Label free quantification of proteins after the infection with M. tuberculosis. Macrophages isolated from seven patients were used in this study. Four conditions were compared.

Tuberculosis (TB) still remains a major public health problem with estimated 9 million incident cases and 1.5 million deaths in 2014 (WHO, Global Tuberculosis Report 2015). More worrisome is the emergence of multi drug resistance (MDR), or even extensively resistant (XDR) M. tuberculosis strains worldwide. The standardized treatment of pan-susceptible tuberculosis is the administration of two antibiotics (rifampicin and isoniazid) for six months, accompanied by two additional antibiotics (pyrazinamid and ethambutol) for the first two months. Although very efficacious, this treatment is very demanding due to the duration and the possible side effects. The treatment of MDR-TB is less standardized, with more toxic and poorly tolerated drugs, resulting in lower cure rates. Therefore, we need not only more molecules with antimycobacterial activity, but also, we urgently need new strategies to increase our therapeutic arsenal for treating MDR-TB. Only three new drugs, bedaquiline, delamanid and PA-824 have been tested in phase2/3 clinical trials.

In this context, the european funded project NAREB has been created. It brings together 14 partners from 8 EU Member and Associated States, and it aims to (i) screen different combinations of antibiotic drugs with nano-carriers (lipid, polymeric, biopolymeric) with and without targeting ligands, (ii) coload antibiotics in order to develop innovative therapeutic combination therapies (iii) test in vitro and in vivo the best therapeutic combinations. In particular, we will analyze more in-depth the effect of bedaquilin, new TB drugs and nano-carriers on the host/bacterial transcriptome using RNAseq.

Tuberculosis (TB), which is caused by Mycobacterium tuberculosis (MTB), is the deadliest disease due to a single infectious agent. Despite considerable efforts to fight the disease, TB remains a major public health problem. Even more worrying for the future, multidrug resistant (MDR) strains of MTB are continually emerging and about 10% of people with MDR-TB have extensively drug-resistant TB (XDR-TB). Drug-sensitive TB can be cured by a 6-month treatment using 4 antibiotics, but MDR-TB and extensively drug-resistant XDR-TB require treatment for up to 2 years with more toxic and costly second- and third-line drugs. Toxicity of these drugs is well described; it includes hepatotoxicity, liver injury, skin reactions, gastrointestinal and neurological disorders. However how MTB drugs influence the host response to MTB infection has been poorly addressed. The main goal of project is to understand how drugs interact with the host in order to improve the treatment.

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.

Without new treatment development tuberculosis could cause about 70 million deaths by 2050, mostly due to the spread of multidrug-resistant strains. The standard drug regimen still builds on the first drugs introduced decades ago, and takes 6 months in the case of drug-sensitive tuberculosis, and up to 2 years in the case of drug-resistant tuberculosis, with heavy side effects. This long therapeutic regimen often results in patients not being able to follow it or complete it correctly, which promotes the chronicity of the infection and ultimately the onset of drug resistance. Although a few new molecules have been discovered, improving both the quality and the duration of tuberculosis chemotherapy remain pressing needs. Furthermore, the failure of chemotherapy is not only due to genetic resistance, which takes relatively long to occur, but also to the intrinsic ability of mycobacteria to diversify in discrete phenotypic states, which can endure drugs even in the absence of genetic mutations. This phenomenon, known as persistence, can eventually favor the onset of resistance, with major repercussions on disease control. In sum, tuberculosis therapy presents many challenges and in our view it is critical to study the ability of a drug or a drug combination to sterilize discrete subpopulations, which may either pre-exist in the population or result from adaptive processes. We found that, prior to drug exposure, phenotypically distinct subpopulations exist that display different drug susceptibility. In light of this, we hypothesized that phenotypic variation from cell to cell favors persistence and can consequently bring about treatment failure. Here we aim to identify molecules that reduce phenotypic variation, making the population more uniformly and rapidly susceptible to standard treatment. To this end, we developed a microfluidic system that allows us to track single cells by live imaging and to carry out a screening at the single-cell level, looking for molecules that homogenize the bacterial population and enhance the effectiveness of the standard treatment. Our approach could ultimately offer original therapeutic strategies towards better control of tuberculosis.