D. Phillips, S. Glaven
U.S. Navy Research Laboratory,
Keywords: membrane sculpting, BAR domain, bacteria, vaccine, scaffolding
Summary:Bacterial membranes and outer membrane vesicles (OMVs) have a range of functionalities in biotech applications, ranging from drug delivery, targeted therapeutics, vaccines, and scaffolds for materials synthesis. Using a novel protein newly identified in bacteria, we have discovered a mechanism to induce higher order curvature structures, such as membrane extensions and tubules, from the bacterial cell membrane. The bacterial membrane sculpting protein BdpA is the first such protein identified and characterized in bacteria. The organism from which the protein was identified, Shewanella oneidensis, is known to produce elongated, narrowing extensions of its outer membrane (OMEs) that are suspected to influence long distance electron transfer and biofilm conductivity. BdpA is enriched in S. oneidensis OME/Vs that are produced naturally when bacteria are attached to a surface. After induction of BdpA expression in response to a small molecule inducer, the S. oneidensis OMEs progress from membrane blebs, to chains of vesicles, and towards narrow, longer tubules. Induced OMEs were recorded to extend beyond 10 µm in length from the body of the cell. The mechanism of inducible OME formation is transferrable to other bacteria on a broad range plasmid vector, including the marine biocathode constituent Marinobacter atlanticus, as well as Escherichia coli. The shape of induced OMEs varies by species, from highly ordered, rigid tubules to a web-like network of OMEs, allowing different membrane extension architectures tailorable to specific applications. BdpA has utility in soft materials production through direct control over membrane curvature formation. When under control of an inducible promoter, BdpA can produce OMEs from organisms in conditions where OMEs would not normally occur, as well as promote OMEs from organisms that do not produce them. This provides unique opportunities for development of novel bacterial membrane-derived living materials, unique scaffolds for materials synthesis, controlled OME/V production under desired growth conditions, and targeted packaging of enzymes for ruggedization. Specifically in the case of S. oneidensis, the OMEs contain proteins capable of electron transfer and are associated with redox activity. BdpA induction for electroactive extension formation could be utilized to bridge breaks in circuits, then catalyze circuit repair in situ. In other bacteria, membrane sculpting BAR proteins have applications in vaccine development, where OME/V production from a pathogen of interest cultured in clinically relevant conditions could produce large membrane particles with surface antigens in their native conformations. This technology is ready to expand testing into other bacteria for utilization of membrane sculpting for the desired downstream industry applications, leading to vaccines against pathogens to which none currently exist.