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International audience; Fragment-based lead discovery has emerged over the last decades as one of the most powerful techniques for identifying starting chemical matter to target specific proteins or nucleic acids in vitro. However, the use of such low-molecular-weight fragment molecules in cell-based phenotypic assays has been historically avoided because of concerns that bioassays would be insufficiently sensitive to detect the limited potency expected for such small molecules and that the high concentrations required would likely implicate undesirable artifacts. Herein, we applied phenotype cell-based screens using a curated fragment library to identify inhibitors against a range of pathogens including Leishmania, Plasmodium falciparum, Neisseria, Mycobacterium, and flaviviruses. This proof-of-concept shows that fragment-based phenotypic lead discovery (FPLD) can serve as a promising complementary approach for tackling infectious diseases and other drug-discovery programs.

International audience; Several phenanthrolinic analogs of quinolones have been synthesized and their antibacterial activity tested against Mycobacterium tuberculosis, other mycobacterial species and bacteria from other genera. Some of them show high activity (of the range observed for rifampicin) against M. tuberculosis replicating in vitro and in vivo (infected macrophages) conditions. These derivatives show the same activity with all or several M. tuberculosis complex bacterial mutants resistant to fluoroquinolones (FQ). This opens the way to the construction of new drugs for the treatment of FQ resistant bacterial infections, including tuberculosis. Several compounds showed also activity against Staphylococcus aureus and probably other species. These compounds do not show major toxicity. We conclude that the novel phenanthrolinic derivatives described here are potent hits for further developments of new antibiotics against bacterial infectious diseases including tuberculosis in particular those resistant to FQ.

International audience; In this study, we aimed to decipher the natural resistance mechanisms of mycobacteria against novel compounds isolated by whole-cell-based high-throughput screening (HTS). We identified active compounds using Mycobacterium aurum. Further analyses were performed to determine the resistance mechanism of M. smegmatis against one hit, 3-bromo-N-(5-nitrothiazol-2-yl)-4-propoxybenzamide (3), which turned out to be an analog of the drug nitazoxanide (1). We found that the repression of the gene nfnB coding for the nitroreductase NfnB was responsible for the natural resistance of M. smegmatis against 3. The overexpression of nfnB resulted in sensitivity of M. smegmatis to 3. This compound must be metabolized into hydroxylamine intermediate for exhibiting antibacterial activity. Thus, we describe, for the first time, the activity of a mycobacterial nitroreductase against 1 analogs, highlighting the differences in the metabolism of nitro compounds among mycobacterial species and emphasizing the potential of nitro drugs as antibacterials in various bacterial species.

The objective of this study was to find molecules with anti-mycobacterial activity from a natural compounds library, investigate their mechanisms of resistance, and assess their synergy with antibiotics. We screened a library of 2582 natural compounds with Mycobacterium aurum with the aim of identifying molecules with anti-mycobacterial activity. The hits with the lowest MICs in M. aurum were also tested for their antimicrobial activity in other mycobacterial species including M. tuberculosis complex strains. The chequerboard titration assay was chosen for determining drug interactions in vitro. Spontaneous resistant mutants were isolated and their whole genome sequences compared to wild type and resistant mutants to identify resistance mechanisms. We found that ionophores show anti-mycobacterial activity in vitro. Resistance mechanism to ionophores is mediated by the MmpL5-MmpS5 transporter overexpression. Ionophore A23187 enhanced beta-lactam activity in M. tuberculosis infected macrophage. It will help in the investigation of new drug combinations against bacterial infections including tuberculosis.

The diagnosis of tuberculosis in developing countries still relies on direct sputum examination by light microscopy, a method that is easy to perform and that is widely applied. However, because of its poor sensitivity and requirement for significant labor and training, light microscopy examination detects the bacilli in only 45 to 60% of all people whose specimens are culture positive for Mycobacterium tuberculosis . Therefore, new diagnostic methods that would enable the detection of the undiagnosed infected population and allow the early commencement of antituberculosis treatment are needed. In this work, the potential use of mycobacterial cyan autofluorescence for the detection of Mycobacterium tuberculosis was explored. The tubercle bacilli were easily visualized as brilliant fluorescent bacilli by microscopy and were easily tracked ex vivo during macrophage infection. Assays with seeded sputum and a 96-well microplate reader fluorimeter indicated that 6 bacilli ml −1 of sputum could be detected. Moreover, the use of microplates allowed the examination of only 200 μl of sputum per sample without a loss of sensitivity. Treatment with heat or decontaminating chemical agents did not interfere with the autofluorescence assay; on the contrary, they improved the level of bacterial detection. Autofluorescence for the detection of bacilli is rapid and easy to perform compared to other methodologies and can be performed with minimal training, making this method suitable for implementation in developing countries.

