Title: 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 DH5α, 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 DH5α 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 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 DH5α, 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 DH5α 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.Beveridge T.J. J. Bacteriol. 1999; 181: 4725-4733Crossref PubMed Google Scholar, 2.Horstman A.L. Kuehn M.J. J. Biol. Chem. 2000; 275: 12489-12496Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 3.Devoe I.W. Gilchrist J.E. J. Exp. Med. 1973; 138: 1156-1167Crossref PubMed Scopus (189) Google Scholar, 4.Keenan J. Day T. Neal S. Cook B. Perez-Perez G. Allardyce R. Bagshaw P. FEMS Microbiol. Lett. 2000; 182: 259-264Crossref PubMed Google Scholar, 5.Fiocca R. Necchi V. Sommi P. Ricci V. Telford J. Cover T.L. Solcia E. J. Pathol. 1999; 188: 220-226Crossref PubMed Scopus (202) Google Scholar, 6.Kato S. Kowashi Y. Demuth D.R. Microb. Pathog. 2002; 32: 1-13Crossref PubMed Scopus (177) Google Scholar, 7.Wai S.N. Takade A. Amako K. Microbiol. Immunol. 1995; 39: 451-456Crossref PubMed Scopus (92) Google Scholar, 8.Wai S.N. Lindmark B. Soderblom T. Takade A. Westermark M. Oscarsson J. Jass J. Richter-Dahlfors A. Mizunoe Y. Uhlin B.E. Cell. 2003; 115: 25-35Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). 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.Beveridge T.J. J. Bacteriol. 1999; 181: 4725-4733Crossref PubMed Google Scholar), 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), 1The abbreviations used are: LT, heat-labile enterotoxin; CHO, Chinese hamster ovary; ETEC, enterotoxigenic E. coli; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; IPTG, isopropyl-1-thio-β-d-galactopyranoside; MBP, maltose binding protein; Omp, outer membrane protein; Tat, twin arginine transporter.1The abbreviations used are: LT, heat-labile enterotoxin; CHO, Chinese hamster ovary; ETEC, enterotoxigenic E. coli; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; IPTG, isopropyl-1-thio-β-d-galactopyranoside; MBP, maltose binding protein; Omp, outer membrane protein; Tat, twin arginine transporter. leukotoxin, and ClyA are associated with vesicles from pathogenic bacteria (2.Horstman A.L. Kuehn M.J. J. Biol. Chem. 2000; 275: 12489-12496Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 4.Keenan J. Day T. Neal S. Cook B. Perez-Perez G. Allardyce R. Bagshaw P. FEMS Microbiol. Lett. 2000; 182: 259-264Crossref PubMed Google Scholar, 5.Fiocca R. Necchi V. Sommi P. Ricci V. Telford J. Cover T.L. Solcia E. J. Pathol. 1999; 188: 220-226Crossref PubMed Scopus (202) Google Scholar, 6.Kato S. Kowashi Y. Demuth D.R. Microb. Pathog. 2002; 32: 1-13Crossref PubMed Scopus (177) Google Scholar, 8.Wai S.N. Lindmark B. Soderblom T. Takade A. Westermark M. Oscarsson J. Jass J. Richter-Dahlfors A. Mizunoe Y. Uhlin B.E. Cell. 2003; 115: 25-35Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar, 9.Yokoyama K. Horii T. Yamashino T. Hashikawa S. Barua S. Hasegawa T. Watanabe H. Ohta M. FEMS Microbiol. Lett. 2000; 192: 139-144Crossref PubMed Google Scholar, 10.Kolling G.L. Matthews K.R. Appl. Environ. Microbiol. 1999; 65: 1843-1848Crossref PubMed Google Scholar). Bacterial outer membrane vesicles interact with both eukaryotic cells and other bacteria via surface-expressed factors to deliver vesicle components and virulence factors (5.Fiocca R. Necchi V. Sommi P. Ricci V. Telford J. Cover T.L. Solcia E. J. Pathol. 1999; 188: 220-226Crossref PubMed Scopus (202) Google Scholar, 6.Kato S. Kowashi Y. Demuth D.R. Microb. Pathog. 