Uncovering Biophysical Properties and Interactions of Bacteria Membrane using An Outer Membrane Supported Bilayer

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Antibiotic resistance is a growing global health concern that has been increasing in prevalence in the past few decades. In Gram-negative bacteria, changes in membrane properties such as surface charge, length of membrane components such as lipopolysaccharide (LPS), and in physical properties of the membrane such as domain clustering, can alter responses to antibiotics and mediate resistance. To study the effect of these properties, model membranes have been used to mimic bacterial membranes and to study changes in membrane properties using quantitative surface techniques. However, most model membrane platforms do not capture the complexity of bacterial membranes, which also contain proteins, LPS, and exhibit asymmetry and diverse lipid compositions. We developed a substrate-supported membrane platform using outer membrane vesicles (OMVs) to characterize biophysical membrane properties and investigate the interactions of bacterial membrane and antibacterial compounds. OMVs are naturally shed from the OM by many Gram-negative bacteria from the outer membrane (OM) and recapitulate OM composition and physical properties. Using five clinical bacterial isolates, we demonstrated the formation of fluid bilayers using a membrane fluorescent dye, octadecyl rhodamine B, and measured diffusion coefficients using fluorescence recovery after photobleaching (FRAP). Using total internal reflection fluorescence microscopy (TIRFM), we also confirmed the presence of outer membrane material such as lipid A of LPS and the proper orientation and asymmetry of the bilayer using genetic modifications involving cleavable fluorescent proteins. To study interactions between bacterial membrane and antibacterial compounds, we use optical techniques to observe changes in membrane properties and quantify membrane interactions using polymyxin B. Findings from this work demonstrate OMV bilayers as a platform for studying membrane interactions while retaining membrane components and asymmetry found in native bacterial membranes.