With the aim to provide a more comprehensive and biologically relevant picture of the drug-membrane interactions and how the effect of these interactions can modify the biophysical properties of the membranes in relation with pharmacological activities, most of the studies are performed by using cells (bacteria or mammalian cells) and membrane models (supported bilayers, liposomes [SUVs, LUVs; GUVs]) mimicking (i) bacterial and (ii) eukaryotic membranes. In close collaboration, we used a range of complementary methods including AFM, 31P NMR, dynamic light scattering, fluorescence spectroscopy (Laurdan, DPH, TMA-DPH, DHE, calcein, octadecylrhodamine B…) and confocal and electronic microscopy.
Combination of existing lipid diversity and functions with biophysics of bacterial membranes is a unique opportunity to discover new antibiotics. Bacteria (as mammalian cells) have capacity to maintain specialized zones in their membranes for fruitfully fill in their biological functions.
In the frame of our work, we focus on areas characterized by high curvature and enriched in cardiolipin, as encountered at poles and division septa of Gram-negative bacteria, with the aim to understand if and how membrane-acting antibiotics (amphiphilic neamine derivatives) might modify bacterial physiological processes.
Intensive medicinal chemistry development was performed in collaboration with Prof. JL Decout and coll. (Grenoble, F) from a group of old antibiotic drugs called aminoglycosides, which target ribosomal RNA. Molecular foundations and structure–activity relationships made on the central backbone (neamine versus neosamine), the nature of the hydrophobic tail (naphtyl, alkyl, alkyl) as well as or the position and the number of substitution on the central backbone to define optimal amphiphilicity, led to the emergence of amphiphilic antibacterial aminoglycosides. More than 80 derivatives were synthesized with very promising compounds active against Gram-positive and Gram-negative sensitive and resistant bacteria. In addition, we did not observe any emergence of resistance in P. aeruginosa treated for 35 days with amphiphilic aminoglycoside derivatves.
To decipher the molecular mechanism involved in their activity, we used both living bacteria (P. aeruginosa) as well as membrane model systems including LUVs (Large Unilamellar Vesicles) for membrane permeability and depolarization, GUVs (Giant Unilamellar vesicles) for confocal microscopy and lipid monolayers, for Langmuir isotherm compression. We demonstrated the interaction of the amphiphilic neamine derivatives with outer membrane’s lipopolysaccharides and inner membrane’s anionic phospholipids mostly cardiolipin leading to membrane permeabilization (NPN and PI assays) and depolarization (DiSC3(5) fluorescence). Targeting cardiolipin bacterial micro- domains mainly located at the cell poles, led to relocation of cardiolipin domains associated with bacterial morphological changes including a severe length decrease.
These results suggest an effect of amphiphilic aminoglycoside antibiotics on cardiolipin domains with in turn changes in the activity of proteins dependent upon cardiolipin and involved in bacterial division (FtsZ) and/or bacterial shape (MreB).
At a glance, our results bring into light fundamental concepts which could be important in membrane-lipid therapy in which the molecular targets are the lipids and the structure they form. The role of lipids can be (i) to facilitate membrane bending and the formation of highly curved intermediates, reducing the energy barriers of fission and fusion and (ii) to recruit specialized proteins. Influencing curvature directly as well as indirectly by targeting negative intrinsic curvature of lipids or in impairing the soft mechanical behavior could be a new approach for antibiotic design.
In this context, we investigated (i) whether enriched lipid domains in cholesterol and sphingomyelin could contribute to function-associated cell (re)shaping, (ii) whether the seminal concept of highly ordered rafts could be refined with the presence of lipid domains exhibiting different enrichment in cholesterol and sphingomyelin and association with erythrocyte curvature areas and (iii) how differences in lipid order between domains and surrounding membrane are regulated and whether changes in order differences could participate to erythrocyte deformation and vesiculation.
For studying the first question, we probed by vital imaging the lateral distribution of cholesterol and sphingomyelin (using either specific Toxin fragments or trace insertion of BODIPY-SM) in relation with: (i) membrane biconcavity of resting red blood cells; (ii) membrane curvature changes and Ca2+ exchanges upon mechanical stretching of healthy red blood cells or in elliptocytes, a red blood cells model of impaired shape; and (iii) membrane vesiculation upon red blood cells aging.
We revealed the specific association of cholesterol- and sphingomyelin-enriched domains with distinct curvature areas of the erythrocyte biconcave membrane. Upon erythrocyte deformation, cholesterol-enriched domains gathered in high curvature areas. In contrast, sphingomyelin-enriched domains increased in abundance upon calcium efflux during shape restoration. Upon erythrocyte storage at 4 °C (to mimick aging), lipid domains appeared as specific vesiculation sites.
The second and third questions benefit from the use of a fluorescent hydration- and membrane packing-sensitive probe, Laurdan, to determine the Generalized Polarization (GP) values of lipid domains vs the surrounding membrane. Sphingomyelin-and cholesterol- enriched domains were less ordered than the surrounding lipids in erythrocytes at resting state. Upon erythrocyte deformation (elliptocytes and stimulation of calcium exchanges) or membrane vesiculation (storage at 4°C), lipid domains became more ordered than the bulk. Upon aging and in membrane fragility diseases (spherocytosis), an increase in the difference of lipid order between domains and the surrounding lipids contributed to the initiation of domain vesiculation.
Altogether, results demonstrated the critical role of domain-bulk differential lipid order modulation for erythrocyte reshaping probably related with the pressure exerted by the cytoskeleton on the membrane.