2002; 32: 1-13Crossref PubMed Scopus (177) Google Scholar, 8.Wai S.N. Lindmark B. Soderblom T. Takade A. Westermark M. Oscarsson J. Jass J. Richter-Dahlfors A. Mizunoe Y. Uhlin B.E. Cell. 2003; 115: 25-35Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar, 11.Demuth D.R. James D. Kowashi Y. Kato S. Cell. Microbiol. 2003; 5: 111-121Crossref PubMed Scopus (51) Google Scholar, 12.Li Z. Clarke A.J. Beveridge T.J. J. Bacteriol. 1998; 180: 5478-5483Crossref PubMed Google Scholar, 13.Beermann C. Wunderli-Allenspach H. Groscurth P. Filgueira L. Cell. Immunol. 2000; 201: 124-131Crossref PubMed Scopus (36) Google Scholar, 14.Shoberg R.J. Thomas D.D. Infect. Immun. 1993; 61: 3892-3900Crossref PubMed Google Scholar, 15.Kadurugamuwa J.L. Beveridge T.J. J. Bacteriol. 1996; 178: 2767-2774Crossref PubMed Scopus (223) Google Scholar, 16.Kadurugamuwa J.L. Beveridge T.J. Antimicrob. Agents Chemother. 1998; 42: 1476-1483Crossref PubMed Google Scholar). 2N. C. Kesty, K. M. Mason, and M. J. Kuehn, submitted for publication.2N. C. Kesty, K. M. Mason, and M. J. Kuehn, submitted for publication. 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. 2N. C. Kesty, K. M. Mason, and M. J. Kuehn, submitted for publication. Other studies have suggested that outer membrane invasins IpaB, C, and D may catalyze the internalization S. flexneri vesicles (16.Kadurugamuwa J.L. Beveridge T.J. Antimicrob. Agents Chemother. 1998; 42: 1476-1483Crossref PubMed Google Scholar). To date, vesicle components have not been altered by genetic manipulation. Previous studies demonstrated that vesicles could be generated containing periplasmic gentamicin by treatment of cells with gentamicin, but they differed from native vesicles in their composition and size (15.Kadurugamuwa J.L. Beveridge T.J. J. Bacteriol. 1996; 178: 2767-2774Crossref PubMed Scopus (223) Google Scholar, 16.Kadurugamuwa J.L. Beveridge T.J. Antimicrob. Agents Chemother. 1998; 42: 1476-1483Crossref PubMed Google Scholar, 17.Kadurugamuwa J.L. Beveridge T.J. J. Bacteriol. 1995; 177: 3998-4008Crossref PubMed Google Scholar). Because vesicles are composed of outer membrane and periplasmic components, we hypothesized that expressed heterologous outer membrane and periplasmic proteins should be packaged in vesicles. Furthermore, we wanted to determine whether vesicle properties could be altered by the expression of proteins into the periplasm and outer membrane of bacteria. For instance, green fluorescent protein (GFP) transported to the periplasm and packaged in vesicles could be used as a lumenal vesicle marker, whereas vesicle incorporation of an outer membrane adhesin/invasin, Ail from Yersinia enterocolitica, could alter the adhesion and internalization properties of the vesicles. In this study, we demonstrate that Ail and periplasmic GFP are packaged in vesicles and that these altered vesicles can be used to track vesicle interactions with mammalian cells. Reagents and Cell Culture—E. coli strains DH5α (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.Miller V.L. Falkow S. Infect. Immun. 1988; 56: 1242-1248Crossref PubMed Google Scholar). Strains were grown in the presence of kanamycin (10 μg/ml) and/or ampicillin (100 μg/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) and maintained in a humidified atmosphere containing 5% CO2 at 37 °C. All chemicals were obtained from Sigma unless otherwise stated. Plasmid Construction—For pNKTG2 (Tat-GFP), the Tat signal sequence and mutGFP3 sequence were cut out of pJDT1 (19.Thomas J.D. Daniel R.A. Errington J. Robinson C. Mol. Microbiol. 