Incorporation of Heterologous Outer Membrane and Periplasmic Proteins into Escherichia coli Outer Membrane Vesicles

Abstract

Gram-negative bacteria shed outer membrane vesicles composed of outer membrane and periplasmic components. Since vesicles from pathogenic bacteria contain virulence factors and have been shown to interact with eukaryotic cells, it has been proposed that vesicles behave as delivery vehicles. We wanted to determine whether heterologously expressed proteins would be incorporated into the membrane and lumen of vesicles and whether these altered vesicles would associate with host cells. Ail, an outer membrane adhesin/invasin from Yersinia enterocolitica, was detected in purified outer membrane and in vesicles from Escherichia coli strains DH5␣, HB101, and MC4100 transformed with plasmidencoded Ail. In vesicle-host cell co-incubation assays we found that vesicles containing Ail were internalized by eukaryotic cells, unlike vesicles without Ail. To determine whether lumenal vesicle contents could be modified and delivered to host cells, we used periplasmically expressed green fluorescent protein (GFP). GFP fused with the Tat signal sequence was secreted into the periplasm via the twin arginine transporter (Tat) in both the laboratory E. coli strain DH5␣ and the pathogenic enterotoxigenic E. coli ATCC strain 43886. Pronase-resistant fluorescence was detectable in vesicles from Tat-GFP-transformed strains, demonstrating that GFP was inside intact vesicles. Inclusion of GFP cargo increased vesicle density but did not result in morphological changes in vesicles. These studies are the first to demonstrate the incorporation of heterologously expressed outer membrane and periplasmic proteins into bacterial vesicles.Gram-negative bacteria secrete proteins solubly and in association with outer membrane vesicles. All Gram-negative bacteria studied to date, including Escherichia coli, Neisseria meningitidis, Pseudomonas aeruginosa, Helicobacter pylori, Borrelia burgdorferi, Shigella flexneri and Actinobacillus actinomycetemcomitans, produce outer membrane vesicles (1-8). Vesicles were first observed by electron microscopy and range in size from 20 -250 nm in diameter. Gram-negative bacteria are bounded by an inner and outer membrane that encloses the periplasmic space. During vesiculation, the outer membrane pinches off (1), resulting in a closed proteoliposome composed of outer membrane lipids and proteins and periplasmic components, but not inner membrane or cytosolic components. Virulence factors such as VacA, shiga toxin, heat-labile enterotoxin (LT), 1 leukotoxin, and ClyA are associated with vesicles from pathogenic bacteria (2, 4 -6, 8 -10).Bacterial outer membrane vesicles interact with both eukaryotic cells and other bacteria via surface-expressed factors to deliver vesicle components and virulence factors (5,6,8,(11)(12)(13)(14)(15)(16).2 For example, LT associated with lipopolysaccharide on the surface of enterotoxigenic E. coli (ETEC) vesicles triggers internalization via caveolae and delivers not only catalytically active LT, which intoxicates the eukaryotic cell, but also other bacterial vesicle components.2 Other s...

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