Visualizing pyrazinamide action by live single cell imaging of phagosome acidification and Mycobacterium tuberculosis pH homeostasis

Abstract

The intracellular population of Mycobacterium tuberculosis (Mtb) is dynamically segregated within multiple subcellular niches with different biochemical and biophysical properties that, upon treatment, may impact antibiotic distribution, accumulation, and efficacy. However, it remains unclear whether fluctuating intracellular microenvironments alter mycobacterial homeostasis and contribute to antibiotic enrichment and efficacy. Here, we describe a dual-imaging approach that allows quantitative monitoring of host subcellular acidification and Mtb intrabacterial pH profiles by live-fluorescence microscopy in a biosafety level 3 laboratory. By combining this live imaging approach with pharmacological and genetic perturbations, we show that Mtb can maintain its intracellular pH independently of the surrounding pH in primary human macrophages. Importantly, we show that unlike bedaquiline (BDQ), isoniazid (INH) or rifampicin (RIF), the front-line drug pyrazinamide (PZA) displays antibacterial efficacy by acting as protonophore which disrupts intrabacterial pH homeostasis in cellulo. By using Mtb mutants with different intra-macrophage localisation, we confirmed that intracellular acidification is a prerequisite for PZA efficacy in cellulo. We anticipate this dual imaging approach will be useful to identify host cellular environments that affect antibiotic efficacy against intracellular pathogens. HighlightsO_LIMtb maintains its intrabacterial pH inside both acidic and neutral subcellular microenvironments of human macrophages C_LIO_LIPyrazinamide, but not other frontline antibiotics, acts as a protonophore in cellulo C_LIO_LIPyrazinamide-mediated intrabacterial pH homeostasis disruption and antibacterial efficacy requires host endolysosomal acidification C_LIO_LICytosolic localisation mediated by ESX-1 contributes to pyrazinamide antibacterial activity resistance C_LIO_LIPyrazinamide conversion into pyrazinoic acid by the pyrazinamidase/nicotinamidase PncA is essential for its protonophore activity and efficacy in cellulo C_LI