Genetic analysis of sRNAs in Chlamydia trachomatis

Bacterial small RNAs (sRNAs) are short transcripts that play critical roles in post-transcriptional gene regulation. In the obligate intracellular pathogen Chlamydia trachomatis, however, sRNAs are poorly understood because genetic tools in Chlamydia have only recently become available. As such, functional studies of two chlamydial sRNAs were limited to an E. coli heterologous system. A major issue with this approach is that Chlamydia lacks the important sRNA chaperone protein, Hfq, that is found in E. coli. The heterologous system is also not suitable for studying the function of a sRNA in the Chlamydia developmental cycle, which involves conversion between the infectious (EB) and the replicative (RB) forms within a eukaryotic host cell.With recent advances in Chlamydia genetics, we have developed an inducible sRNA overexpression system in C. trachomatis. Utilizing our approach, we conducted a genetic screen and identified 4 previously uncharacterized sRNAs (i.e. CtrR3, CtrR7, CtrR0332, and CTIG684) whose individual overexpression reduced the production of infectious chlamydial progeny. We then determined that overexpression of CtrR3 and CtrR0332 both blocked RB-to-EB conversion, whereas CtrR7 overexpression hindered RB replication. To demonstrate how our system can be used to find mRNA targets of a chlamydial sRNA, we took a multi-step approach to identify YtgB and CTL0389 as targets of CtrR3. We also showed the versatility of this genetic approach by applying it to develop a novel sRNA-mediated conditional knockdown system in C. trachomatis. This is the first study to analyze sRNAs in Chlamydia and to experimentally demonstrate that chlamydial sRNAs can regulate gene expression. Our genetic approach for studying sRNAs and for identifying mRNA targets can be readily applied to elucidate the function of other chlamydial sRNAs. In addition, our knockdown system can be utilized to investigate the function of essential genes in C. trachomatis. Overall, the work described in this dissertation has significantly advanced our understanding of sRNAs and post-transcriptional gene regulation in C. trachomatis