Inhibition of mycolic acid transport across the Mycobacterium tuberculosis plasma membrane

Abstract

New chemotherapeutics active against multidrug-resistant Mycobacterium tuberculosis are urgently needed. We report on the identification of an adamantyl urea compound that shows potent bactericidal activity against M. tuberculosis 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 and are in turn 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. tuberculosis 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.

Figure 1. Structure and bactericidal activity of AU1235

(a) Structure of AU1235 (1) [1-(2-adamantyl)-3-(2,3,4-trifluorophenyl)urea] (b) Kill-kinetics showing decrease in viability of M. tb H37Rv in 7H9-OADC-Tween 80 medium containing AU1235 at 0.5 and 1 μg ml⁻¹ (5 and 10 × MIC). The results for each drug concentration are representative of at least two independent experiments.

Figure 2. Effect of AU1235 on mycolic acid biosynthesis and transfer in M. tb

M. tb H37Ra cultured in 7H9-OADC-Tween 80 broth was treated for 5 hr at 37°C with either no inhibitor or with AU1235 at a concentration of 0.05 μg ml⁻¹, 0.5 μg ml⁻¹, or 1 μg ml⁻¹ (0.5x to 10x MIC). [¹⁴C]-acetate was added to the cultures at the same time as the inhibitor. (a) Bacterial cells (B) and culture filtrates (CF) were collected and the lipids contained in each of these fractions extracted as described under the Methods section. The same volume of samples was loaded per lane. The TLC was developed in the solvent system [chloroform:methanol:water] (20:4:0.5, by vol.) and revealed by autoradiography. PE, phosphatidylethanolamine; CL, cardiolipin. (b) Mycolic acid methyl esters were prepared from delipidated cells as described. α–, methoxy- and keto- denote the three forms of mycolic acids produced by M. tb. The same volume of samples was loaded per lane. The TLC was developed thrice in the solvent system [n-hexanes:ethyl acetate] (95:5, by vol.) and revealed by autoradiography. The amount of radioactivity incorporated in the products of interest was semi-quantified using a PhosphoImager and the results (expressed as a % of the value measured in the untreated control) are presented as histograms under their corresponding autoradiograms.

Figure 3. mmpL3 is an essential gene of M. smegmatis

(a) Evidence for allelic replacement at the mmpL3 locus of M. smegmatis mc²155 in the presence of a rescue copy of mmpL3tb expressed from an episomal plasmid. Allelic exchange mutants were rescued with the mmpL3tb gene from M. tb expressed from either pMVGH1-mmpL3tb, pMVGH1-mmpL3tb-G253E or pSETetR-mmpL3tb. Allelic replacement was confirmed by PCR as described under the Methods section. The wild-type (WT) 3,752-bp amplification signal is replaced by a 3,358-bp fragment in the rescued mutants (r-mut) due to the 1,594-bp NotI deletion in the mmpL3 gene and insertion of a 1.2 kb-kanamycin resistance cassette. Only the profiles of two mc²155ΔmmpL3/pMVGH1-mmpL3tb strains are shown here as identical profiles were obtained will all rescued mutant forms. See Supplementary Figure 9 for the image of the full uncut gel. (b) Growth characteristics of wild-type mc²155 (circles) and the conditional mutant, mc²155ΔmmpL3/pSETetR-mmpL3tb (squares), in 7H9-OADC-Tween 80 broth containing either no inducer (open symbols) or 50 ng ml⁻¹ of anhydro-tetracycline (ATc) (full symbols) at 37°C. Reducing ATc concentration in the medium leads to the repression of mmpL3tb in the conditional mutant.

Figure 4. Effect of repressing mmpL3 expression on the mycolic acid content of M. smegmatis.

(a) Analysis of the extractable lipids from wild-type M. smegmatis mc²155 and two independent mc²155ΔmmpL3/pSETetR-mmpL3tb conditional mutants (ΔTet-1 and ΔTet-2) grown on 7H11-OADC agar in the presence of different concentrations of anhydro-tetracycline (ATc). Equal amounts of total cellular lipids from wild-type M. smegmatis mc²155 and the conditional mutants were run in the solvent system [chloroform:methanol:water] (20:4:0.5, by vol.). Left TLC plate: The plate was revealed with cupric sulfate to reveal all organic compounds. Right TLC plate: Samples were run similarly and the plate sprayed with α-naphthol to more specifically reveal glycolipids. Pink/purple-stained compounds correspond to glycolipids; yellow/brown-stained compounds migrating close to the origin are phospholipids. The diffuse or doublet bands at the level of TDM and TMM correspond to various forms of these glycolipids esterified with different types of mycolic acid chains. (b) Analysis of the cell wall-bound mycolates from wild-type M. smegmatis mc²155 and a mc²155ΔmmpL3/pSETetR-mmpL3tb conditional mutant (ΔTet-1). Cell wall-bound mycolic acid methyl esters (MAMEs) prepared from the same amount of wild-type and conditional mutant cells and loaded volume to volume are shown. A decrease in MAME content is seen in the conditional mutant grown at the lowest concentration of ATc. The TLC was developed thrice in the solvent system [n-hexanes:ethyl acetate] (95:5, by vol.) and the plates revealed with cupric sulfate. TMM, trehalose monomycolates; TDM, trehalose dimycolates; PE, phosphatidylethanolamine; CL, cardiolipin; GPLs, glycopeptidolipids.

Figure 5. A model for mycolic acid biosynthesis and transport across the inner membrane

Mycolic acid chains are shown in red. DAT, diacyltrehaloses; PAT, polyacyltrehaloses; PDIM, phthiocerol dimycocerosates. The trehalose released upon transfer of mycolic acids from TMM either onto AG or another molecule of TMM to form TDM is recycled and re-imported inside the cells by the ABC-transporter SugABC and the lipoprotein LpqY; FbpABC are the mycolyltransferases responsible for the transfer of mycolic acids onto their cell envelope acceptors.