2001; 39: 47-53Crossref PubMed Scopus (230) Google Scholar) and inserted into pTrc99A (Amersham Biosciences) using EcoRI and HindIII. The mutGFP3 sequence was replaced with mutGFP2 (20.Cormack B.P. Valdivia R.H. Falkow S. Gene. 1996; 173: 33-38Crossref PubMed Scopus (2474) Google Scholar) using MscI and HindIII. pNKTAT (TatABCE) was constructed by partial digestion of pTatABCE (21.Yahr T.L. Wickner W.T. EMBO J. 2001; 20: 2472-2479Crossref PubMed Scopus (134) Google Scholar) using EcoRI and HindIII and insertion into the multiple cloning site of pK187 (22.Jobling M.G. Holmes R.K. Nucleic Acids Res. 1990; 18: 5315-5316Crossref PubMed Scopus (93) Google Scholar). To induce Tat-GFP or TatABCE, overnight cultures were diluted 1:10 and grown at 37 °C for 6 h, unless otherwise mentioned, in the presence of 0.1 mm IPTG. Plasmids were transformed by electroporation into bacteria using a modified CaCl2 protocol (23.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Press, Cold Spring Harbor, NY1989: 1.82-1.84Google Scholar). Vesicle Purification and Labeling—Vesicles were purified as described previously (2.Horstman A.L. Kuehn M.J. J. Biol. Chem. 2000; 275: 12489-12496Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar) with the following modifications. Bacteria were pelleted (10 min, 10,000 × g), and vesicles were then pelleted from the supernatant (1 h, 40,000 × g), filter-sterilized (0.45 μm; Millipore), and applied to the bottom of a discontinuous Optiprep (Greiner) gradient (2 ml each of 50, 45, 40, 35, 30, and 25% Optiprep in 10 mm HEPES, pH 6.8, supplemented with 0.85% NaCl). Vesicle-containing fractions were combined, and protein concentration was determined using Coomassie Plus (Pierce). Vesicles were fluorescently labeled by diluting 1:1 with fluorescein isothiocyanate (FITC) (1 mg/ml in 50 mm Na2CO3 and 100 mm NaCl, pH 9.2) and incubated for 1 h at 25 °C. Vesicles were pelleted (52,000 × g, 30 min) and washed three times with sterile 50 mm HEPES, pH 6.8. FITC-labeled vesicles were resuspended in Dulbecco's phosphate buffered saline supplemented with 0.2 m NaCl and checked for sterility and protein concentration. Ail Confirmation—To confirm that the 17-kDa band detected in DH5α/Ail vesicles by Coomassie stained SDS-PAGE was Ail, purified DH5α/Ail vesicles (15 μg) were loaded on a 15% SDS-PAGE and transferred to polyvinylidene difluoride, and the N-terminal sequence of the 17-kDa band was determined to be Ala-Ser-Glu-Asn-Ser-Ile-Ser-Ile (Tufts Core Facility). Cargo Quantitation—To determine the percent of GFP or Ail packaged in vesicles, bacteria were pelleted from a culture grown either overnight (Ail) or induced for 6 h (Tat-GFP/TatABCE). Vesicles were pelleted as described above and filter-sterilized. Bacteria and vesicles were precipitated with trichloroacetic acid (20% final concentration; 4 °C, 30 min), and the proteins were pelleted (10 min, 16,000 × g) and washed with acetone. Samples were then resuspended in 1% SDS in phosphate-buffered saline. Vesicle samples and dilutions of the bacterial preparation (1:300 for Ail; 1:1000 for GFP) were analyzed by SDS-PAGE and Coomassie staining (Ail) or immunoblotting (GFP). Densitometry results were quantified using NIH Image. Periplasm and Outer Membrane Preparations—To obtain periplasm from DH5α, the culture was pelleted, resuspended in 20% sucrose in 20 mm Tris, pH 8.0 (4 ml/g cells), and 0.1 m EDTA (0.2 ml/g cells), and lysozyme (600 μg/g cells) were added. Following 40 min incubation on ice, 0.5 m MgCl2 (0.16 ml/g cells) was added, and the spheroplasts were pelleted (20 min, 9500 g). Periplasm was collected from 43886 strains using the polymyxin B method as described previously (2.Horstman A.L. Kuehn M.J. J. Biol. Chem. 2000; 275: 12489-12496Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Outer membranes were purified using the Sarkosyl method as described previously (24.Nikaido H. Methods Enzymol. 