Role of the Mce1 transporter in the lipid homeostasis of Mycobacterium tuberculosis (original) (raw)
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Molecular microbiology, 2015
Mycobacterium tuberculosis proteins that are exported out of the bacterial cytoplasm are ideally positioned to be virulence factors; however, the functions of individual exported proteins remain largely unknown. Previous studies identified Rv0199 as an exported membrane protein of unknown function. Here, we characterized the role of Rv0199 in M. tuberculosis virulence using an aerosol model of murine infection. Rv0199 appears to be a member of a Mce-associated membrane (Mam) protein family leading us to rename it OmamA, for orphaned Mce-associated membrane protein A. Consistent with a role in Mce transport, we showed OmamA is required for cholesterol import, which is a Mce4-dependent process. We further demonstrated a function for OmamA in stabilizing protein components of the Mce1 transporter complex. These results indicate a function of OmamA in multiple Mce transporters and one that may be analogous to the role of VirB8 in stabilizing Type IV secretion systems, as structural simi...
Structural Basis for the Regulation of the MmpL Transporters of Mycobacterium tuberculosis
The Journal of biological chemistry, 2015
The mycobacterial cell wall is critical to the virulence of these pathogens. Recent work shows that the mycobacterial membrane protein large (MmpL) family of transporters contributes to cell wall biosynthesis by exporting fatty acids and lipidic elements of the cell wall. The expression of the Mycobacterium tuberculosis MmpL proteins is controlled by a complex regulatory network, including the TetR-family transcriptional regulators Rv3249c and Rv1816. Here we report the crystal structures of these two regulators, revealing dimeric, two-domain molecules with architecture consistent with the TetR family of regulators. Buried extensively within the C-terminal regulatory domains of Rv3249c and Rv1816 we found fortuitous bound ligands, which were identified as palmitic acid (a fatty acid) and isopropyl laurate (a fatty acid ester), respectively. Our results suggest that fatty acids may be the natural ligands of these regulatory proteins. Using fluorescence polarization and electrophoreti...
Proceedings of the National Academy of Sciences, 2010
Mycobacterium tuberculosis (Mtb) is an exclusively human pathogen that proliferates within phagosomes of host phagocytes. Host lipids are believed to provide the major carbon and energy sources for Mtb, with only limited availability of carbohydrates. There is an apparent paradox because five putative carbohydrate uptake permeases are present in Mtb, but there are essentially no host carbohydrates inside phagosomes. Nevertheless, carbohydrate transporters have been implicated in Mtb pathogenesis, suggesting that acquisition of host sugars is important during some stages of infection. Here we show, however, that the LpqY-SugA-SugB-SugC ATP-binding cassette transporter is highly specific for uptake of the disaccharide trehalose, a sugar not present in mammals, thus refuting a role in nutrient acquisition from the host. Trehalose release is known to occur as a byproduct of the biosynthesis of the mycolic acid cell envelope by Mtb's antigen 85 complex. The antigen 85 complex constitutes a group of extracellular mycolyl transferases, which transfer the lipid moiety of the glycolipid trehalose monomycolate (TMM) to arabinogalactan or another molecule of TMM, yielding trehalose dimycolate. These reactions also lead to the concomitant extracellular release of the trehalose moiety of TMM. We found that the LpqY-SugA-SugB-SugC ATP-binding cassette transporter is a recycling system mediating the retrograde transport of released trehalose. Perturbations in trehalose recycling strongly impaired virulence of Mtb. This study reveals an unexpected accessory component involved in the formation of the mycolic acid cell envelope in mycobacteria and provides a previously unknown role for sugar transporters in bacterial pathogenesis. microbial pathogenesis | mycolic acid biosynthesis | cell wall formation | carbon metabolism T uberculosis, caused by the bacterium Mycobacterium tuberculosis (Mtb), remains a major threat to global health, claiming the life of two million individuals each year (1). Mtb is an obligate human pathogen