"Mycobacterium tuberculosis (Mtb) is now classified by the World Health Organization as the number one killer in the world by a single infectious agent. Mtb is a successful pathogen because of its ability to overcome obstacles raised by the immune system. Sensing and responding to host cues is essential for Mtb pathogenesis. During infection, Mtb encounters a variety of anti-microbial environmental stresses including acidic pH. The two-component regulator system (TCS) PhoPR has been implicated in sensing and responding to acidic pH. Loss of this TCS results in severe attenuation during infection. Therefore it follows, that identification of inhibitors that disrupt this TCS-mediated adaptive response to acidic pH will provide therapeutic leads for further development of novel Mtb treatments and chemical probes to provide a deeper understanding of Mtb physiology at acidic pH. To identify small molecule inhibitors of PhoPR-mediated signaling and Mtb growth at acidic pH, I have taken a high-throughput screening approach using a fluorescent reporter as a synthetic phenotype for PhoPR signaling activity. To analyze this large amount of data I have developed the High throughput screening Automated Analysis and Retrieval of Putative hits (HAARP) custom software suite to provide diagnostic plots and positive hit information. Using this tool, I identified the FDA-approved carbonic anhydrase inhibitor ethoxzolamide (ETZ) as a potent inhibitor of the PhoPR-regulon that attenuates Mtb virulence. Discovery of ETZ established a previously unrecognized link between carbonic anhydrase activity and PhoPRsignaling. We also identified a compound that targets Mtb thiol pools and demonstrated the essentiality of maintaining redox homeostasis through these pools is required for resistance to oxidative stress at acidic pH. These findings highlight that modulation of processes Mtb uses to adapt to acidic environments can provide additional insight into physiological requirements and novel therapeutic targets. While characterizing candidate inhibitors of the PhoPR-regulon and Mtb growth at acidic pH, I discovered a need to analyze or re-analyze transcriptional profiling data (RNA sequencing; RNA-seq). Current workflows available for bacterial RNA-seq analysis are limited and the tools for each step in the process are numerous. Thus, I developed the Simple Program for Automated reference-based bacterial RNA-seq Transcriptome Analysis (SPARTA) as a turn-key software package that can analyze raw Illumina sequencing reads and produce relevant plots and differential gene expression tables."--Pages ii-iii.
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