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- Genetic and chemical biology studies of Mycobacterium tuberculosis pH-driven adaptation
- Dechow, Shelby J.
- Electronic Theses & Dissertations
Mycobacterium tuberculosis (Mtb) endures robust immune responses by sensing and adapting to its host environment. One of the first cues the bacterium encounters during infection is acidic pH, a characteristic of its host niche – the macrophage. Targeting the ability of Mtb to sense and adapt to acidic pH has the potential to reduce survival of Mtb in macrophages. A high throughput screen of a >220,000 compound small molecule library was conducted to discover chemical probes that inhibit Mtb...
Show moreMycobacterium tuberculosis (Mtb) endures robust immune responses by sensing and adapting to its host environment. One of the first cues the bacterium encounters during infection is acidic pH, a characteristic of its host niche – the macrophage. Targeting the ability of Mtb to sense and adapt to acidic pH has the potential to reduce survival of Mtb in macrophages. A high throughput screen of a >220,000 compound small molecule library was conducted to discover chemical probes that inhibit Mtb growth at acidic pH. From this screen, AC2P20 was identified as a chemical probes that kills Mtb at pH 5.7 but is inactive at pH 7.0. Through a combination of transcriptional profiling, mass spectrometry, and free thiol abundance and redox assays, I show that AC2P20 likely functions by depleting intracellular thiol pools and dysregulating redox homeostasis. Findings from this study have helped define new pathways involved in Mtb’s response to acidic pH using a chemical genetic approach.Upon sensing acid stress, Mtb can adapt accordingly by entering a nonreplicating persistent state, resulting in increased tolerance to host immune pressures and antibiotics. During growth in vitro, when given glycerol as a sole carbon source, Mtb responds to acidic pH by arresting its growth and entering a metabolically active state of nonreplicating persistence, a physiology known as acid growth arrest. To answer how Mtb regulates and responds to acidic pH, I performed genetic selections to identify Mtb mutants defective in acid growth arrest. These selections identified enhanced acidic growth (eag) mutants which all mapped to the proline-proline-glutamate ppe51 gene and resulted in distinct amino acid substitutions: S211R, E215K, and A228D. I demonstrated that expression of the PPE51 variants in Mtb promotes significantly enhanced growth at acidic pH showing that the mutant alleles are sufficient to cause the dominant gain-of-function, eag phenotype. Furthermore, I performed single carbon source experiments and radiolabeling experiments showing that PPE51 variants preferentially uptake glycerol at an enhanced rate, suggesting a role in glycerol acquisition. Notably, the eag phenotype is deleterious for growth in macrophages, where the mutants have selectively faster replication but reduced virulence in activated macrophages as compared to resting macrophages. This supports that acid growth arrest is a genetically controlled, adaptive process that could act as a potential targetable physiology in future TB therapeutics. My work with the carbonic anhydrase inhibitor, ethoxzolamide, sought to combine genetic and chemical biology to better understand pH-adaptation in Mtb. Ethoxzolamide is a potent inhibitor of Mtb carbonic anhydrase activity and the PhoPR regulon, suggesting a previously unknown link between carbon dioxide and pH-sensing. We hypothesized that the production of protons from carbonic anhydrase activity could be modulating PhoPR signaling. Mtb has three carbonic anhydrases (CanA, CanB, and CanC), and by using CRISPRi and gene knockout, I show that CanB is required for pathogenesis in macrophages, but I did not observe a function in controlling PhoPR signaling. However, transcriptional profiling at different pH and CO2 concentrations show that PhoPR is induced by high CO2 and also revealed a core subset of CO2 responsive genes independent of PhoPR or acidic pH regulation. Overall, these studies defined new functions for thiol- and redox-homeostasis, glycerol uptake, and CO2-concentration in regulating Mtb adaptation to acidic environments and provide new targets for the development of acidic pH-dependent therapeutics.