Deciphering molecular basis of the mechanism of transport mediated by the MmpL efflux pumps and study of the role of the Eis, N-acetyltransferases in antibiotic resistance in mycobacteria

Le tréhalose monomycolate (TMM) est un glycolipide majeur qui constitue la paroi atypique des mycobactéries. La protéine « Mycobacterial membran protein Large 3 » (MmpL3) transportant le TMM est essentielle à la croissance des bacilles. MmpL3 apparaît donc comme une cible thérapeutique prometteuse à exploiter pour contrer les infections à mycobactéries. Par des approches de biologie structurale et biochimiques, ce projet de thèse visait à comprendre les bases moléculaires du transport du TMM et le mode d'action des inhibiteurs ciblant cette voie. Dans cette finalité, un protocole de purification de MmpL3 a été établi. Nous avons obtenu des cristaux de MmpL3 permettant de résoudre la structure cristalline (S3D) de MmpL3 de Mycobacterium smegmatis. Par ailleurs, des études récentes ont démontré que MmpL3 agit en concert avec d’autres protéines pour le transport du TMM. Nous avons résolu la S3D d’un de ces partenaires, le facteur A de transport du TMM (TtfA) qui est essentiel à la survie des mycobactéries. TtfA possède un repliement unique et notre analyse bioinformatique suggère que la fonction de TtfA ne serait pas uniquement dédiée au transport du TMM. Les mycobactéries peuvent inactiver les antibiotiques de type aminoglycosides (AG) par acétylation. La protéine Eis2 (Enhanced Intracellular Survival) de M. abscessus (Mab) est importante pour la survie du bacille dans les macrophages. Nous avons pu montrer que Eis2, en plus de son rôle important dans la persistance intracellulaire, est capable de modifier les AG et en particulier l' amikacine (AMK), un antibiotique de base du traitement des infections à Mab. La S3D de Eis2, nous a permis d’ identifier plusieurs inhibiteurs de l’ enzyme. Nous avons également démontré que le site actif atypique de Eis1 de Mab, un homologue de Eis2, ne permet pas à l’ enzyme d’ inactiver les AG. Nos études génétiques permettent de conclure que Eis1 contrairement à Eis2 n’ est pas impliqué dans la résistance aux AG.Mycobacteria possess an atypical and hydrophobic cell wall which, limits the penetration of the antibiotics and drug-like molecules. Trehalose monomycolates (TMM) are glycolipids and building blocks of the mycomembrane. The Mycobacterial membrane protein Large 3 (MmpL3) mediates the TMM transport and is essential for bacilli growth. Several studies have highlighted MmpL3 as a promising drug target. To understand by structural and biochemical approaches the molecular basis of TMM transport and the mode of action of inhibitors targeting this pathway, we first established a robust purification protocol of MmpL3. We crystallized MmpL3 from several mycobacterial species, which allowed determination of the MmpL3 crystal structure (S3D) from Mycobacterium smegmatis. Recent studies demonstrated that MmpL3 requires several accessory proteins for efficient TMM transport. We could solve the S3D of one of these MmpL3 partners, the TMM transport factor A (TtfA) that was reported to be essential for the survival of mycobacteria. The TtfA S3D revealed a unique protein fold and bioinformatics analysis suggested that the TtfA function might not be solely dedicated to TMM transport. Mycobacteria can modify and inactivate antibiotics. The enhanced intracellular survival protein (Eis2) from M. abscessus (Mab) is important for invasion of the host and persistence. We could show by structural and biochemical approaches that Eis2, on top of its role in colonization, can modify by acetylation and thus inactivate several aminoglycosides (AG) and particularly amikacin (AMK), one of the cornerstone antibiotics for Mab infection treatment. Furthermore, by exploiting the Eis2 structural data, we found several inhibitors of Eis2 and could propose why apramycin an efficient anti-Mab AG is not inactivated by Eis2. We demonstrated also that the atypical active site of Eis1 from Mab a close homolog of Eis2 is not allowing the inactivation of AG. We could conclude that Eis1 contrary to Eis2 is not involved in AG resistance

MmpL3, the trehalose monomycolate transporter, is stable in solution in several detergents and can be reconstituted into peptidiscs