predominantly growing intracellularly within phagosomes of host phagocytes, although other cell types and niches might also be occupied during different phases of infection. Notwithstanding, there is strong evidence that host lipids provide the main carbon and energy sources for Mtb during infection, with carbohydrates being largely inaccessible for the bacilli (2-5). Support for this, among further findings, comes from the observed up-regulation of lipid catabolism genes of Mtb during intracellular replication in macrophages (4) and from the joint essentiality of the two isocitrate lyase isoforms, icl1 and icl2, for growth of Mtb in mice (6). It has to be mentioned that the importance of some lipid catabolic pathways for in vivo carbon metabolism of Mtb may be somewhat overestimated, as attenuation of mutants might be caused by accumulation of toxic intermediates of incomplete metabolism rather than by blocked utilization of a substrate . Nevertheless, the published literature strongly suggests that Mtb relies on metabolism of lipids from the host via the glyoxylate cycle in vivo. The nature of the lipid substrates used by Mtb during infection, however, remains largely unclear. Recently, there has been growing evidence that cholesterol is a host lipid used by Mtb as one carbon and energy source in vivo, although additional yetunspecified substrates are clearly also important, as blockage of cholesterol uptake and metabolism only partially attenuates Mtb virulence .
Microbiology, 2009
The mce operons constitute four homologous regions in the Mycobacterium tuberculosis genome, each of which has 8-13 ORFs. Although the function of the Mce protein family has not been clearly established, its members are believed to be membrane lipid transporters. Based on functional experiments, we found that the regulator of the mce3 locus, Mce3R, negatively regulates the expression of the Rv1933c-Rv1935c and Rv1936-Rv1941 transcriptional units. These operons are adjacent to one another and divergently transcribed. The predicted functions of most of these genes are related to either lipid metabolism or redox reactions. Bioinformatic analysis of the 59 UTR sequences of the differentially expressed genes allowed us to define a putative Mce3R motif. Importantly, the Mce3R motif was present six and three times in the mce3R-yrbE3A and Rv1935c-Rv1936 intergenic regions, respectively. Two occurrences of this motif mapped within the two regions of the mce3 operon that were protected by Mce3R in a footprinting analysis, thus indicating that this motif is likely to serve as an operator site for the Mce3R regulator in the promoter. In addition, alterations in the lipid content of M. tuberculosis were detected in the absence of Mce3R. Taken together, these results suggest that Mce3R controls the expression of both the putative transport system encoded in the mce3 operon and the enzymes implicated in the modification of the Mce3-transported substrates.
Microbial Pathogenesis, 2020
Mycobacterium tuberculosis (M. tb) is the causative agent of TB and its incidences has been on the rise since 1993. Lipid metabolism is an imperative metabolic process, which grants M. tb the ability to utilize host-derived lipids as a secondary source of nutrition during infection. In addition to degrading host lipids, M. tb is proficient at using lipids, such as cholesterol, to facilitate its entry into macrophages. Mycolic acids, constituents of the mycobacterial cell wall, offer protection and aid in persistence of the bacterium. These are effectively synthesized using a complex fatty acid synthase system. Many pathogenesis studies have reported differences in lipidmetabolism of clinical strains of M. tb that belongs to diverse lineages of the Mycobacterium tuberculosis complex (MTBC). East-Asian and Euro-American lineages possess "unique" cell wall-associated lipids compared to the less transmissible Ethiopian lineage, which may offer these lineages a competitive advantage. Therefore, it is crucial to comprehend the complexities among the MTBC lineages with lipid metabolism and their impact on virulence, transmissibility and pathogenesis. Thus, this review provides an insight into lipid metabolism in various lineages of the MTBC and their impact on virulence and persistence during infection, as this may provide critical insight into developing novel therapeutics to combat TB.
