Summary
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.
Highlights
Mtb maintains its intrabacterial pH inside both acidic and neutral subcellular microenvironments of human macrophages
Pyrazinamide, but not other frontline antibiotics, acts as a protonophore in cellulo
Pyrazinamide-mediated intrabacterial pH homeostasis disruption and antibacterial efficacy requires host endolysosomal acidification
Cytosolic localisation mediated by ESX-1 contributes to pyrazinamide antibacterial activity resistance
Pyrazinamide conversion into pyrazinoic acid by the pyrazinamidase/nicotinamidase PncA is essential for its protonophore activity and efficacy in cellulo
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
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Abbreviations
- (TB)
- tuberculosis
- (Mtb)
- Mycobacterium tuberculosis
- (RIF)
- rifampicin
- (INH)
- isoniazid
- (EMB)
- ethambutol
- (PZA)
- pyrazinamide
- (POA)
- total pyrazinoic acid
- (POA(-))
- pyrazinoate anion
- (HPOA)
- neutral protonated pyrazinoic acid
- (CLEIM)
- correlative light, electron, ion microscopy
- (MDM)
- human monocyte-derived macrophages
- (iPSC)
- human induced pluripotent stem cells
- (iPSDM)
- human induced pluripotent stem cell-derived macrophages
- (ConA)
- Concanamycin A
- (mROI)
- mycobacterial region of interest
- (EC50)
- half maximal effective concentration
- (BDQ)
- bedaquiline
- (Mbv)
- Mycobacterium bovis
- (MOI)
- multiplicity of infection