Discovery of Small RNAs and Characterization of Their Regulatory Roles in Mycobacterium Tuberculosis

Abstract

Mycobacterium tuberculosis is an important global health pathogen and is the leading cause of death due to an infectious disease. The pathogen resides inside of human macrophages and is exposed to a wide array of different bactericidal stresses, yet manages to subvert or adapt to each of these in order to grow, divide, and cause disease in humans. Therefore, there is a need for a deeper understanding of how M. tuberculosis adapts to the stress conditions that are imposed by the human host in order to better understand how to combat this deadly disease. One important way by which bacteria respond to stress and rapidly adapt to changing environments is through the use of trans-encoded small RNAs (sRNAs). These short RNA molecules become highly induced in specific conditions, where they bind directly to a set of mRNA targets to regulate their expression. The interactions between sRNAs and their targets is generally mediated by a protein accessory factor such as Hfq, which acts as an RNA chaperone to allow for sRNA-mRNA binding. Although much is known about how sRNAs function in model bacterial species including Escherichia coli, comparatively little is understood about these regulators in mycobacteria. For example, sRNA discovery studies in M. tuberculosis have focused on sRNAs present in the absence of stress, and not a single sRNA-target interaction has been experimentally validated in mycobacteria. Additionally, mycobacteria contain no obvious homologue of any known accessory factor, and no other accessory factor has been identified in this lineage. After a review of the literature on what is currently known about M. tuberculosis stress adaptation and sRNAs in Chapter 1, we perform large scale sRNA discovery in M. tuberculosis during exposure to host-like stress conditions in Chapter 2. By creating a computational sRNA search tool, we generate a master set of 189 M. tuberculosis sRNA candidates and profiles of their expression. In Chapter 3, we perform the most in-depth characterization of an sRNA in mycobacteria to date by focusing on one sRNA that becomes highly abundant in multiple stress conditions in M. tuberculosis. We show that this sRNA, renamed MrsI, acts as an iron sparing sRNA and binds directly to an mRNA target. We additionally provide evidence that MrsI acts in an anticipatory manner during exposure to oxidative stress to prime M. tuberculosis to rapidly enter an iron sparing state. Finally, in Chapter 4 we perform a variety of screens towards the identification of a mycobacterial sRNA accessory factor. Although these screens do not identify a candidate accessory factor, they do provide important insights into characteristics of the as-of-yet unidentified protein. Taken together, these projects greatly increase our understanding of sRNAs in mycobacteria and how they function in the M. tuberculosis stress response. We provide a compendium of sRNAs and their expression patterns during exposure to host-like stressors, and provide in-depth characterization of how one sRNA, MrsI, adapts the pathogen to iron-limited conditions. The results presented here will be critical for future studies on stress responses in M. tuberculosis, particularly through the pathogen’s use of sRNAs.