key: cord-0283711-9xlugqzj authors: Santucci, Pierre; Aylan, Beren; Botella, Laure; Bernard, Elliott M.; Bussi, Claudio; Pellegrino, Enrica; Athanasiadi, Natalia; Gutierrez, Maximiliano G. title: Visualizing pyrazinamide action by live single cell imaging of phagosome acidification and Mycobacterium tuberculosis pH homeostasis date: 2021-12-20 journal: bioRxiv DOI: 10.1101/2021.12.20.473466 sha: 4bd1ed85ae422b367912f3f6c28d27af107527e6 doc_id: 283711 cord_uid: 9xlugqzj 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 from 30 mg/L to 400 mg/L significantly disrupted bacterial pH homeostasis (Figure 3G-3I) . 249 The absolute changes in Mtb pH-GFP ratio relative to the control condition, confirmed a time- These data highlight that PZA-mediated pH homeostasis disruption within acidic 263 environments is a dynamic process. Endolysosomal acidification and protonophore activity of PZA contribute to Mtb 265 restriction in human macrophages 266 We next hypothesized that functional host intracellular acidification and PZA-mediated pH-267 decrease are required for mycobacterial growth inhibition. In order to test this hypothesis, we ConA co-treatment increased, by approximately 3.5 times, the amount of antibiotic required 275 to efficiently inhibit 50% of Mtb growth in cellulo (49.5 ± 19.2 mg/L and 173.1 ± 35.2 mg/L, 276 respectively) (Figure 4A-4B) . These results agree with our previous observations showing 277 that the use of v-ATPase inhibitors is able to counteract PZA/POA-mediated growth inhibition 278 by impairing POA accumulation within the bacteria (Santucci et al., 2021) . These experiments 279 were also performed in another human macrophage model using iPSDM (Figure S6 ). 280 Notably, in iPSDM antagonistic effects between PZA and ConA were also observed, however EsxA and EsxB (also known as ESAT-6 and CFP-10). A relative growth index was quantified 295 for each strain, and a dose-response analysis using a four-parameter logistic non-linear POA and that such conversion is essential for antibacterial activity within infected 332 macrophages. Here we described a novel live dual imaging approach to monitor pH homeostasis within both 336 the host cell and the pathogen in a biosafety level 3 laboratory. This approach is applicable 337 to both human primary monocyte derived macrophages (MDM) and human iPS-derived 338 macrophages (iPSDM). In both MDM and iPSDM, ConA treatment was able to reduce Mtb-339 associated LysoTracker intensity suggesting that this pharmacological inhibition is a powerful 340 approach to perturb endolysosomal acidification in these two macrophage models. The study of the PZA molecular mechanism(s) of action, and its extensive association with All data reported in this paper will be shared by the lead contact upon reasonable request. This paper does not report original code. where maximum projection of the 3-4 z-planes was used to perform single cell segmentation 887 by using the "Find nuclei" and "Find cells" building blocks. Cells on the edges were excluded 888 from the analysis. 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