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. 2017 Jul 25;114(30):7993-7998.
doi: 10.1073/pnas.1700062114. Epub 2017 Jul 11.

MmpL3 is the flippase for mycolic acids in mycobacteria

Zhujun Xu  1 , Vladimir A Meshcheryakov  1 , Giovanna Poce  2 , Shu-Sin Chng  3   4
Affiliations

MmpL3 is the flippase for mycolic acids in mycobacteria

Zhujun Xu et al. Proc Natl Acad Sci U S A. .

Abstract

The defining feature of the mycobacterial outer membrane (OM) is the presence of mycolic acids (MAs), which, in part, render the bilayer extremely hydrophobic and impermeable to external insults, including many antibiotics. Although the biosynthetic pathway of MAs is well studied, the mechanism(s) by which these lipids are transported across the cell envelope is(are) much less known. Mycobacterial membrane protein Large 3 (MmpL3), an essential inner membrane (IM) protein, is implicated in MA transport, but its exact function has not been elucidated. It is believed to be the cellular target of several antimycobacterial compounds; however, evidence for direct inhibition of MmpL3 activity is also lacking. Here, we establish that MmpL3 is the MA flippase at the IM of mycobacteria and is the molecular target of BM212, a 1,5-diarylpyrrole compound. We develop assays that selectively access mycolates on the surface of Mycobacterium smegmatis spheroplasts, allowing us to monitor flipping of MAs across the IM. Using these assays, we establish the mechanism of action of BM212 as a potent MmpL3 inhibitor, and use it as a molecular probe to demonstrate the requirement for functional MmpL3 in the transport of MAs across the IM. Finally, we show that BM212 binds MmpL3 directly and inhibits its activity. Our work provides fundamental insights into OM biogenesis and MA transport in mycobacteria. Furthermore, our assays serve as an important platform for accelerating the validation of small molecules that target MmpL3, and their development as future antituberculosis drugs.

