Microbial biosynthesis of designer outer membrane vesicles

Abstract

Outer membrane vesicles (OMVs) are nanoscale proteoliposomes that are ubiquitously secreted by Gram-negative bacteria. Interest in these bioparticles has escalated over the years, leading to discoveries regarding their composition, production, and vaccine potential. Given that many steps in vesicle biogenesis are 'engineerable,' it is now possible to tailor OMVs for specific applications. Such tailoring involves modifying the OMV-producing bacterium through protein, pathway, or genome engineering in a manner that specifically alters the final OMV product. For example, targeted deletion or upregulation of genes associated with the cell envelope can modulate vesicle production or remodel the composition of vesicle components such as lipopolysaccharide. Likewise, bacteria can be reprogrammed to incorporate heterologously expressed proteins into either the membrane or lumenal compartment of OMVs. We anticipate that further research in the field of OMV engineering will enable continued design and biosynthesis of specialized vesicles for numerous biotechnological purposes ranging from the delivery of vaccines to the deconstruction of cellulosic substrates.

Figure 1. Engineering of bacterial OMVs

Efforts to engineer bacterial OMVs are focused in three main areas: (1) vesiculation, or the process of membrane blebbing to produce vesicles; (2) protein decoration, or the targeting of natural or engineered biomolecules to specific subcompartments (i.e., lumen, membrane, exterior) of vesicles; and (3) detoxification, or the modification of the lipid A portion of bacterial LPS. These areas are visualized in the context of OMV structure and biogenesis. Biomolecules relevant to this review are depicted and labeled in the key.

Figure 2. Decorating the interior and exterior of bacterial OMVs

Recombinant proteins can be targeted to the lumen, the membrane, and the outer surface of OMVs. Localizing proteins in the lumen is accomplished using signal peptides that target proteins to either the (A) Tat (for folded proteins; post-translational) or (B) Sec (for unfolded proteins; post- or co-translational) export pathways. Incorporation of proteins in the membrane is accomplished using proteins that natively transit the (B) Sec pathway followed by the (C) outer membrane protein (OMP) assembly pathway. Display of proteins on the exterior is accomplished by fusion to a canonical outer membrane protein that transits the (B) Sec and (C) OMP assembly pathways, or to a non-canonical outer membrane protein (e.g., ClyA) whose export pathways (D) are undetermined.