Abstract
Mycobacterium abscessus has recently emerged as one of the most difficult-to-manage Non-Tuberculous mycobacteria (NTM), causing severe pulmonary infections, especially in cystic fibrosis patients. Mycobacteria contain several genes encoding proteins belonging to the mycobacterial membrane protein large (MmpL) family, acting as lipid transporters and proposed to work as efflux pumps. The MmpS4-MmpL4a-MmpL4b complex mediates the biosynthesis/transport of glycopeptidolipids (GPL) across the plasma membrane. M. abscessus exhibits either a smooth (S) morphotype when GPL are associated at the bacterial surface or a rough (R) morphotype when GPL are lacking. In addition to lipid transport, MmpL proteins can mediate the transport of a wide panel of substrates, including drugs, and can also be considered as important virulence factors in pathogenic mycobacteria. Therefore, they can be viewed as a promising drug targets to be further exploited against M. abscessus.The research conducted during this thesis focused mainly in the contribution of MmpL in the S-to-R transition and in drug resistance mechanisms. We focused also on the development of genetic tools that allow to easily manipulate the M. abscessus genome, particularly to generate mutants, based on a single and double homologous recombination events. The system comprises a red fluorescence marker (tdTomato) that simplifies the selection of the positive clones that have undergone gene disruption. The mmpSL4ab gene cluster was used to validate these techniques due to their participation in production/transport of GPL. Using this method, inactivation of mmpL4a in the S variant was associated with a switch to the R morphotype and absence of GPL production. Since the S-to-R transition has been proposed to occur within the host, we next described the heterogeneity of isolates from two cystic fibrosis patients pulmonary co-infected with both morphotypes using whole genome sequencing. This allowed to identified new single nucleotide polymorphism in the GPL locus which may be involved in the S-to-R conversion.Previous work reported the contribution of the M. abscessus MmpS5/MmpL5 efflux pump system in resistance to thiacetazone analogues. Herein, we further elucidated the mechanism of resistances involving the TetR transcriptional regulator (MAB_4384) in the regulation of this efflux pump.Due to the extreme difficulty encountered in the treatment of M. abscessus, alternative therapeutic options have recently been proposed, including for instance the use clofazimine and bedaquiline in current treatments. Our work was aimed at describing the mechanisms of resistance to these drugs and identified mutations in a putative TetR transcriptional regulator (MAB_2299c). Genetic and biochemical approaches demonstrated that mutations in this regulator were associated with increased expression of two efflux pump systems, MAB_2300-2301 and MAB_1135c-1134c, both contributing to the intrinsic resistance level of M. abscessus to clofazimine and bedaquiline. Therefore, MAB_2299c may represent a useful marker of clofazimine and bedaquiline resistance in clinical isolates.The double crossing-over gene inactivation technique developed during these studies represent a powerful tool to easily and efficiently generate unmarked mutants and offers the possibility of delete multiple genes in the same strain. It opens the way to future studies aimed at validating novel drug targets and/or virulence genes not only in M. abscessus but perhaps also in other NTM.