Identification of Essential Metabolic and Genetic Adaptations to the Quiescent State in Mycobacterium Tuberculosis: A Dissertation

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

Next generation perfusion process development for production of biologics

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Upstream control strategy development for afucosylated species in mAb biomanufacturing

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The normalcy of dormancy: common themes in microbial quiescence

All microorganisms are exposed to periodic stresses that inhibit growth. Many bacteria and fungi weather these periods by entering a hardy, nonreplicating state, often termed quiescence or dormancy. When this occurs during an infection, the resulting slowly growing pathogen is able to tolerate both immune insults and prolonged antibiotic exposure. While the stresses encountered in a free-living environment may differ from those imposed by host immunity, these growth-limiting conditions impose common pressures, and many of the corresponding microbial responses appear to be universal. In this review, we discuss the common features of these growth-limited states, which suggest new approaches for treating chronic infections such as tuberculosis