Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2012 Dec 18.
Published in final edited form as: J Biol Chem. 2003 Oct 24;279(3):2069–2076. doi: 10.1074/jbc.M307628200

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

Nicole C Kesty 1, Meta J Kuehn 1,‡
PMCID: PMC3525464  NIHMSID: NIHMS420868  PMID: 14578354

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 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, 11–16).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 studies have suggested that outer membrane invasins IpaB, C, and D may catalyze the internalization S. flexneri vesicles (16).

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 (15–17). 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.

EXPERIMENTAL PROCEDURES

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). 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) and inserted into pTrc99A (Amersham Biosciences) using EcoRI and HindIII. The mutGFP3 sequence was replaced with mutGFP2 (20) using MscI and HindIII. pNKTAT (TatABCE) was constructed by partial digestion of pTatABCE (21) using EcoRI and HindIII and insertion into the multiple cloning site of pK187 (22). 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).

Vesicle Purification and Labeling

Vesicles were purified as described previously (2) 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).

Outer membranes were purified using the Sarkosyl method as described previously (24). 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 freeze-thawed. 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). 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 micro-titer 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 ProLong Antifade kit (Molecular Probes), and the cells were observed using a Nikon Eclipse TE200 laser-scanning confocal microscope.

RESULTS

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 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. 1. Ail is detected in the outer membrane and in vesicles produced by DH5α/Ail.

Fig. 1

Open in a new tab

Coomassie-stained SDS-PAGE of 1-μg outer membrane (OM) fraction (A) and purified vesicles (B) from DH5α (1.5 μg) and DH5α/Ail (2 μg). Arrowheads indicate OmpF/C (39 kDa), OmpA (35 kDa), and Ail (17 kDa).

Fig. 2. Ail copurifies with vesicle markers and does not increase vesicle density.

Fig. 2

Open in a new tab

Coomassie-stained SDS-PAGE of fractions of increasing density (left to right) of DH5α (A) and DH5α/Ail (B) vesicles from a discontinuous Opti-prep 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.

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 DH5αvesicles (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: DH5αvesicles 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, 27). 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.

Fig. 3. Ail and endogenous Omps are packaged equally into vesicles.

Fig. 3

Open in a new tab

Coomassie-stained SDS-PAGE of DH5α/Ail cell lysate (C) and DH5α/Ail vesicles (V). Positions of OmpF/C, OmpA and Ail are indicated. Cargo incorporation was determined by densitometry.

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, 28).2 Because Ail is a known adhesin/invasin that can confer an invasive phenotype to HB101 (18), 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.

Fig. 4. FITC-labeled DH5α/Ail vesicles associate with CHO cells.

Fig. 4

Open in a new tab

Confocal microscopy of CHO cells incubated for 3 h with FITC-DH5α (A) and FITC-DH5α/Ail (B) vesicles. Selected cells from top panels (box) are magnified in bottom panels. Magnification is indicated for pairs of upper and lower panels. C, quantitation of cell-associated vesicles (ng of vesicles per well) following a 3-h incubation of CHO cells with 5 μg of FITC-DH5α/Ail (gray) and FITC-DH5α (black) vesicles. Error bars are ± S.E. Significant difference (*) was determined by Student’s t test (p < 0.001, n ≥ 2). Data are representative of two trials done in duplicate.

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); 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, 31).

DH5α and 43886, a pathogenic E. coli strain, were transformed with a plasmid encoding IPTG-inducible GFP (20) fused to the Tat signal sequence (Tat-GFP). As shown previously for Tat-GFP expressed in MC4100 (19, 31), 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, 32). 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.

Fig. 5. Tat-GFP is transported to the periplasm in DH5α and 43886.

Fig. 5

Open in a new tab

A, TatABCE increases Tat-GFP transport to the periplasm of DH5α. Data are represented as fold increase of fluorescence (RFU, relative fluorescence unit) in DH5α/Tat-GFP periplasm ± S.E. with induction of TatABCE using 0.1 mM IPTG for 6 h. Significant difference (*) was determined by Student’s t test (p < 0.002; n = 6). B, anti-GFP (upper) and anti-MutL (lower) immunoblot analysis of periplasm and spheroplasts for DH5α/Tat-GFP/TatABCE and spheroplasts for DH5α following induction with 0.1 mM IPTG for indicated times. C, anti-GFP (upper) and anti-MutL (lower) immunoblot analysis of periplasm and spheroplasts for 43886/Tat-GFP/TatABCE and spheroplasts for 43886 following induction as described for panel B. Positions of mature GFP (27 kDa) and MutL (68 kDa) are indicated.

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. 6. GFP is packaged into vesicles from bacteria overexpressing TatABCE.

Fig. 6

Open in a new tab

A, anti-OM (upper) and anti-GFP (lower) immunoblot of 1-μg vesicles from DH5α/Tat-GFP, DH5α/Tat-GFP/TatABCE, and DH5α. Hours of induction with 0.1 mM IPTG are indicated. B, Coomassie-stained SDS-PAGE (upper) and anti-GFP immunoblot (lower) of 10 μg vesicles from 43886/Tat-GFP, 43886/Tat-GFP/TatABCE, and 43886. Positions of an outer membrane antigen (OM Ag, 34 kDa), OmpF/C, OmpA, and GFP are indicated.

Fig. 7. GFP cofractionates with vesicle Omps, and GFP-containing vesicles are slightly denser than native vesicles.

