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Results from clinical strains and knockouts of the H37Rv and CDC1551 laboratory strains demonstrated that ndh (Rv1854c) is not a resistance-conferring gene for isoniazid, ethionamide, delamanid, or pretomanid in Mycobacterium tuberculosis. This difference in the susceptibility to NAD-adduct-forming drugs compared with other mycobacteria may be driven by differences in the absolute intrabacterial NADH concentration., Funding Agencies|National Institute of Health Grant [ANR-20-PAMR-0005]; PNG National Tuberculosis Programme [AI26170]; National Institute of Health

Globisporangium ultimum is an oomycetal pathogen causing damping-off on over 300 different plant hosts. Currently, as for many phytopathogens, its control relies in the use of chemicals with negative impact on health and ecosystems. Therefore, many biocontrol strategies are under investigation to reduce the use of fungicides. In this study, the soil bacterium Pseudomonas sp. NCIMB 10586 demonstrates a strong iron-repressed in vitro antagonism against G. ultimum MUCL 38045 caused by the secretion of a novel non-ribosomal peptide synthetase (NRPS) siderophore named mupirochelin. Its putative structure obtained by MS/MS analysis bears similarities to other siderophores and bioactive compounds. Besides mupirochelin, we observed the production of a third and novel NRPS-independent siderophore (NIS), here termed triabactin. The iron-responsive transcriptional repression of the two newly identified siderophore gene clusters corroborates their role as iron scavengers. However, their respective contributions to the strain fitness are dissimilar. Bacterial growth in iron-deprived conditions is greatly supported by pyoverdine production and, to a lesser extent, by triabactin. On the contrary, mupirochelin does not contribute to the strain fitness under the studied conditions. Besides a possible repression induced by the ferric uptake regulator (Fur), the expression of mupirochelin gene cluster has been shown to be activated by the AraC-like transcriptional regulator MchR. RT-qPCR results suggest that MchR activates mupirochelin biosynthesis when it binds the ferri-mupirochelin chelate. Therefore, the regulation of mupirochelin pathway is tightly controlled by iron availability. The predictive structure of MchR generated with Alphafold2 indicates the presence of a N-terminal effector-binding domain containing a large hydrophobic cavity buried in its centre and presumed to accommodate mupirochelin. Purification of a recombinant MBP-MchR fusion protein was achieved to characteriz, Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie), info:eu-repo/semantics/nonPublished

The sensitivity of Mycobacterium tuberculosis, the pathogen that causes tuberculosis (TB), to antibiotic prodrugs is dependent on the efficacy of the activation process that transforms the prodrugs into their active antibacterial moieties. Various oxidases of M. tuberculosis have the potential to activate the prodrug ethionamide. Here, we used medicinal chemistry coupled with a phenotypic assay to select the N-acylated 4-phenylpiperidine compound series. The lead compound, SMARt751, interacted with the transcriptional regulator VirS of M. tuberculosis, which regulates the mymA operon encoding a monooxygenase that activates ethionamide. SMARt751 boosted the efficacy of ethionamide in vitro and in mouse models of acute and chronic TB. SMARt751 also restored full efficacy of ethionamide in mice infected with M. tuberculosis strains carrying mutations in the ethA gene, which cause ethionamide resistance in the clinic. SMARt751 was shown to be safe in tests conducted in vitro and in vivo. A model extrapolating animal pharmacokinetic and pharmacodynamic parameters to humans predicted that as little as 25 mg of SMARt751 daily would allow a fourfold reduction in the dose of ethionamide administered while retaining the same efficacy and reducing side effects., SCOPUS: ar.j, info:eu-repo/semantics/published

Killing more than one million people each year, tuberculosis remains the leading cause of death from a single infectious agent. The growing threat of multidrug-resistant strains of Mycobacterium tuberculosis stresses the need for alternative therapies. EthR, a mycobacterial transcriptional regulator, is involved in the control of the bioactivation of the second-line drug ethionamide. We have previously reported the discovery of in vitro nanomolar boosters of ethionamide through fragment-based approaches. In this study, we have further explored the structure-activity and structure-property relationships in this chemical family. By combining structure-based drug design and in vitro evaluation of the compounds, we identified a new oxadiazole compound as the first fragment-based ethionamide booster which proved to be active in vivo, in an acute model of tuberculosis infection., SCOPUS: ar.j, info:eu-repo/semantics/published

