Abstract
Mycobacterium tuberculosis is an obligate human pathogen that causes tuberculosis, a disease resulting in approximately 10 million deaths in 2022, making it the primary cause of death from a single infectious agent. The treatment regime for TB is extensive and involves administration of up to four drugs daily for six months. The emergence of multi-drug resistance to these first-line antibiotics further complicates treatment. Drug resistance mechanisms of M. tuberculosis include modification of pro-drug activating enzymes, modification or upregulation of the drug target, and drug efflux. Numerous mycobacterial membrane transporters have been annotated as drug efflux pumps based on homology to characterized efflux pumps in other bacteria or based on increased drug susceptibility after deletion of the transporter. Neither are sufficient to conclude that a transporter actively exports drugs. Chapter 2 discusses the experimental approaches required to establish a transporter’s function as an efflux pump and reviews the literature on putative mycobacterial efflux pumps. A lack of data for many transporters prevents differentiation between direct drug efflux and indirect effects on drug resistance. Some transporters implicated in drug efflux were found to export lipids that are important for the integrity of the mycobacterial cell envelope, such as the phthiocerol dimycocerosates exporter MmpL7, thereby affecting drug influx. Data strongly suggests that MmpL5, Rv1258c (Tap), Mmr and Rv2686c-2688c actively export drugs, although only overproduction of MmpL5 is associated with drug resistance in clinical M. tuberculosis strains. MmpL5 and MmpL4 are redundant putative drug efflux pumps and export siderophores, iron chelating molecules secreted by bacteria to obtain this essential nutrient during infection. Further, they are involved in the efflux of bedaquiline, a key drug to treat multidrug resistant M. tuberculosis. MmpL4 and MmpL5, and their associated periplasmic proteins MmpS4 and MmpS5, are required for growth of M. tuberculosis under iron limited conditions in vitro and in tuberculosis mouse models. The main aim of this work was to structurally characterize these transporters, given the lack of mechanistic insights into siderophore and drug export. In Chapter 3, we determined the structure of a truncated form of MmpL4 in a closed conformation by cryo-electron microscopy. We identified a siderophore binding site at the inner leaflet of the cytoplasmic membrane in a nanodisc-reconstituted sample of MmpL4. A functional assay capitalizing on the self-poisoning phenotype observed in mycobacteria when siderophore secretion is disrupted, i.e. through deletion of mmpL4/mmpL5, was used to investigate molecular features of MmpL4 and MmpL5. This assay revealed the importance of MmpL4's and MmpL5’s unique coiled-coil domain, a cluster of residues within the transmembrane domain implicated in proton translocation and the mycobactin binding site for transporter function. The same structural elements are also important to confer bedaquiline resistance, suggesting that MmpL4 and MmpL5 directly efflux drugs. Additionally, we discovered an interaction between MmpL4 and acyl carrier proteins, including MbtL which participates in the synthesis of siderophores. The purification of stable and homogeneous MmpL4 is complicated by the unusual coiled-coil domain, which negatively affects the biochemical behavior of the protein. Chapter 4 details the bioinformatic and biochemical characterization of MmpL4 and homologues of M. smegmatis, culminating in the development of the truncated construct (lacking the coiled-coil domain) used for the high-resolution structure determination in Chapter 3. In addition, AlphaFold2 predictions were obtained of a MmpS4-MmpL4 trimer and a complex of MmpL4 and the periplasmic protein Rv0455c, an essential component of the siderophore export machinery with an unknown function. These predictions await experimental conformation but suggest that MmpS4 stabilizes the MmpL4 trimer, and that the coiled-coil domain of MmpL4 serves as a scaffold for the assembly of other periplasmic and outer membrane components of the siderophore export machinery, such as Rv0455c. This thesis provides the first structural exploration of MmpL4 and functional investigation of M. tuberculosis MmpL4 and MmpL5 mutants, thereby broadening our understanding of the two crucial processes of siderophore and drug export across the inner membrane. Beyond fundamental insights, these molecular findings pave the way for structure-based drug design targeting the siderophore export pathway and set the stage for further exploration of this unique export machinery.
Earp, Jennifer