Structure-fonction des transporteurs transmembranaires de la famille MmpL3 de Mycobacterium tuberculosis

The emergence of resistance to a multitude of antimicrobial agents in pathogenic bacteria is considered a major threat to public health (2). These strains are recognized as multidrug resistant organisms (MDR) (4). Research is progressing in Gram positive, Gram positive high GC and Gram negative bacteria, and given the scale of the public health threat, these MDR have become potential targets for this research. The objectives of the present study consist of the structural and functional characterization of various transmembrane transporters of the still enigmatic RND (Resistance-Nodulation-Division) family, namely: MmpL3 in Mycobacterium tuberculosis (Mtb) via the study of its ortholog CmpL1 in Corynebacterium glutamicum (Cgl) and TriAxBC in Pseudomonas aeruginosa (P. aeruginosa).  The first component of this project, presented in Chapter 2, studies the structure of the transporter MmpL3 Mtb (a TMM mycolic acid transporter essential for the viability of Mtb (5) and that of its CmpL1 Cgl orthologue, which have been predicted via the I- Tasser Pack (6-8). These structures were subsequently validated by comparing to the electronic map generated for CmpL1 (18 Å) by negative staining transmission electron microscopy (TEM). Characterization of the purified CmpL1 transporter by size exclusion chromatography confirmed the trimeric complex size around 315 KDa (including the detergent crown) corroborated by SDS-PAGE gel analyses. Collaborative genetic and biochemical studies have also identified residues involved in the transport of TMM in MmpL3 as well as those residues conferring antibiotic resistance. This data highlighted the location of the essential residues of transport and resistance in the central channel of the trimeric Mmpl3 model. The MmpL3 region activating proto-motor transport has been located at a central cavity, which is an intrinsic feature of the RND family. The low-resolution electronic maps obtained for the protein CmpL1 may serve as the foundation of future studies.     In Chapter 3 we explore the therapeutic potential of the targeting of the transmembrane transporter MmpL3 in different Mycobacterium strains. Our collaborators studied the therapeutic effect of the most promising inhibitors of the MmpL3 Mtb transporter on certain multi-resistant mycobacterial non-tuberculous (NTB) strains. Based on our comparative structural modeling obtained by I-TASSER (6-8), we supplemented the biochemical data by highlighting the structural similarities and differences between the TB and NTB strains as well as their functional impacts. This chapter highlights the interest of direct or indirect targeting of MmpL3 in NTB species. Indeed, the efficacy of certain selected MmpL3 Mtb inhibitors on the treatment of NTB pulmonary infection have potential as generalizable treatment options for other NTB multi-resistant strains, or even to help address the problem of resistance to antibiotics and simplify current combination approaches.                                                                                Other collaborative studies undertaking the same structural approaches were carried out for TriAxBC tripartite carriers (P. aeruginosa), efflux pumps belonging to the RND family. The purpose of Chapter 4 was to generate a structure of the complex and decipher its mode of assembly and expulsion of antibiotics from the intracellular environment. A quaternary structure model of TriAxBC was predicted by I-TASSER (6-8) and validated against its 4.3 Å electronic map generated by Cryo-EM. The TriAxBC complex was also characterized by gel filtration confirming an approximate size of 620 KDa and its trimer composition by SDS-PAGE.  In conclusion, this study is combining different biochemical, genetic and structural approaches to highlight the need for a multidisciplinary approach to characterizing the structure function of RND transporters. The latter remain enigmatic; however, our contribution and the progress of others will allow the generation of new specific drugs targeting multiresistant strains.

Keyphrases: Mycobacterium, Tuberculosis, MmpL3, RND, TMM, Mycobacterium non tuberculeux, TriAxBC, I-TASSER, Cryo-EM, Mycolic acid, Non tuberculosis Mycobacterium, Efflux pump, Electronic microscopy