Although the chemical composition of mycobacterial cell walls is well known, the 3D organization of the various constituents is not fully understood. In particular, it is unclear whether the major wall component lipoarabinomannan (LAM) is exposed on the outermost surface or hindered by other constituents such as mycolic acids. To address this pertinent question, we used atomic force microscopy (AFM) with tips bearing anti-LAM antibodies to detect single LAM molecules on Mycobacterium bovis BCG cells. First, we showed the ability of anti-LAM tips to detect isolated, purified LAM molecules. We then mapped the distribution of LAM on mycobacteria, prior to and after treatment with the drug isoniazid. We found that LAM was not exposed on the surface of native cells, pointing to the presence of a homogeneous layer of mycolic acids, whereas it was greatly exposed on isoniazid-treated cells, in agreement with the action mode of the drug. This single-molecule study provides novel insight into the architecture of mycobacterial cell walls and offers promising perspectives for understanding the action modes of antimycobacterial drugs.

The biosynthesis of the Mycobacterium tuberculosis cell wall is targeted by some of the most powerful antituberculous drugs. To date, the molecular mechanisms by which these antibiotics affect the cell wall characteristics are not well understood. Here, we used atomic force microscopy - in three different modes - to probe the nanoscale surface properties of live mycobacteria and their modifications upon incubation with four antimycobacterial drugs: isoniazid, ethionamide, ethambutol, and streptomycine. Topographic imaging, combined with quantitative surface roughness analysis, demonstrated that all drugs induce a substantial increase of surface roughness to an extent that correlates with the localization of the target (i.e., synthesis of mycolic acids, arabinogalactans, or proteins). Chemical force microscopy with hydrophobic tips revealed that the structural alterations induced by isoniazid and ethambutol were correlated with a dramatic decrease of cell surface hydrophobicity, reflecting the removal of the outermost mycolic acid layer. Consistent with this finding, tapping mode imaging, combined with immunogold labeling, showed that the two drugs lead to the massive exposure of hydrophilic lipoarabinomannans at the surface. Taken together, these structural, chemical, and immunological data provide novel insight into the action mode of antimycobacterial drugs, as well as into the spatial organization of the mycobacterial cell wall.

Traditionally, cell surface properties have been difficult to study at the subcellular level, especially on hydrated, live cells. Here, we demonstrate the ability of chemical force microscopy to map the hydrophobicity of single live cells with nanoscale resolution. After validating the technique on reference surfaces with known chemistry, we probe the local hydrophobic character of two medically important microorganisms, Aspergillus fumigatus and Mycobacterium bovis, in relation with function. Applicable to a wide variety of cells, the chemically sensitive imaging method presented here provides new opportunities for studying the nanoscale surface properties of live cells and for understanding their roles in mediating cellular events.

Although hydrophobic forces are of great relevance in biological systems, quantifying these forces on complex biosurfaces such as cell surfaces has been difficult owing to the lack of appropriate, ultrasensitive force probes. Here, chemical force microscopy (CFM) with hydrophobic tips was used to measure local hydrophobic forces on organic surfaces and on live bacteria. On organic surfaces, we found an excellent correlation between nanoscale CFM and macroscale wettability measurements, demonstrating the sensitivity of the method toward hydrophobicity and providing novel insight into the nature of hydrophobic forces. Then, we measured hydrophobic forces associated with mycolic acids on the surface of mycobacteria, supporting the notion that these hydrophobic compounds represent an important permeation barrier to drugs.

Progress in understanding the structure-function relationships of the mycobacterial cell wall has been hampered by its complex architecture as well as by the lack of sensitive, high-resolution probing techniques. For the first time, we used atomic force microscopy (AFM) to image the surface topography of hydrated Mycobacterium bovis bacillus Calmette Guérin cells and to investigate the influence of the antimycobacterial drug ethambutol on the cell wall architecture. While untreated cells showed a very smooth and homogeneous surface morphology, incubation of cells in the presence of ethambutol caused dramatic changes of the fine surface structure. At 4 micro g mL(-1), the drug created concentric striations at the cell surface and disrupted a approximately 8 nm thick cell wall layer, attributed to the outer electron-opaque layer usually seen by electron microscopy, while at 10 micro g mL(-1) an underlying approximately 12 nm thick layer reflecting the thick electron-transparent layer was also altered. These noninvasive ultrastructural investigations provide novel information on the macromolecular architecture of the mycobacterial envelope as well as into the destructuring effects of ethambutol.