(380h) Gram-Positive Bacterial Membrane Model Developed Using Membrane Vesicles to Study Biophysical Properties and Antibiotic Interactions

Extracellular vesicles (EVs) produced by eucaryotic and procaryotic cells provide a useful platform to study biophysical properties of the membrane and the function of membrane components or vesicle cargo in cellular and pathophysiological processes. Membrane vesicles (MVs) secreted by gram-positive bacteria typically retain many of the native components, including, lipids, proteins, nucleic acids, and even virulence factors in some cases. Thus, MV-derived membrane platforms present a realistic model of bacterial membrane recapitulating the composition and physical properties of the membrane of gram-positive bacteria. We developed a substrate-supported membrane platform using MVs to characterize biophysical membrane properties and investigate the interactions between bacteria membrane and antibacterial compounds. We isolated MVs from Bacillus subtilis WT strain using established protocols and used these vesicles to form planar bilayers on glass and quartz crystal sensors. We monitored the MV rupturing process and evaluated the two-dimensional diffusion co-efficient of the bilayer on glass using membrane-intercalating fluorescent dye, octadecyl rhodamine B. We used quartz crystal microbalance with dissipation (QCM-D) to determine other physical properties, such as, viscosity, of the membrane as well. For identifying the presence of native membrane components, such as, lipoteichoic acids and primary amines on the developed bilayer platform via antibody binding and amine conjugation, we used total internal reflection fluorescence microscopy (TIRFM). We further investigated whether we could simulate the known interaction patterns of antimicrobial compounds with bacterial membrane with these MV-derived platforms. To this end, we studied the changes in membrane properties upon daptomycin and bacitracin addition using fluorescence microscopy and QCMD. This work endorses the use of supported bilayers constructed from MVs secreted from gram-positive bacteria for studying antibiotic interactions. The application of this platform can be extended towards studying underlying biophysical differences in strains with varying antibiotic resistances or for screening novel membrane-acting antibiotics.