Keywords: Mycobacterial membrane protein Large; drug binding and inhibition; lipid transport; membrane biogenesis; trehalose monomycolate.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
TMM biosynthesis is intact in mycobacterial spheroplasts. (A) Schematic diagram illustrating the processes important for MA transport across the cell envelope. Following synthesis, TMMs must be flipped across the IM, released from the IM, and then transported across the periplasm (presumably via a chaperone). MmpL3 is implicated in TMM transport at the IM, but its exact role has not been elucidated. At the OM, the Ag85 complex transfers the mycolate chain from TMM to cell wall-linked AG polysaccharides or to another TMM to form TDM. Other known lipid species found in the OM and IM are omitted for simplicity. PL, phospholipid. (B) TLC analysis of newly synthesized [14C]-labeled lipids extracted from wild-type M. smegmatis cells (WC) and spheroplasts (SP), visualized by phosphor imaging. Lipids were radiolabeled in the presence or absence of isoniazid as indicated. The developing solvent system comprises chloroform-methanol-water (30:8:1). A mycolate-based species that appears only in the presence of glucose is indicated with an asterisk. CL, cardiolipin; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PIM, phosphatidylinositol mannoside.
Fig. 2.
Fig. 2.
Newly synthesized TMMs in mycobacterial spheroplasts are accessible to degradation by LysB, indicating that these TMMs reside in the outer leaflet of the IM. (A) TLC analyses of newly synthesized [14C]-labeled lipids extracted from M. smegmatis spheroplasts treated with functional or nonfunctional (S82A) LysB. Lipids were resolved on TLCs developed using solvent systems comprising either chloroform-methanol-water (30:8:1) (Left) or hexane-diethylether-acetic acid (70:30:1) (Right), followed by phosphor imaging. In addition to MA, treatment with functional LysB resulted in the release of an unidentified apolar lipid, annotated with an asterisk. TAG, triacylglycerol. (B) α-GroEL2 and α-His immunoblot analyses of pellet and supernatant fractions obtained from sedimentation of M. smegmatis spheroplasts treated with functional or nonfunctional (S82A) LysB.
Fig. 3.
Fig. 3.
Antimycobacterial compounds BM212 and AU1235 reduce TMM accessibility to LysB in spheroplasts, indicating inhibition of TMM flipping across the IM. Representative TLC analyses of [14C]-labeled lipids newly synthesized in the presence of indicated concentrations of BM212 and AU1235 (A) and SQ109 (B), and extracted from M. smegmatis spheroplasts following treatment with or without purified LysB. The effects of pmf disruptors, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and nigericin were also tested. At higher concentrations, these uncouplers affected lipid synthesis, consistent with the depletion of ATP. DMSO and methanol were used to dissolve the respective compounds, and thus serve as negative controls. Equal amounts of radioactivity were spotted for each sample. The developing solvent system comprises chloroform-methanol-water (30:8:1). (C) Graphical plot showing the effects of various compounds on the amounts of LysB-accessible TMMs in spheroplasts. The percentage of TMMs accessible to LysB is given by the difference in TMM levels between samples with or without LysB treatment, normalized against the level in control samples without LysB treatment. TMM levels in each sample were quantified as a fraction of total mycolates (TMM + MA). Average percentages and SDs from three biological replicates are plotted. The average background of TMM hydrolysis due to random cell lysis during the experiment (∼40%) is indicated. Student’s t test: *P < 0.05 compared with the corresponding DMSO or methanol controls.
Fig. 4.
Fig. 4.
Antimycobacterial compounds BM212 and AU1235 reduce surface display of 6-azido-TMMs in spheroplasts, indicating inhibition of TMM flipping across the IM. (A) Schematic diagram illustrating the 6-azido-TMM surface display assay. Spheroplasts were incubated with 6-azido-trehalose to allow synthesis of 6-azido-TMMs (22), which were subsequently labeled with alkyne-containing biotin (DIBO-biotin) via click chemistry (23). Surface-exposed biotin-TMMs were recognized by Alexa Fluor 488-conjugated streptavidin and visualized by fluorescence microscopy. Representative bright-field and fluorescence microscopy images are shown following DIBO-biotin/Alexa Fluor 488-streptavidin labeling of spheroplasts synthesizing TMM (B), or 6-azido-TMM in the presence of DMSO (C), BM212 (D, twofold MIC), and AU1235 (E, twofold MIC). (Scale bars: 3 μm.) (F) Fluorescence intensity per unit area for individual spheroplasts (n = 100) in each condition in B–E is plotted, with the medians and interquartile ranges indicated. Mann–Whitney test: ****P < 0.0001 compared with the “no drug treatment” control.
Fig. 5.
Fig. 5.
Mutations in MmpL3 render BM212 less effective in the inhibition of TMM flipping across the IM. Representative TLC analyses of [14C]-labeled lipids newly synthesized in the presence of indicated concentrations of BM212 and extracted from wild-type (WT) (A), mmpL3V197M (B), and mmpL3A326T (C) M. smegmatis spheroplasts following treatment with or without purified LysB are shown. Equal amounts of radioactivity were spotted for each sample. The developing solvent system comprises chloroform-methanol-water (30:8:1). (D) Graphical plot showing the dose-dependent effects of BM212 on the percentage of TMMs accessible to LysB in the respective spheroplasts (quantification as per Fig. 3). Average percentages and SDs from three biological replicates are plotted. The average background of TMM hydrolysis due to random cell lysis during the experiment (∼40%) is indicated. Student’s t test: *P < 0.05, **P < 0.01 compared with the corresponding DMSO controls for each respective strain. Representative bright-field and fluorescence microscopy images are shown following DIBO-biotin/Alexa Fluor 488-streptavidin labeling of mmpL3V197M spheroplasts synthesizing TMM (E), 6-azido-TMM in the presence of DMSO (F) and 6-azido-TMM in the presence of BM212 (G, twofold MIC). (Scale bars: 3 μm.) (H) Fluorescence intensity per unit area for individual spheroplasts (n = 100) in each condition in E–G is plotted, with the medians and interquartile ranges indicated. Mann–Whitney test: N.S., not significant (P > 0.5 compared with the “no treatment” control).
Fig. 6.
Fig. 6.
BM212 binds MmpL3 in vitro in a specific manner. (A) Representative Clear Native-PAGE analyses of purified MmpL3-His samples incubated with increasing concentrations of [14C]-BM212, visualized separately by phosphor imaging and Coomassie blue (CB) staining. The amount of [14C]-BM212 bound to MmpL3 at each concentration was quantified, normalized to the amount at maximum binding, averaged across triplicates, and plotted. Error bars represent SDs. The data were fitted to a “one-site–specific binding” model using nonlinear regression (Kd = 65.5 ± 17.9 μM). (B) Clear Native-PAGE analyses of purified MmpL3-His samples incubated with a fixed concentration of [14C]-BM212, but in the presence of increasing concentrations of cold BM212. Gels were visualized separately by phosphor imaging and CB staining. (C) Mutations that confer resistance against BM212 cluster on a structural model of MmpL3, suggesting a possible binding site. A Phyre2 (35) structural model for M. smegmatis MmpL3 without its C-terminal cytoplasmic domain is shown in side (Left) and top (Right) views. For clarity, periplasmic domains are removed from the top-view image. Residues important for passage of protons are highlighted in black. Residues that conferred resistance against BM212 (10) when mutated in MmpL3 from M. smegmatis, Mycobacterium bovis bacillus Calmette–Guérin, and M. tuberculosis are highlighted in red, purple and cyan, respectively.
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