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.

Lipids dictate membrane properties to modulate lateral membrane organization, lipid/protein diffusion and lipid-protein interactions, thereby underpinning proper functioning of cells. Mycobacterium tuberculosis harnesses the power of its atypical cell wall lipids to impact immune surveillance machinery centered at the host cell membrane. However, the role of specific virulent lipids in altering host cellular functions by modulating membrane organization and the associated signaling response are still pertinent unresolved questions. Here, combining membrane biophysics and cell biology, we elucidate how virulent Mtb sulfoglycolipids hijack the host cell membrane, affecting its order, fluidity, and stiffness along with manipulating the linked cytoskeleton. The functional outcome of this perturbation was assayed by monitoring membrane-associated autophagy signaling. These actions form a part of the overall response to commandeer host membrane-associated immune processes during infection. The findings on the mechanism of action of Mtb lipids on host cell membrane structure and downstream signaling will deepen the collective understanding of their functional aspects in membrane-dictated bacterial survival, pathogenesis and drug resistance and reveal suitable membrane driven-therapeutic intervention points and diagnostic tools.

Cholesterol induced mechanical effects on artificial lipid bilayers are well known and have been thoroughly investigated by AFM force spectroscopy. However, dynamics of cholesterol impingement into bilayers at various cholesterol concentrations and their effects have not been clearly understood. In this paper we present, the effect of cholesterol as a function of its concentration in a simple single component dioleoylphosphatidylcholine (DOPC) bilayer. The nature of measured breakthrough forces on a bilayer with the addition of cholesterol, suggested that it is not just responsible to increase the mechanical stability but also introduces irregularities across the leaflets of the bilayer. This cholesterol induced asymmetry across the (in the inner and outer leaflets) bilayer is related to the phenomena of interleaflet coupling and is a function of cholesterol concentration probed by AFM can provide an unprecedented direction on mechanical properties of lipid membrane as it can be directly correlated to biophysical properties of a cell membrane.

The mycobacterial cell envelope acts as a multilayered barrier to drugs. However, the role of lipid composition in the properties of different mycobacterial membranes, otherwise dictating their interactions with drugs, is poorly understood. In this study, we found that hydration states, solvation relaxation kinetics, rotational lipid mobility, and lateral lipid diffusion differed between inner and outer mycobacterial membranes. Molecular modeling showed that lipid clustering patterns governed membrane dynamics in the different layers of the cell envelope. By regulating membrane properties, lipid composition and structure modulated water abundance and interactions with lipid head groups. These findings can help deepen our understanding of the physical chemistry underlying membrane structure and function, as well as the interaction of mycobacterial membranes with drugs and host membranes.

Synthetic channels with high ion selectivity are attractive drug targets for diseases involving ion dysregulation. Achieving selective transport of divalent ions is highly challenging due their high hydration energies. A small tripeptide amphiphilic scaffold installed with a pybox ligand selectively transports Cu II ions across membranes. The peptide forms stable dimeric pores in the membrane and transports ions by a Cu 2 + /H + antiport mechanism. The ligand-induced excellent Cu II selectivity as well as high membrane permeability of the peptide is exploited to promote cancer cell death. The peptide's ability to restrict mycobacterial growth serves as seeds to evolve antibacterial strategies centred on selectively modulating ion homeostasis in pathogens. This simple peptide can potentially function as a universal, yet versatile, scaffold wherein the ion selectivity can be precisely controlled by modifying the ligand at the C terminus.