1994; 235: 225-234Crossref PubMed Scopus (87) Google Scholar). To purify outer membrane from DH5α strains with and without Ail, spheroplasts collected from the periplasm preparation were resuspended in ice-cold 10 mm Tris (pH 8) and sonicated, and the cells were pelleted (5 min, 8,000 × g). Whole membranes were pelleted from the supernatants (60 min; 40,000 × g) and washed in 10 mm Tris, pH 8, before being resuspended in distilled water and freezethawed. The membranes were then incubated in 0.5% Sarkosyl (sodium N-lauroylsarcinosinate; 25 °C, 20 min), and the outer membrane was pelleted (90 min, 40,000 × g). Equal volumes of sample were separated by SDS-PAGE and immunoblotted for GFP or MutL. Pronase Protection Assay—Bacterial membrane integrity was determined using a RNase detection assay as described previously (25.Bernadac A. Gavioli M. Lazzaroni J.C. Raina S. Lloubes R. J. Bacteriol. 1998; 180: 4872-4878Crossref PubMed Google Scholar). To detect lumenal GFP, 3 μg of 43886/Tat-GFP/TatABCE vesicles were incubated (37 °C, 30 min) with 0.1 mg/ml Pronase (Roche Molecular Biochemicals) followed by Complete protease inhibitor (Roche; 37 °C, 5 min). As a control, vesicles were solubilized with 1% SDS prior to incubation with Pronase. Immunoblot Analysis—Samples of vesicles or outer membranes were separated using 15% SDS-PAGE. Proteins were transferred to polyvinylidene difluoride (Bio-Rad) and blocked in phosphate-buffered saline with 1% Tween 20 and 3% nonfat dry milk. Membranes were immunoblotted for GFP (rabbit anti-GFP; 1:1000), MutL (rabbit anti-MutL; 1:1000), maltose binding protein (rabbit anti-MBP; 1:6000; New England Biolabs), or E. coli outer membrane antigens (rabbit anti-OM; 1:1000) and detected using horseradish peroxidase-anti-rabbit IgG antibody (1:10,000), ECL reagents (Amersham Biosciences), and autoradiography. Negative Staining Electron Microscopy—To visualize vesicles, samples were applied to carbon-coated 400-mesh copper grids (Electron Microscopy Sciences) and stained with 2% uranyl acetate. Quantitative Fluorescence Assay—To quantitate the amount of vesicles bound by cells, a 96-well fluorescent assay was used. CHO cells were plated at a concentration of 8 × 104 cells/well and incubated overnight (37 °C, 5% CO2) to allow cell adherence. Cells were washed with Hanks' buffer (2×) and incubated with FITC-labeled vesicles (1 μg) in serum- and antibiotic-free media (100 μl). Following incubation, wells were washed with Hanks' (2×) to remove unbound vesicles, and 100 μl of Hanks' containing 1% Triton X-100 was added. Fluorescence was quantitated using a 96-well plate FLUOstar Galaxy fluorometer (BMG Lab Technologies) with excitation at 485 nm and emission at 520 nm. Relative fluorescence units were converted to micrograms of vesicle protein using separate standard curves that established the relative fluorescence units per microgram values of FITC-DH5α vesicles and FITC-DH5α Ail vesicles (r = 0.99), which were present on each microtiter plate tested. Fluorescence and Immunofluorescence Microscopy—CHO or HT29 (1.6 × 105 cells/well) cells were plated on Permanox microwell chamber