Dynamical Organization of Compositionally Distinct Inner and Outer Membrane Lipids of Mycobacteria

Abstract

Mycobacterium species, including Mycobacterium tuberculosis, employs atypical long (C_60-90) and branched lipids to produce a complex cell wall and localizes these toward distinct spatial locations, inner membrane (IM) and outer membrane (OM), thus forming a robust permeability barrier. The properties and functional roles of these spatially orchestrated membrane platforms remain unknown. Herein, we report the distinctive lateral organization, fluidity, and lipid domain architecture of protein-free membranes reconstituted from IM and OM lipids in vitro from M. smegmatis (Msm) underscored by their lipid packing and lipid dynamics. We show that Msm OM, against common notion, is more dynamic and fluid compared with IM and reveal the role of cell wall-associated peptidoglycans and lipoarabinomannan on the Msm OM organization. Overall, these studies indicate that mycobacterial species may regulate their overall membrane functionality by regulating the synthesis of these complex arrays of lipids. Based on the structure-function relationship drawn here, documented alteration in the mycobacterial lipidome during cellular infection and/or drug treatment could reflect a mechanism to fine-tune M. tuberculosis membrane properties to its advantage. These findings are expected to inspire development of lipid-centric therapeutic approaches targeted toward its membrane.

Figure 1

Structures of representative lipids from spatially distinct membrane regions in mycobacterial species. Sulfolipids (SL-1), trehalose dimycolate (TDM), and phthiocerol dimycocerosate (PDIM) reflect the noncovalent lipids in the Mtb outer membrane (OM) along with glycopeptidolipids (GPLs). Lipoarabinomannan (LAM)-lipomannan (LM) and phosphatidylinositol mannosides (PIMs) transverse the peptidoglycan layer. α-Mycolic acids (α-MAs) represent the major component of peptidoglycan-associated lipids (PALs). Tetra-acylated phospho-myo-inositol dimannosides and Ac₂PIM₂ (and other acylated phosphomannosides) are most abundant in the Mtb inner membrane (IM).

Figure 2

Visualization and size variation of mycobacterial OM and IM lipid vesicles. Shown are cryo-TEM images for IML, OML, OL, OP, and OLP lipid vesicles. cryo-TEM images of TDM and the three-component model membrane of TDM:DAG:DPPG (9.1:18.2:72.7 mol%) are shown as an aid to explain the facetation behavior. The inset images show the enlarged image of a single lipid vesicle. Scale bars, 500 nm. DLS provided the hydrodynamic diameter range observed for IML, OML, OL, OP, and OLP vesicles at 25°C.

Figure 3

Bilayer depth-dependent changes in membrane order and fluidity of mycobacterial lipid membranes. Temperature-dependent changes in (A) Laurdan GP and (B) DPH anisotropy shows microviscosity changes in the deep hydrophobic acyl chain region. (C) TMA-DPH anisotropy is indicative of microviscosity changes in the interfacial region. (D) Laurdan anisotropy depicts microviscosity changes at the lipid headgroup region. (E) Fluorescence lifetimes of the indicated lipid probes are shown. Data presented are mean (±SEM) of three independent experiments. (F–I) Temperature-dependent changes in the acyl chain conformational dynamics of the indicated mycobacterial membranes were measured using FT-IR monitoring ν_asymCH₂. (F) Asymmetric CH₂ stretching mode wavenumber in IML is shown. (G–I) Shown are comparisons between the temperature-dependent asymmetric CH₂ stretching of (G) OML and OLP, (H) OML and OL, and (I) OML and OP. Black arrows indicate changes in slope, which can be ascribed to the onset of phase transitions. Solid arrows indicate main phase transition, and dashed arrows indicate minor slope change and the appearance of additional phases. Data presented are mean (±SD) of three independent experiments.

Figure 4

Visualization of the domain architecture in mycobacterial model membranes. (A and B) Shown is AFM imaging (A) and force spectroscopy (B) of IML, OML, and OLP. The topographical images show phase separation of these independent lipid systems into ordered and disordered phase at 25°C. Scale bars, 5 μm for IML and 2 μm for OML and OLP. For IML and OLP, the height difference between the two phases is ∼3 nm, whereas for OML, it is 2 nm, suggesting differential lateral packing of these lipids. The BrF distribution shows the mean values centered at 4.24 ± 3.5 nN and 19.9 ± 3.8 nN for IML (n = 500), 0.93 ± 0.46 nN for OML (n = 194), and 5.82 ± 1.1 nN and 19.7 ± 4.61 nN for OLP (n = 144), showing nanomechanical heterogeneity in these samples. Data represented are mean (±SD). (C) Shown is super-resolution AFM image of IML and associated line profile at 25°C. (D) Confocal fluorescence microscopy images of indicated lipid systems are shown. Fluorescence intensity was collected in two channels at 25°C. N-Rh-DPPE was used as a marker labeling preferentially the disordered domains (detected by red channel), TopFluor-Cholesterol partitions preferentially into l_o phase of the membrane. Line profile of the GUVs shows differential incorporation of N-Rh-DPPE (red line), and TopFluor-Cholesterol (green line) into GUVs are plotted on the right. Scale bars, 20 μm. To see this figure in color, go online.

Figure 5

Membrane lipid order maps of mycobacterial model membrane revealing phase segregation and lipid microdomain formation. (A) Pseudocolored Laurdan GP images of indicated GUVs at 25°C are shown. (B) Shown is the GP distribution from the stack of GP images deconvoluted by fitting Gaussian distribution. Scale bars, 64 μm (n of IML = 39, OML = 85, and OLP = 69). To see this figure in color, go online.