Mutually Exclusive Interactions of Rifabutin with Spatially Distinct Mycobacterial Cell Envelope Membrane Layers Offer Insights into Membrane-Centric Therapy of Infectious Diseases

Abstract

The mycobacterial cell envelope has spatially resolved inner and outer membrane layers with distinct compositions and membrane properties. However, the functional implication and relevance of this organization remain unknown. Using membrane biophysics and molecular simulations, we reveal a varied interaction profile of these layers with antibiotic Rifabutin, underlined by the structural and chemical makeup of the constituent lipids. The mycobacterial inner membrane displayed the highest partitioning of Rifabutin, which was located exclusively in the lipid head group/interfacial region. In contrast, the drug exhibited specific interaction sites in the head group/interfacial and hydrophobic acyl regions within the outer membrane. Altogether, we show that the design of membrane-active agents that selectively disrupt the mycobacterial outer membrane structure can increase drug uptake and enhance intracellular drug concentrations. Exploiting the mycobacterium-specific membrane-drug interaction profiles, chemotypes consisting of outer membrane-disruptive agents and antitubercular drugs can offer new opportunities for combinational tuberculosis (TB) therapy.

Figure 1

Schematic representation of the structure of the key lipids present in the mycobacterial cell envelope layers. SL-1, sulfolipids; TDM, trehalose dimycolate; PDIM and MA, noncovalent lipids in the outer membrane. The inner membrane consists of tetra- and monoacylated phospho-myo-inositol dimannosides (ACPIM₂, AC₂PIM₂) and higher-order phosphomannosides such as ACPIM₆ along with standard phospholipids including phosphatidylinositol (PI) and phosphatidylethanol (PE) amine. Lipoarabinomannans (LAMs) with the outer membrane-free lipids constitute the mycomembrane.

Figure 2

Nonlinear least-squares regression curves of the partitioning of 25 μM Rifabutin into different Msm membranes obtained from the third derivative UV absorption intensities ranging from 284 to 297 nm measured using UV–visible spectroscopy. Below: partition coefficient (K_p) and distribution coefficient (log D) of Rifabutin within different Msm membrane systems calculated from the fitted curves.

Figure 3

(A) Fluorescence probe quenching to determine the location of Rifabutin within each lipid bilayer. Static quenching (red circle) and dynamic quenching (black square) of DPH and TMA–DPH probes were measured against increasing concentrations of Rifabutin (0–30 μM) in each Msm membrane system. (B) Stern–Volmer quenching constant (K_SV), dynamic quenching constant (K_D), and bimolecular quenching constant (K_q) were derived from the static and lifetime fluorescence plots to determine the extent of quenching by, and the accessibility of, Rifabutin to DPH/TMA–DPH fluorescence probes embedded in different Msm membrane systems, respectively. (C) Number of Rifabutin molecules in the regions at different z-direction distances from the membrane center.

Figure 4

(A) Mycobacterial membrane fluidity perturbations over temperature in the absence (black square) and presence (red circle) of 10 mol % Rifabutin indicated by fluorescence probes TMA–DPH and DPH. (B) Head-group modulations in the presence of 10 mol % Rifabutin tracked with the help of water-sensitive fluorescence probe Laurdan in the indicated Msm systems. Laurdan deconvolution of the normalized and baseline-corrected fluorescence intensity curves at 25 °C (—) into charge-transfer states (---) and solvent-relaxed state (blue dashed curves) by the log-normal (LN) deconvolution method (red curves). (C) Generalized polarization (GP) of the lipid systems across various temperatures in the absence (black square) and presence (red circle) of 10 mol % Rifabutin.

Figure 5

(A) Confocal imaging of the N-Rh-DHPE-labeled Msm systems. Both ordered (devoid of N-Rh-DHPE signal) and disordered regions (with N-Rh-DHPE signal) in the membranes are visualized. Rifabutin addition did not alter the abundance or distribution of disordered domains in three replicate studies. (B) Topography of inner membrane observed as solid-supported bilayers (SLBs) with atomic force microscopy. (C) Inner membranes displaying at least two significant lipid domains of ∼5.8 nm (domain 1) and ∼6.3 nm (domain 2) in height. (D) Temporal changes in the height of lipid domains after the addition of 25 μM Rifabutin.

Figure 6

Molecular dynamics (MD) simulations of the mycomembrane and inner membrane to determine the membrane properties. (A) Domain distribution and (B) height heterogeneity in the absence and presence of Rifabutin to study the influence of Rifabutin on the membranes. The Δ ratio of different size clusters of lipids in (C) mycomembrane and (D) inner membrane. The Δ ratio values are calculated by the ratio changes influenced by Rifabutin. Diffusion rate of the lipids in (E) outer membrane/mycomembrane and (F) inner membrane with or without Rifabutin molecules calculated from the mean-square displacement (MSD) of these molecules.