slides (Nunc Inc.) and incubated (37 °C) with vesicles (1–5 μg/well) for various times in serum- and antibiotic-free media. Unbound vesicles were then removed, and the cells were washed. Cells were fixed with 4% paraformaldehyde. For immunofluorescence, cells were then permeabilized and blocked for 30 min with 0.1% Triton-X, 5% goat serum, and 0.1% bovine serum albumin. Cells were then incubated for 1 h with rabbit anti-GFP antibody (2.5 μg/ml) and visualized with rhodamine red-X-labeled goat anti-rabbit antibody (2.5 μg/ml; Jackson ImmunoResearch Laboratories). Slides were mounted with a coverslip and Pro-Long Antifade kit (Molecular Probes), and the cells were observed using a Nikon Eclipse TE200 laser-scanning confocal microscope. 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.Miller V.L. Falkow S. Infect. Immun. 1988; 56: 1242-1248Crossref PubMed Google Scholar). Ail was detected in purified outer membrane preparations and in vesicles produced by DH5α/Ail (Fig. 1, A and B), MC4100/Ail, and HB101/Ail (data not shown), and its presence in vesicles was confirmed by N-terminal sequencing. Co-fractionation in Optiprep density gradients of Ail with OmpA and OmpF/C, outer membrane components of native vesicles (Fig. 2, A and B), showed that Ail was shed into the culture supernatant in association with other outer membrane vesicle components. Electron microscopy examination of vesicles in low density Optiprep fractions revealed that >90% were closed vesicles (Fig. 2, C and D), not membrane whirls or fragments of lysed vesicles. These data demonstrated that the heterologous outer membrane proteins expressed in E. coli are present in native-like vesicles.Fig. 2Ail copurifies with vesicle markers and does not increase vesicle density. Coomassie-stained SDS-PAGE of fractions of increasing density (left to right) of DH5α (A) and DH5α/Ail (B) vesicles from a discontinuous Optiprep gradient (25–50%). Positions of OmpF/C (39 kDa), OmpA (35 kDa), and Ail (17 kDa) are indicated. Negative staining electron microscopy of purified DH5α (C) and DH5α/Ail (D) vesicles. Size bar applies to panels C and D.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To investigate the effect of a heterologous protein on yield, we compared vesicle production as a function of protein concentration per colony forming unit. After overnight growth, DH5α/Ail produced 2.3-fold more vesicles per colony forming unit than DH5α. A difference in vesicle density was not detected, because vesicles with Ail migrated to the same density fraction as DH5α vesicles (see peak vesicle fractions 4 and 5, Fig. 2, A and B). In addition, no distinguishable difference was detected in the vesicles by negative staining electron microscopy: DH5α vesicles ranged from 22–90 nm (Fig. 2C), and DH5α/Ail vesicles ranged from 22–77 nm (Fig. 2D) in diameter. Therefore, outer membrane vesicle density and size were unaltered due to the incorporation of a heterologous protein into the membrane, but vesicle yield increased. To determine whether Ail was incorporated differently into vesicles than endogenous outer membrane cargo, we compared the amounts of Ail, OmpF/C, and OmpA packaged into vesicles (Fig. 3). Of the total amount in the bacteria, 0.32% of the Ail, 0.23% of OmpF/C, and 0.14% of the OmpA were packaged into vesicles, which falls in the previously reported range of protein packaged in E. coli vesicles (26.Mug-Opstelten D. Witholt B. Biochim. Biophys. Acta. 1978; 508: 287-295Crossref PubMed Scopus (46) Google Scholar, 27.Hoekstra D. van der Laan J.W. de Leij