Abstract

Mycobacterium tuberculosis (Mtb) causes the disease tuberculosis (TB) and is one of the leading causes of death worldwide by an infectious agent. Furthermore, the emergence and evolution of drug resistance has significantly hindered global health efforts to combat this deadly epidemic. Thus, there is a critical need for development of new antibiotics with unique mechanisms of action and design of novel strategies for optimizing the efficacy of existing therapeutic regimens. It is estimated that 10.7% of new infected and 27.2% of previously treated TB cases are resistant to the frontline antibiotic isoniazid (INH), a central component of TB chemotherapeutic regimens. Resistance to INH most commonly occurs through mutations in the katG gene that reduce its ability to convert INH to its active form. We previously reported the discovery and characterization of the compound C10, a small molecule inhibitor that enhances killing of wild-type (WT) Mtb by INH and re-sensitizes a collection of INH-resistant mutants to killing by INH. This was the first instance of re-sensitization of a genetically resistant mutant that has been reported in Mtb, making C10 an incredibly valuable tool we can leverage to design new strategies for reversing drug resistance. Thus, we sought to elucidate the mechanism of C10 potentiation of INH in order to pinpoint promising candidates for enhancing the efficacy of INH and other antibiotics. Prior transcriptional profiling implicated metabolism as a potential target of C10 activity. To dissect the effect of C10 on Mtb metabolism, we performed metabolic profiling of Mtb treated with C10 in the presence and absence of INH. We found that C10 and INH together led to significant depletion of tricarboxylic acid (TCA) cycle metabolites and dysregulation of gluconeogenesis and the methylcitrate cycle (MCC). I discovered that metabolic supplementation with either propionate or pyruvate could block the ability of C10 to potentiate INH in WT or the INH-resistant katGW328L Mtb strain. To dissect how pyruvate was metabolically rescuing Mtb from C10, I performed metabolic flux analysis of katGW328L Mtb using isotopically labeled glycerol or pyruvate to map carbon flux during treatment with C10 and INH. I discovered that C10 diverts carbon flux to synthesis of α-ketoisovalerate and valine and inhibits glycerol assimilation into multiple cell wall precursors, leading me to examine the effects on the Mtb cell wall. Using a combination of biochemical assays and imaging techniques to investigate the Mtb cell wall, I determined that C10 and INH together perturb the cell wall architecture and integrity, which is restored by metabolic supplementation with propionate or pyruvate. Utilizing orthogonal approaches to investigate the effects of C10 on Mtb physiology, I also observed that C10 blocked biofilm formation and induced hypersusceptibility to low pH, suggesting that C10 inhibits the ability of Mtb to adapt to host-relevant stresses. We previously used two Mtb mutants that are resistant to C10 to uncouple the effects of C10 on energy metabolism from the ability of C10 to potentiate INH. Using these same mutants, I determined disruption of energy homeostasis by C10 is not responsible for increased sensitivity to low pH and only partially contributes to inhibition of biofilm formation. Furthermore, consistent with my other findings that C10 exposes conditional vulnerabilities in cell wall integrity, I also showed that C10 rendered Mtb hypersusceptible to osmotic stress. My contributions expand upon our understanding of how C10 impacts Mtb physiology and reveals novel vulnerabilities between metabolism and cell wall synthesis. Although the precise target of C10 remains elusive, our findings provide a robust framework for guiding future efforts to identify the mechanism of action of C10 and other potential druggable targets.

Committee Chair

Christina Stallings

Committee Members

Gary Patti; Jeffrey Henderson; Jennifer Philips; Tamara Doering

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Molecular Microbiology & Microbial Pathogenesis)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

3-13-2026

Language

English (en)

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Microbiology Commons

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