Composition and organization of outer membrane lipid transport systems in mycobacteria – MYCOLT

Mycobacteria include important pathogens, such as Mycobacterium tuberculosis, the causative agent of human tuberculosis. These bacteria are surrounded by an unusually thick lipid-rich cell envelope containing very long-chain fatty acids called mycolic acids that interact with a variety of atypical surface-exposed lipids to form an outer membrane, known as the mycomembrane. Metabolic pathways involved in the formation of outer membrane lipids have been extensively scrutinized but the mechanisms governing their transport from the cytoplasmic compartment where they are synthetized, through the cell envelope are far less well understood. A family of transporters called MmpL (Mycobacterial membrane protein Large) has been shown to play a key role in the translocation of these lipids across the plasma membrane but there are still a lot of uncertainties regarding their exact function, structural organization, and the involvement of other proteins in lipid transport.
This project aims to fill this knowledge gap by addressing fundamental questions on the composition and organization of the molecular machinery underlying transport of outer membrane lipids in mycobacteria. To this end, we have selected two families of lipids, namely the trehalose monomycolates/trehalose dimycolates (TMM/TDM) and trehalose polyphleates (TPP), and the mycobacterial model organism Mycobacterium smegmatis, as working models. TMM/TDM are key components of the mycomembrane and we found that the TPP pathway could serve as an excellent informative model to describe export of other major classes of surface-exposed lipids specific to M. tuberculosis. We will use structural and genetic approaches to depict the structure and the supramolecular organization of the MmpL transporter involved in TPP export and gain further insight into the mechanism of lipid transport by MmpL. We will also implement a combination of global and hypothesis-driven approaches to identify new proteins associated with TPP or TMM/TDM transport across and beyond the plasma membrane. We will investigate in-depth the topological organization, subcellular localization, structure, and function of these novel transport proteins using a combination of cutting-edge approaches to provide organizational and mechanistic information on the lipid transport machinery. Finally, we will extend and validate the information gained on lipid transport in M. smegmatis to Mycobacterium abscessus, an emerging opportunistic pathogen, which produces both TMM/TDM and TPP.
This project will provide strong knowledge on the nature and organization of the TMM/TDM and TPP transport machineries. This information will serve as a guide to depict the organization of lipid transport pathways and mycomembrane assembly in other mycobacterial pathogens, including M. tuberculosis, and in related mycolata species whose cell envelope resembles that of mycobacteria. Understanding the biogenesis of the mycomembrane is an issue of major importance because 1) this structure is a key element in the virulence of pathogenic mycobacteria; 2) it contributes substantially to the high intrinsic antibiotic resistance of mycobacteria; and 3) its biogenesis is the target of many antituberculosis drugs available on the market or currently under development. Owing the importance of outer membrane lipids in the survival and virulence of pathogenic mycobacteria, the identification of new proteins involved in lipid export may lead to the discovery of new druggable targets for the treatment of mycobacterial infections, including tuberculosis that remains a major public health problem.