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Abstract

Treatment for tuberculosis disease (TB) is failing on a global scale, necessitating new drug discovery efforts to identify direct and adjunctive therapies that reduce treatment complexity, length and side effects. Mycobacterium tuberculosis (Mtb), the obligate human pathogen and causative agent of TB, resides inside alveolar macrophages and combats acidic stress by residing in phagosomes and inhibiting their maturation. Mtb secretes a protein tyrosine phosphatase, PtpA, which binds to subunit H of the macrophage’s v-ATPase pump to block acidification. Improved understanding of the role of acidification in Mtb pathogenesis is needed and can assist in the development of better TB therapies. Following the hypothesis that acidification affects Mtb intracellular survival, I pursued the development of a phenotypic assay to identify compounds that modulate phagosome acidification in Mtb infected macrophages. The developed method uses Mtb intraphagosomal acidification to accurately correlate the proportion of live Mtb during infection. To evaluate the assay suitability for screening purposes, I screened two compound libraries and found that early acidification does not always correlate with inhibition of Mtb growth over time. Yet, early acidification occurs when directly interfering with microbial processes like DNA and protein synthesis over inhibition of cell wall synthesis. The link between phagosome acidification and Mtb viability was further studied using chemical inhibitors of v-ATPase function: Bafilomycin A1 (BafA1), Bafilomycin D (BafD), and Cladoniamide B (ClaB). I showed that all three limit the growth of intracellular mycobacteria in a bacteriostatic manner and that ClaB is metabolized by macrophages into a product with direct antimicrobial activity. I also found that the antimicrobial effect of BafA1 is abrogated in the Mtb ptpA mutant compared to parental strain and demonstrated PtpA:BafA1 binding. These findings suggest that although inhibition of phagosome acidification permits Mtb replication within macrophages, chemical inhibition may modulate host cellular processes leading to mycobacterial clearance. By assessing Mtb’s response to phagosome acidification and deacidification, this work sheds light on the complexity of host-pathogen interactions in early infection and provides a foundational assay that can be used to further investigate this phenomenon to uncover new direct and adjunctive treatments to eliminate TB.