Supramolecular organization and dynamics of mannosylated phosphatidylinositol lipids in the mycobacterial plasma membrane

Abstract

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), a disease that claims ~1.6 million lives annually. The current treatment regime is long and expensive, and missed doses contribute to drug resistance. Therefore, development of new anti-TB drugs remains one of the highest public health priorities. Mtb has evolved a complex cell envelope that represents a formidable barrier to antibiotics. The Mtb cell envelop consists of four distinct layers enriched for Mtb specific lipids and glycans. Although the outer membrane, comprised of mycolic acid esters, has been extensively studied, less is known about the plasma membrane, which also plays a critical role in impacting antibiotic efficacy. The Mtb plasma membrane has a unique lipid composition, with mannosylated phosphatidylinositol lipids (phosphatidyl-myoinositol mannosides, PIMs) comprising more than 50% of the lipids. However, the role of PIMs in the structure and function of the membrane remains elusive. Here, we used multiscale molecular dynamics (MD) simulations to understand the structure-function relationship of the PIM lipid family and decipher how they self-organize to shape the biophysical properties of mycobacterial plasma membranes. We assess both symmetric and asymmetric assemblies of the Mtb plasma membrane and compare this with residue distributions of Mtb integral membrane protein structures. To further validate the model, we tested known anti-TB drugs and demonstrated that our models agree with experimental results. Thus, our work sheds new light on the organization of the mycobacterial plasma membrane. This paves the way for future studies on antibiotic development and understanding Mtb membrane protein function.

Keywords: antibiotics diffusion; multi-scale molecular dynamics; mycobacteria inner membrane; phosphatidyl-myoinositol mannosides.

Fig. 1.

Structure of the mycobacterial lipids. (A) Schematic of an asymmetrical model of the mycobacterial plasma membrane and composition as previously defined (13). (B) Schematic of the core of the PIM lipids found in mycobacteria with the groupings for CG beads. The inositol core is highlighted in red. The bead types for MARTINI 3 are shown. (C) Overlay of the AT (sticks) and CG (spheres) models for each lipid, with chemical characteristics shown to the Right.

Fig. 2.

Mycobacterial membrane model. (A) Side view of the membrane with each lipid type depicted in a different color as shown in Fig. 1A. (B) A density plot showing the density of water, sugar groups, phosphate groups, tail groups, and ions over the simulation box. (C) Snapshots of the periplasmic and cytoplasmic leaflets whose compositions are AcPIM₂ 22%, AcPIM₆ 11%, Ac₂PIM₆ 10%, CL 24%, PE 20% and PI 13% (periplasmic leaflet), and AcPIM₂ 10% and Ac₂PIM₂ 90% (cytoplasmic leaflet). The system size is 50 × 50 × 15 nm. (D) Relative number of neighbors of each lipid type for the periplasmic membrane. (E) Mean squared displacement (MSD_xy) (nm²) as a function of lagtime (ns) for each lipid type in the periplasmic and cytoplasmic leaflets. The inserts show the position of the phosphate group of each lipid type over the last 500 ns of the simulation. (F) Contour plots of the area per lipid (nm²) in each leaflet at the starting frame (Upper) and final frame (Lower) of the simulation. A darker color indicates a larger area per lipid.

Fig. 3.

Asymmetry of the mycobacterial membrane. (A) A simplified workflow for the lipid contacts analysis from all membrane proteins. (B) An example of a protein (E. coli protein MraY UniProt id: P0A6W3 and Mtb transporter MmpL3 UniProt id: P9WJV5) embedded in either an E. coli or mycobacterial membrane. The orange spheres represent phosphates. For the E. coli membrane, the cyan sticks show PG and the gray sticks PE. For mycobacterial membrane, the lipid sticks are shown in the colors illustrated in Fig. 1A. Selected residues are colored according to the key in (C). (C) Graphs for E. coli and Mtb (Top and Bottom respectively) showing the relative abundance of selected residues within 8 Å of lipids. The gray lines show the position of the phosphates. The cytoplasmic region is shown by negative z values, the periplasmic region by positive values.

Fig. 4.

Behavior of the antibiotics with mycobacterial membrane and proteins. (A) Chemical structures of BDQ and ISZ with the CG groupings overlaid and bead types shown. (B) PMFs of the two antibiotics being pulled through either a mycobacterial or simple membrane in the z-direction. BDQ is shown in blue and ISZ in red, with the mycobacterial membrane results having a solid line and simple membrane having a dashed line. The error is shown in gray. A schematic of the PMF is shown to the Left. (C) Structure of Mycobacterium smegmatis ATP synthase (PDB: 7JG5) with the c-subunits shown in gray, the a-subunit shown in cyan, and the other components shown as a gold surface. (D) Density in the x and y dimensions of selected lipids and BDQ when starting in the cytoplasmic leaflet relative to the protein shown in gray. (E) Density of the phosphates (orange) and BDQ over the course of the simulations where the antibiotic started in either the periplasmic leaflet (green) or the cytoplasmic leaflet (blue). (F) Snapshot of a single simulation containing a Mtb ATPase model and 8 × BDQ models showing the main positions BDQ occupied. Phosphates are shown in orange, BDQ shown in blue, c-subunits are shown in gray, and the a-subunit is shown in cyan. The lipid sticks are shown in the colors illustrated in Fig. 1A. (G) Comparison of the highest occupancy sites identified with PyLipID (surface) and BDQ from the cryo-EM structure (PDB: 7JGC) (sticks). (H) PMF of BDQ moving through a mycobacterial plasma membrane with the error shown in gray. A schematic of the PMF is shown as an Insert.