Periodic Reporting for period 1 - SpaTime_AnTB (Single-cell spatiotemporal analysis of Mycobacterium tuberculosis responses to antibiotics within host microenvironments)
Reporting period: 2020-04-01 to 2022-03-31
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a global health issue and one of the deadliest diseases caused by a single infectious agent worldwide. Drug-sensitive TB requires treatment with a minimum of four antibiotics over a course of at least 6 months. The duration and toxicity of the current anti-TB regimens affect compliance, leading to treatment failure, relapse and emergence of resistant strains which constitute a real challenge. Therefore, the design of new drug regimens or alternative therapeutic strategies are desperately needed to reduce treatment duration and ultimately eradicate TB. In that context, understand how lung physiopathology and microenvironments affect antibiotic distribution, accumulation and efficacy is crucial, specially within macrophages, that represent one of the principal niches for Mtb survival and replication during infection. In this project entitled SpaTime_AnTB, we aimed at (i) implementing robust and innovative high-resolution quantitative microscopy approaches, in order to visualize the spatial distribution of antibiotics within infected human macrophages, (ii) determine how multiple intracellular niches and their respective microenvironments impact the accumulation and efficiency of antibiotics, and finally (iii) develop a model of host-pathogen interaction based on the use bacterial reporter strains, subcellular compartments labeling techniques and real-time microscopy approaches to define Mtb responses to different subcellular compartments in the presence or absence of front-line drugs.
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)
Infectious respiratory diseases, which include TB, are leading causes of death and disability in the world. Antimicrobial resistance (AMR) is also a serious global challenge for public health and sustainable development. Drug-resistant Mtb strains are increasingly becoming resistant to numerous drugs available making it more difficult to treat infections. Therefore, the study of intracellular pharmacokinetics and antibiotic mode of action may uncover new potential targets for the development of alternative therapeutic strategies.
However, such investigations are extremely challenging and require the development of cutting-edge technologies. Throughout the 24 months of this action, we have implemented several quantitative microscopy approaches to investigate antibiotic subcellular distribution, characterize their inhibitory activities and better define their molecular mode of action within innate immune cells. These unvaluable tools allowed us to decipher in detail, the long-time argued mode of action of Pyrazinamide (PZA), a front-line drug clinically used against TB for more than 50 years.
In the short term, such technological tools and biological findings will directly benefit academic scientists and clinicians working on bacterial pathogenesis, respiratory infections and antibiotic mode of actions. We have tried to reach as many colleagues as possible, and our laboratory is committed to make all the results fully available upon reasonable request. Moreover, we think that sharing our expertise, expanding our experimental-analytical tools and datasets might contribute to collegial groundbreaking discoveries in the years to come. In that context, our laboratory will welcome national and international collaborations to further broaden these investigations and keep benefiting the biomedical researchers and TB community.
In the long term, these concepts associated with innovative experimental approaches aim at identifying new bacterial therapeutic targets, discovering new bioactive molecules and uncovering novel antibiotic mode of action with the ultimate goal of reducing the global AMR problem and undoubtedly changing patients’ life.
However, such investigations are extremely challenging and require the development of cutting-edge technologies. Throughout the 24 months of this action, we have implemented several quantitative microscopy approaches to investigate antibiotic subcellular distribution, characterize their inhibitory activities and better define their molecular mode of action within innate immune cells. These unvaluable tools allowed us to decipher in detail, the long-time argued mode of action of Pyrazinamide (PZA), a front-line drug clinically used against TB for more than 50 years.
In the short term, such technological tools and biological findings will directly benefit academic scientists and clinicians working on bacterial pathogenesis, respiratory infections and antibiotic mode of actions. We have tried to reach as many colleagues as possible, and our laboratory is committed to make all the results fully available upon reasonable request. Moreover, we think that sharing our expertise, expanding our experimental-analytical tools and datasets might contribute to collegial groundbreaking discoveries in the years to come. In that context, our laboratory will welcome national and international collaborations to further broaden these investigations and keep benefiting the biomedical researchers and TB community.
In the long term, these concepts associated with innovative experimental approaches aim at identifying new bacterial therapeutic targets, discovering new bioactive molecules and uncovering novel antibiotic mode of action with the ultimate goal of reducing the global AMR problem and undoubtedly changing patients’ life.