Inhibition of mycolic acid transport across the Mycobacterium tuberculosis plasma membrane

Grzegorzewicz, Anna E.; Phạm, Hà; Gundi, Vijay A.K.B.; Scherman, Michael S.; North, E. Jeffrey; Hess, Tamara; Jones, Victoria; Gruppo, Veronica; Born, Sarah E M; Korduláková, Jana; Chavadi, Sivagami Sundaram; Morisseau, Christophe; Lenaerts, Anne J.; Lee, Richard; McNeil, Michael; Jackson, Mary

doi:10.1038/nchembio.794

Cited by 393 publications

(602 citation statements)

References 48 publications

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“…Interestingly, promising new compounds, which recently emerged from different screening programs, also target various steps in cell envelope metabolism (reviewed in reference 7). These are exemplified by (i) the inhibitors of MmpL3 (8)(9)(10)(11)(12), the trehalose monomycolate transporter required for the proper localization of mycolic acids, and (ii) the inhibitors of the decaprenyl-phosphoribose 2=-oxidoreductase DprE1 (11,(13)(14)(15), involved in the biosynthesis of mycobacterial AG and LAM. Specifically, DprE1 inhibitors block the biosynthetic pathway of the arabinan precursor and, therefore, the buildup of a covalently linked complex of mycolic acids, AG, and peptidoglycan, which form the so-called cell wall core (16).…”

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confidence: 99%

The Phosphatidyl- myo -Inositol Mannosyltransferase PimA Is Essential for Mycobacterium tuberculosis Growth In Vitro and In Vivo

et al. 2014

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The cell envelope of Mycobacterium tuberculosis contains glycans and lipids of peculiar structure that play prominent roles in the biology and pathogenesis of tuberculosis. Consequently, the chemical structure and biosynthesis of the cell wall have been intensively investigated in order to identify novel drug targets. Here, we validate that the function of phosphatidyl-myo-inositol mannosyltransferase PimA is vital for M. tuberculosis in vitro and in vivo. PimA initiates the biosynthesis of phosphatidyl-myoinositol mannosides by transferring a mannosyl residue from GDP-Man to phosphatidyl-myo-inositol on the cytoplasmic side of the plasma membrane. To prove the essential nature of pimA in M. tuberculosis, we constructed a pimA conditional mutant by using the TetR-Pip off system and showed that downregulation of PimA expression causes bactericidality in batch cultures. Consistent with the biochemical reaction catalyzed by PimA, this phenotype was associated with markedly reduced levels of phosphatidyl-myo-inositol dimannosides, essential structural components of the mycobacterial cell envelope. In addition, the requirement of PimA for viability was clearly demonstrated during macrophage infection and in two different mouse models of infection, where a dramatic decrease in viable counts was observed upon silencing of the gene. Notably, depletion of PimA resulted in complete clearance of the mouse lungs during both the acute and chronic phases of infection. Altogether, the experimental data highlight the importance of the phosphatidyl-myo-inositol mannoside biosynthetic pathway for M. tuberculosis and confirm that PimA is a novel target for future drug discovery programs.T he lack of proper treatment for serious infectious diseases due to the emergence of multidrug resistance (MDR) underlines the need for the discovery of novel antibiotics (1). This is particularly true for tuberculosis (TB), for which 3.7% of new cases and 20% of previously treated cases are estimated to have MDR-TB. In several countries in Eastern Europe and central Asia, these numbers rise to 9 to 32% and more than 50%, respectively, making the situation particularly worrisome. In addition, in the case of TB, which claimed 1.3 million lives in 2012, the treatment of the leastcomplicated, drug-sensitive cases is lengthy and uncomfortable, and new drugs are urgently needed to shorten the regimen and make it more acceptable to the patients (2).The cell envelope of Mycobacterium tuberculosis represents a highly attractive target for the discovery of novel TB drugs. Supporting this notion, several key medicines used in the current TB therapy are inhibitors of essential enzymes participating in cell envelope biosynthesis. The first-line drug ethambutol has been shown to target at least three unique integral membrane-associated arabinofuranosyltransferases (EmbA, EmbB, and EmbC) involved in the biosynthesis of arabinogalactan (AG) and lipoarabinomannan (LAM) (3, 4). The inhibition of InhA by isoniazid (INH) in M. tuberculosis leads to the specific depl...

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confidence: 99%

The Phosphatidyl- myo -Inositol Mannosyltransferase PimA Is Essential for Mycobacterium tuberculosis Growth In Vitro and In Vivo

et al. 2014

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“…Because DAT, TAT, TetraAT, and PAT are all found at the cell surface, this model implies that MmpL10 transports all of these lipids across the cytoplasmic membrane. This substrate promiscuity would distinguish MmpL10 from previously characterized MmpLs such as MmpL7 and MmpL3, which exclusively transport phthiocerol mycocerosate (PDIM) and trehalose monomycolate (TMM), respectively (41)(42)(43). On the other hand, in sulfolipid 1 biosynthesis, when Chp1 is absent, the diacylated sulfotrehalose intermediate SL 1278 (also known as Ac 2 SGL) is detected at the cell surface, suggesting that MmpL8 is capable of transporting this intermediate in addition to SL-1 and shares the ability to transport multiple substrates with MmpL10, which is a closely related homologue (58% sequence identity).…”

