Project #12997
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#12997 : Identifying subpopulation-specific signatures of tuberculosis persistence for enhanced diagnostics.
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Name of Applicant : Giulia MANINA
Date of application : 26-02-2019
Unit : Microbial Individuality and Infection
Location : 62-04-15
Phone : +33 01 45 68 87 45
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Project context and summary :

Microbes are prone to rapid changes and they can either exploit or countervail their variation in a context-dependent manner. To this purpose, both genetic diversity and non-genetic phenotypic variation exist. However, while the overall mutational evolution occurs over lengthy timescales, epigenetic changes take place on a large scale and more rapidly. Collectively this implies that the diversity we observe is profoundly driven by non-genetic variation. This is particularly relevant for the WHO Priority Pathogen Mycobacterium tuberculosis, whose lack of lateral gene transfer and low mutation rate make phenotypic variation an important means of adaptation to stressful conditions. A few studies, including ours, have begun to explore this phenomenon at the single-cell level in M. tuberculosis in axenic and host conditions, which are technically very challenging. This project is based on the assumption that M. tuberculosis can successfully endure harsh environmental conditions thanks to its phenotypic variation. In our view a better understanding of the drivers of phenotypic variation will improve the design and development of original strategies for tuberculosis control. Here we investigate the physiology of M. tuberculosis at the single-cell and subpopulation scale, striving to demystify the bases of phenotypic diversity and the implications for adaptation and persistence. Previously we examined by real-time imaging a fluorescent reporter of ribosomal expression (rRNA-GFP) as a gauge for cellular activity, and found that M. tuberculosis displays phenotypic heterogeneity under optimal growth conditions, which is enhanced in the host, in long-term stationary phase and upon drug exposure. Remarkably we could also detect subpopulations of quiescent bacilli, whose molecular characteristics have yet to be determined, which is the aim of this project. Here we constructed a dual fluorescent reporter of metabolic activity/quiescence in M. tuberculosis, by using our rRNA-GFP reporter as a background strain, further modified with a red fluorescent marker of cellular quiescence. We carried out snapshot microscopy and single-cell analysis during optimal growth conditions as compared with stressful conditions. We found that the cell-activity marker decreases, whereas the cell-quiescence marker is induced under different host-mimetic conditions. We also observed significant intracellular variation during infection assays. Now we envision carrying out a comprehensive analysis of M. tuberculosis phenotypic variation by RNA sequencing. We aim to reveal the molecular differences between subpopulations of bacilli that exhibit discrete metabolic potential, based on their fluorescence output. We have recreated the most interesting conditions on a bulk scale, and sorted active versus quiescent subpopulations, aiming to compare their transcriptional profiles, and to ultimately identify subpopulation-specific biomarkers of persistence towards more accurate diagnostics.

Related team publications :
Stress and host immunity amplify Mycobacterium tuberculosis phenotypic heterogeneity and induce nongrowing metabolically active forms, PMID: 25543231, doi: 10.1016/j.chom.2014.11.016.
Phenotypic Heterogeneity in Mycobacterium tuberculosis, PMID: 27837741, doi: 10.1128/microbiolspec.TBTB2-0021-2016.
Preexisting variation in DNA integrity predicts the fate of single mycobacterial cells under stress (Submitted to the EMBO Journal).
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Project Type : Medium
Status : Awaiting Publication

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