Comparative Phenotypic Screening Identifies Protein Synthesis Inhibitors as Compounds That Enhance Early Acidification of <i>Mycobacterium tuberculosis</i> in Macrophages

(Mtb), the obligate human pathogen and causative agent of TB, resides within alveolar macrophages and combats acidic stress by inhibiting phagosome maturation. Understanding the role of acidification in Mtb pathogenesis can aid the development of improved TB therapies. Aligning with the hypothesis that acidification affects Mtb intracellular survival, we developed a phenotypic assay to identify compounds that enhance acidification of Mtb in infected macrophages. To evaluate the assay suitability for screening purposes, we screened two compound libraries and found that early acidification does not necessarily correlate with inhibition of Mtb growth over time. Further, we found that compounds interfering with bacterial protein and DNA synthesis induced early phagosome acidification. However, other compounds, including cell wall synthesis inhibitors, did not consistently correlate with acidification or growth inhibition. Using early acidification could therefore provide a potential avenue to inform antimicrobial kinetics. By assessing Mtb's response to phagosome acidification, this work highlights the complexity of host-pathogen interactions in early infection and provides a valuable tool for investigating Mtb survival mechanisms and accelerating drug discovery efforts.