Section: Discussionmentioning

confidence: 99%

The rv1184c Locus Encodes Chp2, an Acyltransferase in Mycobacterium tuberculosis Polyacyltrehalose Lipid Biosynthesis

et al. 2015

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Trehalose glycolipids are found in many bacteria in the suborder Corynebacterineae, but methyl-branched acyltrehaloses are exclusive to virulent species such as the human pathogen Mycobacterium tuberculosis. In M. tuberculosis, the acyltransferase PapA3 catalyzes the formation of diacyltrehalose (DAT), but the enzymes responsible for downstream reactions leading to the final product, polyacyltrehalose (PAT), have not been identified. The PAT biosynthetic gene locus is similar to that of another trehalose glycolipid, sulfolipid 1. Recently, Chp1 was characterized as the terminal acyltransferase in sulfolipid 1 biosynthesis. Here we provide evidence that the homologue Chp2 (Rv1184c) is essential for the final steps of PAT biosynthesis. Disruption of chp2 led to the loss of PAT and a novel tetraacyltrehalose species, TetraAT, as well as the accumulation of DAT, implicating Chp2 as an acyltransferase downstream of PapA3. Disruption of the putative lipid transporter MmpL10 resulted in a similar phenotype. Chp2 activity thus appears to be regulated by MmpL10 in a relationship similar to that between Chp1 and MmpL8 in sulfolipid 1 biosynthesis. Chp2 is localized to the cell envelope fraction, consistent with its role in DAT modification and possible regulatory interactions with MmpL10. Labeling of purified Chp2 by an activity-based probe was dependent on the presence of the predicted catalytic residue Ser141 and was inhibited by the lipase inhibitor tetrahydrolipstatin (THL). THL treatment of M. tuberculosis resulted in selective inhibition of Chp2 over PapA3, confirming Chp2 as a member of the serine hydrolase superfamily. Efforts to produce in vitro reconstitution of acyltransferase activity using straight-chain analogues were unsuccessful, suggesting that Chp2 has specificity for native methyl-branched substrates.

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“…9 Instead, there is strong evidence that these MmpL transporters and their MmpS accessory proteins are responsible for shuttling fatty acid and lipid components of the cell wall, such as trehalose monomycolate, sulfolipids, phthiocerol dimycocerosate, diacyltrehalose, monomeromycolyl diacylglycerol, and mycolate wax ester. 3,9,11,12,[19][20][21][22][23] The regulation of MmpL protein expression and the role of MmpLs in cell wall remodeling in different environmental conditions has not been explored. Thus we capitalized on data made available by the TB Systems Biology Consortium to begin an in-depth analysis of how mmpL and mmpS genes are regulated.…”

Section: Introductionmentioning

confidence: 99%

“…MmpL3 is essential; MmpL4, MmpL5, MmpL7, MmpL8, MmpL10, and MmpL11 are required for full Mtb virulence. [9][10][11][12] MmpL3 transports the trehalose dimycolate (TDM) precursor trehalose monomycolate to the mycobacterial surface. 12 MmpL3 is therefore essential since TDM biosynthesis and incorporation into the mycobacterial cell wall is required for mycobacterial replication and viability.…”

Section: Introductionmentioning

confidence: 99%

“…[9][10][11][12] MmpL3 transports the trehalose dimycolate (TDM) precursor trehalose monomycolate to the mycobacterial surface. 12 MmpL3 is therefore essential since TDM biosynthesis and incorporation into the mycobacterial cell wall is required for mycobacterial replication and viability. 13,14 Based on the genomic sequence of H37Rv, 15 Mtb harbors 14 different MmpL proteins, belonging to the resistance-nodulation-cell division (RND) superfamily of transporters.…”

Section: Introductionmentioning

confidence: 99%

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Crystal structure of the Mycobacterium tuberculosis transcriptional regulator Rv0302

et al. 2015

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Mycobacterium tuberculosis is a pathogenic bacterial species, which is neither Gram positive nor Gram negative. It has a unique cell wall, making it difficult to kill and conferring resistance to antibiotics that disrupt cell wall biosynthesis. Thus, 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 complicated regulatory network system. Here we report crystallographic structures of two forms of the TetR-family transcriptional regulator Rv0302, which participates in regulating the expression of MmpL proteins. The structures reveal a dimeric, two-domain molecule with architecture consistent with the TetR family of regulators. Comparison of the two Rv0302 crystal structures suggests that the conformational changes leading to derepression may be due to a rigid body rotational motion within the dimer interface of the regulator. Using fluorescence polarization and electrophoretic mobility shift assays, we demonstrate the recognition of promoter and intragenic regions of multiple mmpL genes by this protein. In addition, our isothermal titration calorimetry and electrophoretic mobility shift experiments indicate that fatty acids may be the natural ligand of this regulator. Taken together, these experiments provide new perspectives on the regulation of the MmpL family of transporters.

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Inhibition of mycolic acid transport across the Mycobacterium tuberculosis plasma membrane

Cited by 393 publications

References 48 publications

The Phosphatidyl- myo -Inositol Mannosyltransferase PimA Is Essential for Mycobacterium tuberculosis Growth In Vitro and In Vivo

The Phosphatidyl- myo -Inositol Mannosyltransferase PimA Is Essential for Mycobacterium tuberculosis Growth In Vitro and In Vivo

The rv1184c Locus Encodes Chp2, an Acyltransferase in Mycobacterium tuberculosis Polyacyltrehalose Lipid Biosynthesis

Crystal structure of the Mycobacterium tuberculosis transcriptional regulator Rv0302

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