Genetics of Antibiotic Synergy in Mycobacterium tuberculosis

Abstract

Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis (Mtb), which has been around for millennia and continues to affect millions of people every year. Successful TB treatment requires months of combination antibiotics, which complicates the ability to treat all cases quickly and effectively. Expanding our antibiotic arsenal against TB and optimizing drug combinations are two important steps to reducing treatment failure. In Chapter One of this dissertation, we examine the research tools available to identify new antibiotic targets in Mtb and to characterize the mechanisms of action of anti-tubercular compounds. New targets can be discovered through small molecule screens as well as target-based discovery using genetic methods. We can furthermore apply these tools to identify synergies between experimental treatments and optimize the effects of different antibiotics. We have applied multiple genetic tools to identify and characterize promising candidates for target-based drug discovery against Mtb. The bulk of our work presented here focuses on a specific enzyme, peptidyl tRNA hydrolase (Pth). We find that while Pth could be investigated as a new antibiotic target on its own, its added appeal is as an enzyme whose inhibition would lend additional efficacy to existing drugs. In Chapter Two, we identify two distinct biological roles for Pth in Mtb. We demonstrate that while one is required for bacterial survival, the other is not. Understanding the essential function of a protein is critical for assessing whether cells could quickly develop resistance against an inhibitor of that protein simply by reducing the need for it. In Chapter Three, we dive deeper into Pth’s essential role in Mtb and probe the vulnerability of protein synthesis in this pathogen by assessing how Pth interacts with tRNA in cells. We develop new experimental strategies to study tRNA in Mtb and lay a groundwork for future work on tRNA dynamics. By examining Pth through a therapeutic lens, we next discover that drugs targeting this enzyme would synergize with two existing classes of antibiotics that are not currently used to treat TB: macrolides and aminoacyl tRNA synthetase inhibitors. Macrolides are inexpensive, well-tolerated drugs used to treat other bacterial infections but are not effective on their own against TB. Aminoacyl tRNA synthetase inhibitors have seen success in other types of infections and are currently being developed to target Mtb. In the final chapter of this thesis, we discuss the implications of our work and lingering questions that would further improve our understanding of Mtb biology. We also present ongoing work that builds on tools and findings presented in this dissertation. Altogether, our goal is to contribute to a growing body of knowledge on Mtb physiology and the development of new strategies against an age-old disease.