Student Theses and Dissertations

Date of Award

2025

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Thesis Advisor

Jeremy M. Rock

Abstract

Tuberculosis (TB) is the world’s deadliest infectious disease, though many in the rich world consider it an illness of Victorian nobility and poets. This is because, despite killing more than one million people annually, TB is most prevalent in low- and middle[1]income countries. TB is caused by the bacterium Mycobacterium tuberculosis (Mtb), and treatment for drug-sensitive TB consists of four antibiotics taken in combination for between four and six months. Treatment is long, costly, and is often inaccessible to those in TB endemic regions of the world. This pandemic is worsened by the emergence of drug resistant Mtb, particularly Mtb that is resistant to the first line antibiotic rifampicin (Rif). Rif resistant (RifR) Mtb caused up to 450,000 cases of TB and 264,000 deaths worldwide in 2021. Treatment for drug resistant TB is even more arduous, consisting of expensive second- and third-line antituberculars with side effects such as deafness, hepatotoxicity, and peripheral neuropathy, taken for six to nine months. RifR is caused by mutations in rpoB, the gene encoding the β subunit of RNA polymerase (RNAP), the target of Rif. These mutations block Rif binding but also alter the shape of the RNA exit channel, potentially disrupting transcription elongation. As is true for many other bacteria, drug resistance in Mtb is often associated with a fitness cost in the absence of antibiotics, and Rif resistance is no exception. RifR Mtb is frequently less fit than RifS Mtb and displays altered transcription dynamics, cell wall and lipid composition, and collateral sensitivity to some antibiotics. RifR Mtb is known to evolve second site compensatory mutations in the α and β’ subunits of RNAP that rescue the fitness of the strain to near Rif sensitive (RifS) levels. These compensatory mutations also ameliorate some of these biochemical and physiological defects of the resistant strain. A deeper understanding of the trade-offs encountered by RifR in Mtb is needed to develop effective treatment for resistant infections and potentially limit the evolution of drug resistance in a drug sensitive population. Here, we took a functional genomics approach to identify mechanisms underlying double-edged sword of RifR in βS450L Mtb, the most clinically common RifR conferring mutation, which accounts for over 70% of all RifR Mtb in the clinic. Employing genome[1]wide CRISPR interference (CRISPRi) screens, we identified genes with differential sensitivity to inhibition in RifR Mycobacterium smegmatis (Msmeg; a model organism of Mtb) and Mtb, compared to a RifS strain. We identified genes involved in translation[1]related processes as bolstering the fitness of RifR mycobacteria. This includes the essential transcription factor nusG, which has dual roles in promoting transcription elongation and transcription pausing/termination. Given the opposing roles of NusG, with the former potentially boosting the fitness of the slow βS450L RNAP, and the latter exacerbating the hyper-termination defect of βS450L RNAP, we investigated how nusG may be evolving in the clinic. Utilizing a genome-wide association study, we discovered novel mutations in nusG which buffer the fitness of βS450L Mtb by minimizing the pro[1]pausing activity of NusG. This ameliorates the slow transcription elongation and hyper[1]pausing/termination of βS450L Mtb. These results define hyper-termination as a source of the fitness defect of βS450L Mtb, identify a new series of compensatory mutations, and may inform new therapies to limit the evolution of drug-resistance. We also conducted differential vulnerability screening in two fast, hypo-terminating RifR mutants, βH445Y and βD435V Mtb. We found that many of the same genes and pathway that are more sensitive to inhibition in βS450L Mtb were less sensitive to inhibition in these fast RNAP mutants. For example, thiS, the gene encoding the thiamine diphosphate (TPP) synthase, is a top collateral vulnerability in βS450L Mtb, and a top collateral invulnerability in these fast, hypo-terminating RifR mutants. We determined that the enhanced vulnerability of thiS in βS450L Mtb is due to the depletion of branch chain amino acids (BCAA) upon TPP limitation. This led us to investigate ilvB1, the most differentially vulnerable TPP dependent enzyme in our dataset. ilvB1 encodes the large subunit of the acetohydroxyacid synthase enzyme, which catalyzes the first step of BCAA biosynthesis. Mtb ilvB1 gene expression is likely regulated by transcription attenuation, as it is in other bacteria. We utilized a series of reporter strains to determine that the inability of the slow, pause-prone βS450L RNAP to read through the regulatory terminator upstream of ilvB1 in the absence of BCAA plays a role in mediating the enhanced vulnerability of ilvB1. These data also highlight hyper[1]termination as a source of the fitness defect of βS450L Mtb and suggest that other genes controlled by attenuation mechanisms may be more vulnerable to inhibition in βS450L Mtb. The data presented in the text not only identify a new class of compensatory mutations in βS450L Mtb and define hyper-termination as a mediator of the fitness defect of βS450L but provide insights into the nature of other compensatory mutations and suggest rational ways to exacerbate the fitness cost of RifR Mtb for treatment and the prevention of the evolution of RifR. Known compensatory mutations in rpoA and rpoC, which encode the α and β’ subunits of RNAP, may rescue the fitness of βS450L Mtb by similar mechanisms as nusG and β protrusion clinical variants- that is by limiting the swiveling of the βS450L RNAP and minimizing hyper-termination. By targeting a collateral vulnerability, such as the pro-elongation activity of NusG or BCAA biosynthesis, one may be able to treat βS450L Mtb infections. Additionally, targeting a collateral vulnerability in tandem with Rif in a RifS population may be able to limit the evolution of RifR in the first place. Finally, these data suggest compounds that enhance transcription pausing and termination may also be able to treat RifR Mtb and shift the evolutionary path of resistance towards less fit RifR mutants that may be more easily cleared by the immune system.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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Available for download on Wednesday, March 10, 2027

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