Therapeutic potential of the Mycobacterium tuberculosis mycolic acid transporter, MmpL3
Antimicrobial agents and chemotherapy, 2016
In recent years, whole cell-based screens for novel small molecule inhibitors active against Mycobacterium tuberculosis in culture followed by the whole genome sequencing of spontaneous resistant mutants have identified multiple chemical scaffolds thought to kill the bacterium through the inactivation of the mycolic acid transporter, MmpL3. Consistent with the fact that MmpL3 is required for the formation of the mycobacterial outer membrane, we here conclusively show, using conditionally regulated knock-down mutants, that mmpL3 is required for the replication and viability of M. tuberculosis, both under standard laboratory growth conditions and during the acute and chronic phases of infection in mice. Speaking for the vulnerability of this target, silencing mmpL3 had a rapid bactericidal effect on actively replicating cells in vitro and reduced by three to five logs in less than four weeks the bacterial load of acutely and chronically infected mouse lungs, respectively. Depletion of...
The Journal of biological chemistry, 2017
Mycobacterium tuberculosis causes tuberculosis in humans and predominantly infects alveolar macrophages. To survive inside host lesions and to evade immune surveillance, this pathogen has developed many strategies. For example, M. tuberculosis uses host-derived lipids/fatty acids as nutrients for prolonged persistence within hypoxic host microenvironments. M. tuberculosis imports these metabolites through its respective transporters, and in the case of host fatty acids, a pertinent question arises: does M. tuberculosis have the enzyme(s) for cleavage of fatty acids from host lipids? We show herein that a previously uncharacterized membrane-associated M. tuberculosis protein encoded by Rv2672 is conserved exclusively in Actinomycetes, exhibits both lipase and protease activities, is secreted into macrophages, and catalyzes host lipid hydrolysis. In light of these functions, we annotated Rv2672 as Mycobacterial Secreted Hydrolase 1 (Msh1). Further, we found that this enzyme is upregul...
Inhibition of mycolic acid transport across the Mycobacterium tuberculosis plasma membrane
Nature Chemical Biology, 2012
New chemotherapeutics active against multidrug-resistant Mycobacterium tuberculosis (M. tb) are urgently needed. We report on the identification of an adamantyl urea compound displaying potent bactericidal activity against M. tb and a unique mode of action, namely the abolition of the translocation of mycolic acids from the cytoplasm where they are synthesized to the periplasmic side of the plasma membrane where they are transferred onto cell wall arabinogalactan or used in the formation of virulence-associated outer membrane trehalose-containing glycolipids. Whole genome sequencing of spontaneous resistant mutants of M. tb selected in vitro followed by genetic validation experiments revealed that our prototype inhibitor targets the inner membrane transporter, MmpL3. Conditional gene expression of mmpL3 in mycobacteria and analysis of inhibitor-treated cells validate MmpL3 as essential for mycobacterial growth and support the involvement of this transporter in the translocation of trehalose monomycolate across the plasma membrane. Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
A vitamin B₁₂ transporter in Mycobacterium tuberculosis
Open biology, 2013
Vitamin B₁₂-dependent enzymes function in core biochemical pathways in Mycobacterium tuberculosis, an obligate pathogen whose metabolism in vivo is poorly understood. Although M. tuberculosis can access vitamin B₁₂ in vitro, it is uncertain whether the organism is able to scavenge B₁₂ during host infection. This question is crucial to predictions of metabolic function, but its resolution is complicated by the absence in the M. tuberculosis genome of a direct homologue of BtuFCD, the only bacterial B₁₂ transport system described to date. We applied genome-wide transposon mutagenesis to identify M. tuberculosis mutants defective in their ability to use exogenous B₁₂. A small proportion of these mapped to Rv1314c, identifying the putative PduO-type ATP : co(I)rrinoid adenosyltransferase as essential for B₁₂ assimilation. Most notably, however, insertions in Rv1819c dominated the mutant pool, revealing an unexpected function in B₁₂ acquisition for an ATP-binding cassette (ABC)-type prot...