International audienceMycobacteria possess a complex and waxy cell wall comprising a large panel of glycolipids. Among these, trehalose monomycolate (TMM) represents abundant and crucial components for the elaboration of the mycomembrane. TMM is synthesized in the cytoplasmic compartment and translocated across the inner membrane by the MmpL3 transporter. Inhibitors impeding TMM transport by targeting MmpL3 show great promises as new antimycobacterials. The recent X-ray or Cryo-EM structures of MmpL3 complexed to TMM or its inhibitors have shed light on the mechanisms of TMM transport and inhibition. So far, purification procedures mainly involved the use of n-Dodecyl-ß-d-Maltopyranoside to solubilize and stabilize MmpL3 from Mycobacterium smegmatis (MmpL3Msm) or Lauryl Maltose Neopentyl Glycol for MmpL3 from Mycobacterium tuberculosis. Herein, we explored the possibility to solubilize and stabilize MmpL3 with other detergents. We demonstrate that several surfactants from the ionic, non-ionic and zwitterionic classes are prone to solubilize MmpL3Msm expressed in Escherichia coli. The capacity of these detergents to stabilize MmpL3Msm was evaluated by size-exclusion chromatography and thermal stability. This study unraveled three new detergents DM, LDAO and sodium cholate that favor solubilization and stabilization of MmpL3Msm in solution. In addition, we report a protocol that allows reconstitution of MmpL3Msm into peptidiscs

Structural analysis of the N ‐acetyltransferase Eis1 from Mycobacterium abscessus reveals the molecular determinants of its incapacity to modify aminoglycosides

International audienceEnhanced intracellular survival (Eis) proteins belonging to the superfamily of the GCN5-related N-acetyltransferases play important functions in mycobacterial pathogenesis. In Mycobacterium tuberculosis, Eis enhances the intracellular survival of the bacilli in macrophages by modulating the host immune response and is capable to chemically modify and inactivate aminoglycosides. In nontuberculous mycobacteria (NTM), Eis shares similar functions. However, Mycobacterium abscessus, a multidrug resistant NTM, possesses two functionally distinct Eis homologues, Eis1Mab and Eis2Mab . While Eis2Mab participates in virulence and aminoglycosides resistance, this is not the case for Eis1Mab, whose exact biological function remains to be determined. Herein, we show that overexpression of Eis1Mab in M. abscessus fails to induce resistance to aminoglycosides. To clarify why Eis1Mab is unable to modify this class of antibiotics, we solved its crystal structure bound to its cofactor, acetyl-CoA. The structure revealed that Eis1Mab has a typical homohexameric Eis-like organization. The structural analysis supported by biochemical approaches demonstrated that while Eis1Mab can acetylate small substrates, its active site is too narrow to accommodate aminoglycosides. Comparison with other Eis structures showed that an extended loop between strands 9 and 10 is blocking the access of large substrates to the active site and movement of helices 4 and 5 reduces the volume of the substrate-binding pocket to these compounds in Eis1Mab . Overall, this study underscores the molecular determinants explaining functional differences between Eis1Mab and Eis2Mab, especially those inherent to their capacity to modify aminoglycosides

Crystal structure of the aminoglycosides N -acetyltransferase Eis2 from Mycobacterium abscessus

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Biochemical, structural, and functional studies reveal that MAB_4324c from Mycobacterium abscessus is an active tandem repeat N ‐acetyltransferase

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Slow release of a synthetic auxin induces formation of adventitious roots in recalcitrant woody plants

Clonal propagation of plants by induction of adventitious roots (ARs) from stem cuttings is a requisite step in breeding programs. A major barrier exists for propagating valuable plants that naturally have low capacity to form ARs. Due to the central role of auxin in organogenesis, indole-3-butyric acid is often used as part of commercial rooting mixtures, yet many recalcitrant plants do not form ARs in response to this treatment. Here we describe the synthesis and screening of a focused library of synthetic auxin conjugates in Eucalyptus grandis cuttings and identify 4-chlorophenoxyacetic acid–L-tryptophan-OMe as a competent enhancer of adventitious rooting in a number of recalcitrant woody plants, including apple and argan. Comprehensive metabolic and functional analyses reveal that this activity is engendered by prolonged auxin signaling due to initial fast uptake and slow release and clearance of the free auxin 4-chlorophenoxyacetic acid. This work highlights the utility of a slow-release strategy for bioactive compounds for more effective plant growth regulation