L. Witholt B. Biochim. Biophys. Acta. 1976; 455: 889-899Crossref PubMed Scopus (166) Google Scholar). In addition, the Omp to Ail ratio appeared constant in each fraction (0.5 + 0.03 S.E., fractions 2–7; Fig. 2B). Therefore, heterologous Ail is neither selectively enriched nor selectively excluded from vesicles, and Ail appears to be included in every vesicle. Ail Induces Vesicle Internalization by Eukaryotic Cells— Our previous work has demonstrated that LT, which is externally bound to vesicles because of its association with lipopolysaccharide, induces the association and internalization of vesicles by eukaryotic cells (2.Horstman A.L. Kuehn M.J. J. Biol. Chem. 2000; 275: 12489-12496Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 28.Horstman A.L. Kuehn M.J. J. Biol. Chem. 2002; 277: 32538-32545Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar).2 Because Ail is a known adhesin/invasin that can confer an invasive phenotype to HB101 (18.Miller V.L. Falkow S. Infect. Immun. 1988; 56: 1242-1248Crossref PubMed Google Scholar), we wanted to determine whether Ail is able to catalyze the internalization of DH5α vesicles. We used fluorescently labeled vesicles to study cell association. Wild type DH5α vesicles displayed minimal eukaryotic cell association (Fig. 4A); however, the presence of Ail in vesicles increased cell association dramatically (Fig. 4B). Quantitation of cell-associated fluorescence revealed that the presence of Ail increased DH5α vesicle-cell association 10-fold (Fig. 4C) and that Ail-dependent vesicle-cell association was vesicle concentration-dependent (data not shown). Interestingly, the unbound DH5α/Ail vesicles removed from the eukaryotic cells contained Ail as shown by Coomassie staining (data not shown). This further supported the finding that Ail was present in every vesicle, because Ail would be expected to be depleted from this fraction if the vesicles contained heterogeneous cargo populations. These data demonstrate that an exogenous outer membrane protein packaged in vesicles can alter the adherence properties of the vesicles. GFP Transport to the Periplasm Is Limited by Endogenous Levels of TatABCE—Next we wanted to determine whether an exogenous periplasmic protein, which could be utilized in future studies as a lumenal vesicle marker, would also be packaged in vesicles. Due to the reducing environment of the periplasm, GFP transported via the Sec pathway is unable to fold in the periplasm (29.Feilmeier B.J. Iseminger G. Schroeder D. Webber H. Phillips G.J. J. Bacteriol. 2000; 182: 4068-4076Crossref PubMed Scopus (284) Google Scholar); however, GFP folded in the cytoplasm can be transported to the periplasm by the twin arginine transporter system (Tat) using the Tat signal sequence (19.Thomas J.D. Daniel R.A. Errington J. Robinson C. Mol. Microbiol. 2001; 39: 47-53Crossref PubMed Scopus (230) Google Scholar, 31.Santini C.L. Bernadac A. Zhang M. Chanal A. Ize B. Blanco C. Wu L.F. J. Biol. Chem. 2001; 276: 8159-8164Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). DH5α and 43886, a pathogenic E. coli strain, were transformed with a plasmid encoding IPTG-inducible GFP (20.Cormack B.P. Valdivia R.H. Falkow S. Gene. 1996; 173: 33-38Crossref PubMed Scopus (2474) Google Scholar) fused to the Tat signal sequence (Tat-GFP). As shown previously for Tat-GFP expressed in MC4100 (19.Thomas J.D. Daniel R.A. Errington J. Robinson C. Mol. Microbiol. 