GSBS Dissertations and Theses

Approval Date

12-1-2016

Document Type

Doctoral Dissertation

Academic Program

Molecular Genetics and Microbiology

Department

Microbiology and Physiological Systems

First Thesis Advisor

Christopher Sassetti

Keywords

Mycobacterium tuberculosis, quiescence, metabolism, drug resistance

Abstract

Mycobacterium tuberculosis stably adapts to respiratory limited environments by entering into a nongrowing but metabolically active state termed quiescence. This state is inherently tolerant to antibiotics due to a reduction in growth and activity of associated biosynthetic pathways. Understanding the physiology of the quiescent state, therefore, may be useful in developing new strategies to improve drug efficiency. Here, we used an established in vitro model of respiratory stress, hypoxia, to induce quiescence. We utilized metabolomic and genetic approaches to identify essential and active pathways associated with nongrowth. Our metabolomic profile of hypoxic M. tuberculosis revealed an increase in several free fatty acids, metabolite intermediates in the oxidative pathway of the tricarboxylic acid (TCA) cycle, as well as, the important chemical messenger, cAMP. In tandem, a high-throughput transposon mutant library screen (TnSeq) revealed that a cAMP-regulated protein acetyltransferase, MtPat, was conditionally essential for survival in the hypoxic state. Via 13C-carbon flux tracing we show an MtPat mutant is deficient in re-routing hypoxic metabolism away from the oxidative TCA cycle and that MtPat is involved in inhibiting fatty-acid catabolism in hypoxia. Additionally, we show that reductive TCA metabolism is required for survival of hypoxia by depletion of an essential TCA enzyme, malate dehydrogenase (Mdh) both in in vitro hypoxia and in vivo mouse infection. Inhibition of Mdh with a novel compound resulted in a significantly greater killing efficiency than the first-line anti-M. tuberculosis drug isoniazid (INH). In conclusion, we show that understanding the physiology of the quiescent state can lead to new drug targets for M. tuberculosis.

DOI

10.13028/M2NK5S

Rights and Permissions

Copyright is held by the author, with all rights reserved.

Available for download on Tuesday, December 05, 2017

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