneumoniae A-03 data set. Both genes were significantly down-regulated in persistent C. trachomatis, adding to a growing body of evidence for key differences among chlamydial species in their persistent gene expression patterns.

This project has contributed significantly to our understanding of the molecular basis of the important persistent phase of chlamydial development.