Background The establishment of the cellular localization of proteins in M. tuberculosis will provide of valuable information for the identification of new drug/vaccine/diagnostic targets. Cytolocalization by inmunofluorescence microscopy has been limited in mycobacteria because to difficulties in effectively permeabilize it. Results A treatment combining lysozyme with triton X-100 was found to be an effective permeabilization method of the mycobacterial envelope. Conclusion A rapid and simple permeabilization protocol has been successfully assessed in pure cultures of both Mycobacterium smegmatis and Mycobacterium tuberculosis H37Rv. This method can be successful used in the cytolocalization of proteins by immunolabeling.

Mycobacterium tuberculosis has the remarkable capacity to survive within the hostile environment of the macrophage, and to resist potent antibacterial molecules such as reactive oxygen species (ROS). Thus, understanding mycobacterial resistance mechanisms against ROS may contribute to the development of new anti-tuberculosis therapies. Here we identified genes involved in such mechanisms by screening a high-density transposon mutant library, and we show that several of them are involved in the intracellular lifestyle of the pathogen. Many of these genes were found to play a part in cell envelope functions, further strengthening the important role of the mycobacterial cell envelope in protection against aggressions such as the ones caused by ROS inside host cells.

BACKGROUND: The Mycobacterium tuberculosis PhoP/PhoR two-component signal transduction system controls the expression of about 2% of the genome and plays a major role in pathogenicity. However, its regulon has not been well characterized. METHODOLOGY/PRINCIPAL FINDINGS: The binding site of PhoP transcription regulator was identified in the upstream regions of msl3, pks2, lipF and fadD21 genes, by using gene fusions, electrophoretic mobility shift assays and DNase I footprinting experiments. A consensus sequence for PhoP binding was deduced. It consists of two direct repeats, DR1/DR2, associated with a third repeat, DR3, important in some cases for PhoP binding to DR1/DR2 but located at a variable distance from these direct repeats. DR1/DR2 and DR3 consensus sequences were used to screen the whole-genome sequence for other putative binding sites potentially corresponding to genes directly regulated by PhoP. The identified 87 genes, encoding transcription regulators, and proteins involved in secondary metabolites biosynthesis, transport and catabolism are proposed to belong to the PhoP regulon. CONCLUSIONS/SIGNIFICANCE: A consensus sequence derived from the analysis of PhoP binding to four gene promoter regions is proposed. We show for the first time the involvement of a third direct repeat motif in this binding reaction. The consensus sequence was instrumented to study the global regulation mediated by PhoP in M. tuberculosis. This analysis leads to the identification of several genes that are potentially regulated by this key player.

We thank Julien Bortoli Chapalay, Damiano Banfi, Antoine Gibelin, and Gerardo Turcatti from EPFL’s Biomolecular Screening Facility for help with screening and compound management; Valérie Briolat and Marc Monot from the Institut Pasteur Biomics Platform for RNA-Seq support; and Priscille Brodin, Tony Maxwell, Claudia Sala, and Anthony Vocat of the MM4TB consortium for advice and technical support.; International audience; Screening of a diversity-oriented compound library led to the identification of two 6,11-dioxobenzo[f ]pyrido[1,2a]indoles (DBPI) that displayed low micromolar bactericidal activity against the Erdman strain of Mycobacterium tuberculosis in vitro. The activity of these hit compounds was limited to tubercle bacilli, including the nonreplicating form, and to Mycobacterium marinum. On hit expansion and investigation of the structure activity relationship, selected modifications to the dioxo moiety of the DBPI scaffold were either neutral or led to reduction or abolition of antimycobacterial activity. To find the target, DBPIresistant mutants of M. tuberculosis Erdman were raised and characterized first microbiologically and then by whole genome sequencing. Four different mutations, all affecting highly conserved residues, were uncovered in the essential gene rv0338c (ispQ) that encodes a membrane-bound protein, named IspQ, with 2Fe−2S and 4Fe-4S centers and putative iron−sulfur-binding reductase activity. With the help of a structural model, two of the mutations were localized close to the 2Fe−2S domain in IspQ and another in transmembrane segment 3. The mutant genes were recessive to the wild type in complementation experiments and further confirmation of the hit−target relationship was obtained using a conditional knockdown mutant of rv0338c in M. tuberculosis H37Rv. More mechanistic insight was obtained from transcriptome analysis, following exposure of M. tuberculosis to two different DBPI; this revealed strong upregulation of the redox-sensitive SigK regulon and genes induced by oxidative and thiol-stress. The findings of this investigation pharmacologically validate a novel target in tubercle bacilli and open a new vista for tuberculosis drug discovery.