To be effective, chemotherapy against tuberculosis (TB) must kill the intracellular population of the pathogen, Mycobacterium tuberculosis (Mtb). However, how host cell microenvironments affect antibiotic accumulation and efficacy remains unclear. In a first study, we used correlative light, electron, and ion microscopy (CLEIM) to investigate how various microenvironments within human macrophages affect the distribution and accumulation profile of pyrazinamide/pyrazinoic acid molecules (PZA/POA), a key antibiotic against TB. Our results showed that PZA/POA accumulate heterogeneously among host-cells and individual intracellular bacteria. By using a Mtb mutant strain lacking the ESX-1 Type VII secretion system, unable to damage phagosomal membranes, we demonstrated that restriction to membrane-bound compartments positively impact PZA/POA accumulation. Alternatively, by using pharmacological inhibitors of the proton H+ v-ATPase, we showed that phagosomal acidification is crucial for intrabacterial PZA/POA accumulation. Using this innovative experimental system, we also demonstrated that correlative ion-electron imaging can be used to identify anti-TB drugs distribution and interaction at a subcellular resolution, opening new avenues to design and assess alternative drug regimens within Mtb-infected human macrophages. Our results showed that Bedaquiline enhances PZA/POA enrichment in cellulo by increasing host-cell lysosomal activity. In a second study, we developed a dual-live imaging approach in BSL-3 conditions that allow to dynamically visualize in real time phagosomal acidification, Mtb pH homeostasis and PZA/POA mode of action. By combining pharmacological and genetic perturbations, we showed that Mtb can maintain its intracellular pH independently of the surrounding pH in primary human macrophages. We also demonstrated that unlike bedaquiline (BDQ), isoniazid (INH) or rifampicin (RIF), the front-line drug PZA/POA displays antibacterial efficacy by disrupting intrabacterial pH homeostasis within infected macrophages. By using Mtb mutants with different subcellular localisation, we confirmed that intracellular acidification is a prerequisite for PZA/POA efficacy in cellulo. Overall, our results showed that intracellular localization and host-environments drive antibiotic efficacy within infected human macrophage. Such technological tools, biological systems and experimental approaches can be applicable to the treatment of other intracellular pathogens and help to inform the development of more effective combined therapies that target heterogenous bacterial populations within the host. The research work that has been carried during the course of the action has been published in peer-reviewed open access journals to make data rapidly accessible for the scientific community. Moreover, The Francis Crick Institute is committed to an open culture where ideas can be tested and challenges shared to accelerate the creation and use of knowledge. Therefore, this work has also been presented or will be presented at different national and international conferences. Our work was also promoted on the homepages of the host laboratory/institutions and through social media (i.e. team and institute websites, researchers and laboratory twitter accounts, The Crick newsletters, Nature Microbiology Portofolio). Indeed, the results generated during this action have benefited from the continuous support of the Crick Communication Team, was subjected to multiple press releases. Finally, this work has been also awarded 2nd best oral communication award at the UK Cellular Microbiology Network (2021) and selected as Article of the Month by the French learning society SFBBM (2022)
