Molecular mechanisms of cell wall lipid transport in Mycobacterium tuberculosis

Presently, Tuberculosis (Tb), caused by Mycobacterium tuberculosis (Mtb), remains a global health threat, particularly with the emergence of drug-resistant strains and HIV and Tb co-infection that challenges the current Tb treatment. Preventative measures and several anti-Tb drugs target the lipids of the mycobacterial outer membrane consisting of mycolic acids amongst an array of glycolipids. In Mtb, there are thirteen outer membrane lipid transporters designated mycobacterial membrane protein large (MmpL) proteins. These proteins are also involved in heme acquisition and possibly drug resistance and nitrosative stress adaptation, contributing to mycobacterial survival and pathogenesis. MmpL proteins are structurally and phylogenetically classed as resistance-nodulation-division (RND) transporters that use protonmotive force (PMF) to mediate substrate translocation. Unlike their RND counterparts in Gram-negative bacteria the structure and mechanisms of these transporters in Mycobacteria are yet to be determined. This work aimed to investigate the mechanism of three transport systems: MmpL3, MmpL7 and DrrABC (encoded by mmpL3, mmpL7 and drrABC genes) using molecular modelling, genetics and lipid and protein biochemistry. mmpL3 is essential for trehalose monomycolate (TMM) transport. While mmpL7 is required for the export of two structurally related lipids, phthiocerol dimycocerosates (PDIM) and phenolic glycolipids (PGL). With the use of different membrane extraction agents, we were able to identify and propose improvements for the structural characterisation of MmpL3 and MmpL7. Molecular analysis and the MmpL7 homology model facilitated the identification of residues in the transmembrane and periplasmic domain that were verified by genetics and lipid analysis as critical for PDIM synthesis and transport. Even though MmpL7 is an RND transporter, there was no evidence of conserved PMF sites or a network forming proton translocation channel. Conveniently within the same genetic context is drrC, part of the drrABC cluster, that has a co-dependent relationship with mmpL7 in PDIM transport. Interestingly the overexpression of only drrAB genes function as a multi-drug ATP-binding cassette (ABC) pump. Structural modelling and molecular analysis revealed that drrABC encodes a heterodimeric ABC transporter where drrB and drrC encode the transmembrane spanning domains that create a pore or channel for substrate entry/exit, while drrA encodes the nucleotide binding domain for ATP binding and hydrolysis mediated transport suggesting a potential mechanism for PDIM/PGL transport. Genetics and biochemical analyses verified that all drrABC genes were required for PDIM transport across the membrane. Indeed, this work provides evidence that MmpL7 does not operate as an independent transporter but instead serves as a scaffold linking lipid synthesis and transport. The knowledge gained from such investigations related to MmpL proteins endeavours to aid in better understanding of anti-Tb drug development