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

Nicole C Kesty,Meta J Kuehn

doi:10.1074/jbc.m307628200

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 DH5alpha, HB101, and MC4100 transformed with plasmid-encoded 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 DH5alpha 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.

Highlights

Gram-negative bacteria secrete proteins solubly and in association with outer membrane vesicles
Ail Is Present in E. coli Vesicles—To determine whether exogenous outer membrane proteins are secreted via vesicles, purified vesicles were prepared from several laboratory strains of E. coli transformed with a plasmid encoding the Y. enterocolitica outer membrane protein Ail under the control of its native promoter [18]
Ail was detected in purified outer membrane preparations and in vesicles produced by DH5␣/Ail (Fig. 1, A and B), MC4100/Ail, and HB101/Ail, and its presence in vesicles was confirmed by N-terminal sequencing

Summary

EXPERIMENTAL PROCEDURES

Reagents and Cell Culture—E. coli strains DH5␣ (Stratagene), MC4100, and ETEC (ATCC strain 43886) were grown in LB or CFA broth (1% casamino acids, 0.15% yeast extract, 0.005% MgSO4, and 0.005% MnCl2), respectively. HB101/pVM102 (Ail) was kindly provided by Dr Virginia Miller [18]. Strains were grown in the presence of kanamycin (10 ␮g/ml) and/or ampicillin (100 ␮g/ml) as required. Human colorectal HT29 cells (ATCC HTB-38) were grown in McCoy’s 5a media supplemented with 10% bovine calf serum. CHO-K1 cells (ATCC CCL-61) were grown in Ham’s F12K media supplemented with 10% bovine calf serum. All cell lines were grown in the presence of penicillin/ streptomycin/amphotericin B antibiotic-antimycotic solution (Sigma).

Heterologous Proteins in Bacterial Vesicles

RESULTS

DISCUSSION