Bacterial Outer Membrane Vesicles as Antibiotic Delivery Vehicles

Abstract

Bacterial outer membrane vesicles (OMVs) are nanometer-scale, spherical vehicles released by Gram-negative bacteria into their surroundings throughout growth. These OMVs have been demonstrated to play key roles in pathogenesis by delivering certain biomolecules to host cells, including toxins and other virulence factors. In addition, this biomolecular delivery function enables OMVs to facilitate intra-bacterial communication processes, such as quorum sensing and horizontal gene transfer. The unique ability of OMVs to deliver large biomolecules across the complex Gram-negative cell envelope has inspired the use of OMVs as antibiotic delivery vehicles to overcome transport limitations. In this review, we describe the advantages, applications, and biotechnological challenges of using OMVs as antibiotic delivery vehicles, studying both natural and engineered antibiotic applications of OMVs. We argue that OMVs hold great promise as antibiotic delivery vehicles, an urgently needed application to combat the growing threat of antibiotic resistance.

Keywords: Gram-negative bacteria; antibiotic resistance; antibiotics; drug delivery; outer membrane vesicles.

Figure 1

OMV Biogenesis. OMVs are formed due to blebbing of the bacterial outer membrane. The vesicle contains outer membrane-associated proteins and lipids (including lipopolysaccharide), as well as periplasmic components such as peptidoglycan.

Figure 2

Natural Delivery Functions of OMVs. (A) Protein Delivery. Proteins derived from a donor cell are encapsulated within OMVs and delivered to recipient cells. (B) Gene Delivery. DNA (plasmid, chromosomal, and/or phage-associated) is encapsulated within OMVs and delivered to recipient cells. In some cases, this new gene is expressed by daughter cells. (C) C16-HSL and CAI-1. Hydrophobic quorum sensing molecules, such as C16-HSL and CAI-1, have been observed to be delivered to bacterial cells via OMVs. (D) Quorum sensing. PQS is a hydrophobic quorum sensing molecule. As it intercalates into the outer leaflet of the bacterial membrane, a wedge-like force promotes the formation of PQS-containing OMVs.

Figure 3

Engineered OMVs. (A) The Tol-Pal System. The Tol-Pal system consists of five proteins, TolA, TolQ, and TolR located in the inner membrane (IM), TolB located in the peptidoglycan layer, and Pal located in the outer membrane. Mutation of any of these components has been shown to affect vesiculation. (B) Structure of LPS. LPS consists of a hydrophobic lipid A, which is commonly hexaacylated and di-phosphorylated (P). The polysaccharide portion of the molecule consists of a well conserved inner and outer core and a nonconserved O-antigen.

Figure 4

Strategies for Drug Loading. (A) Bacterial Incubation. Bacteria grown in the presence of antibiotics have been found to release antibiotic-containing OMVs. (B) Electroporation and Sonication. Electroporation and sonication can enhance drug loading within the OMV lumen.

Figure 5

Surface Engineering of OMVs. Several genetic strategies have been used to localize certain proteins on the surface of OMVs. Fusion proteins between cytolysin A (ClyA) and several cargos, including GFP have been created in E. coli. Ice nucleation protein (INP) was used to tether an antibody on the surface of the OMV by creating a fusion between INP, a cohesin-containing Scaf3 domain, and an antibody-binding Z-domain. Simultaneously, SlyB was used to localize luciferase to the OMV lumen. Up to four bacterial antigens were tethered to the surface of OMVs using the hemoglobin protease (Hbp). This protein was also used in combination with the SpyCatcher/Tag and SnoopCatcher/Tag systems to display heterologous proteins on the OMV surface.