2001; 39: 47-53Crossref PubMed Scopus (230) Google Scholar, 31.Santini C.L. Bernadac A. Zhang M. Chanal A. Ize B. Blanco C. Wu L.F. J. Biol. Chem. 2001; 276: 8159-8164Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar), we found that induction of Tat-GFP expression increased the concentration of periplasmic GFP (data not shown). Although GFP-associated fluorescence was detectable, the amount of periplasmic GFP was saturable and could not be increased with longer induction (data not shown). The Tat machinery (TatABCE) has previously been shown to limit the transport of Tat substrate SufI into the periplasm (21.Yahr T.L. Wickner W.T. EMBO J. 2001; 20: 2472-2479Crossref PubMed Scopus (134) Google Scholar, 32.Sargent F. Gohlke U. De Leeuw E. Stanley N.R. Palmer T. Saibil H.R. Berks B.C. Eur. J. Biochem. 2001; 268: 3361-3367Crossref PubMed Scopus (125) Google Scholar). To determine whether this was the limiting factor in Tat-GFP transport, bacteria expressing Tat-GFP were transformed with a plasmid encoding IPTG-inducible TatABCE. Both periplasmic fluorescence (Fig. 5A) and immunoblot analysis for GFP (data not shown) revealed a significant increase of periplasmic GFP when exogenous TatABCE expression was induced. Periplasmic GFP was detectable in DH5α and 43886 after induction of Tat-GFP and TatABCE (Fig. 5, B and C). In the periplasm, the Tat signal sequence is cleaved from Tat-GFP to yield the mature 27-kDa form of GFP. Bands with slightly higher molecular weights were seen in the spheroplasts, which may represent immature forms of GFP (Fig. 5B). After induction for 6 h, 91 and 86% of the GFP was periplasmic in DH5α/Tat-GFP/TatABCE and 43886/Tat-GFP/TatABCE, respectively. Immunoblot analysis for MutL, a cytoplasmic protein, confirmed that the spheroplasts were intact and did not leak cytoplasmic components into the periplasmic preparation (Fig. 5, B and C). These results demonstrated a periplasmic localization of fluorescent GFP in laboratory and pathogenic strains of E. coli that is limited by the expression level of the Tat transporter. GFP in the Periplasm Is Packaged into Vesicles—Because Tat-GFP was transported and properly folded in the periplasm, we wanted to determine whether it was packaged into vesicles. Vesicles were purified from cultures of strains expressing Tat-GFP with and without TatABCE after 6 and 12 h of induction. GFP was detectable in both DH5α- and 43886-derived vesicles when TatABCE was expressed (Fig. 6, A and B). Low levels of GFP were detected in the vesicles of 43886 cells expressing only endogenous levels of TatABCE (Fig. 6B). Furthermore, GFP cofractionated with vesicles on an Optiprep gradient during vesicle purification, as observed by the presence of OmpF/C, OmpA, and GFP in the same fractions (Fig. 7A, fractions 4–6). In contrast, soluble GFP and maltose-binding protein (MBP) (Fig. 7B, fractions 8 and 9) or GFP and MBP from lysed vesicles (data not shown) remained in the heavy fractions in an Optiprep gradient. These data showed that heterologously expressed periplasmic GFP copurified during vesicle purification is associated with intact E. coli vesicles.Fig. 7GFP cofractionates with vesicle Omps, and GFP-containing vesicles are slightly denser than native vesicles. Fractions of increasing density (left to right) of vesicles (A) from 43886/Tat-GFP/TatABCE and 43886 or soluble periplasm (B) from 43886/Tat-GFP/TatABCE using a discontinuous Optiprep gradient (25–50%) were applied to SDS-PAGE and immunoblotted with anti-GFP (upper panel; A and B), Coomassie stained (lower panels; A), or immunoblotted with anti-MBP (lower panel; B). Positions of OmpF/C, OmpA, GFP, and MBP are indicated. Negativ