Fig. 7

Open in a new tab

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. Negative staining electron microscopy of purified vesicles from DH5α (C) and 43886 (D) strains without (left) and with (right) TatGFP/TatABCE is shown. Size bars are shown for each pair.

43886 vesicles containing GFP were slightly more dense than those without GFP, because GFP and OmpF/C and OmpA were detected in more dense fractions of the gradient (Fig. 7A; compare Omp peak in fractions 4 and 5, middle panel, with peak in fraction 3, lower panel). Similar results were seen with DH5α vesicles containing GFP (data not shown). Although the difference in density due to the presence of lumenal GFP in vesicles suggested that the lipid to protein ratio was altered (Fig. 7A), we did not detect a significant difference in the size of vesicles purified from strains with and without Tat-GFP/TatABCE (Fig. 7, C and D). 43886 vesicles ranged from 43–165 nm and 43886/Tat-GFP/TatABCE ranged from 20 –165 nm in diameter, whereas DH5α vesicles ranged from 22–90 nm and DH5α/Tat-GFP/TatABCE ranged from 25–90 nm in diameter. Note that native DH5α vesicles are approximately one-half the size of 43886 vesicles (Fig. 7, C and D, left panels). Unlike the effect of a heterologous outer membrane protein, inclusion of GFP did not affect bacterial vesicle yield.

We determined that GFP in the culture supernatant was not present as a result of reduced membrane integrity. Unlike bacterial cultures of strains expressing cytoplasmic GFP (data not shown), an RNase detection assay confirmed that bacteria expressing Tat-GFP/TatABCE were not leaking periplasm into the culture supernatant. We had previously determined that vesicles remain intact and do not leak periplasmic components when stored in 0.2 M NaCl or when osmotically buffered in Optiprep (data not shown). To establish whether GFP was a lumenal component of vesicles rather than attached to their extracellular surface, Pronase was added to intact and solubilized vesicles. GFP was protected from degradation in unsolubilized vesicles, demonstrating that periplasmic GFP is in the lumen of 43886 vesicles (Fig. 8A). Similar results were observed with the endogenous lumenal vesicle component, MBP (Fig. 8A).

Fig. 8. GFP is a lumenal component of vesicles.

Fig. 8

Open in a new tab

A, anti-GFP immunoblot analysis (upper panel), anti-MBP immunoblot analysis (middle panel), and Coomassie-stained SDS-PAGE (middle panel) of 10 μg 43886/Tat-GFP/TatABCE vesicles incubated with and without Pronase for 30 min. As a control, vesicles were lysed with 1% SDS before Pronase treatment. B, anti-GFP (upper panel) and anti-MBP (lower panel) immunoblot analysis of 43886/Tat-GFP/TatABCE cell lysates (C) and vesicles (V). Arrowheads indicate OmpF/C, GFP, and MBP (42 kDa). Cargo packaging was determined by densitometry.

To determine whether GFP was incorporated into vesicles to the same extent as endogenous periplasmic cargo, we compared the amounts of GFP and MBP packaged in vesicles (Fig. 8B). Of the total amount in the bacteria, 0.1% of both GFP and MBP were packaged into 43886 vesicles. As with Ail, we were unable to detect any vesicles that did not contain GFP, and subsets of vesicles from 43886/Tat-GFP/TatABCE fractionated by density (lanes 3–6; Fig. 7A) all contained the same ratio of Omps to GFP. Therefore, periplasmic GFP is neither selectively enriched nor selectively excluded from vesicles, and GFP appears to be included in every vesicle.

GFP-containing 43886 Vesicles Interact with Eukaryotic Cells

In previous studies, we have examined the internalization of ETEC vesicles by human colorectal (HT29) cells.2 ETEC vesicles interact with HT29 cells via LT, and FITC-labeled outer membrane vesicle components other than LT were internalized. One goal of packaging GFP inside vesicles was to utilize GFP as a lumenal marker during internalization and trafficking experiments of ETEC vesicles. GFP-containing vesicles were fluorescent; however, the cell-associated fluorescence of GFP-containing vesicles was barely above background, unlike the amount of cell-associated fluorescence of vesicles externally labeled with FITC used in the quantitative assay for Ail-containing vesicles. The fluorescence of the GFP was also too low for confocal microscopy experiments, although punctate green staining was visible (data not shown). Nevertheless, we wanted to determine whether GFP, a lumenal vesicle component, was associated with cells. Therefore, immunofluorescence was used to enhance the GFP signal. Bright punctate staining was observed in HT29 cells incubated with purified vesicles from 43886/Tat-GFP/TatABCE (Fig. 9A), but not from DH5α/Tat-GFP/TatABCE (Fig. 9B). These results demonstrated that lumenal contents are present in cell-associated vesicles and that GFP can be utilized as a lumenal vesicle marker for intracellular trafficking.

Fig. 9. GFP-containing 43886 vesicles associate with eukaryotic cells.

Fig. 9

Open in a new tab

Confocal microscopy of HT29 cells incubated for 8 h with 5 μg of GFP-containing 43886 (A) and DH5α (B) vesicles visualized using anti-GFP and a rhodamine-labeled secondary antibody. Selected cells from upper panels (boxed) are magnified in bottom panels. Magnification is indicated for pairs of upper and lower panels.

DISCUSSION

Vesicles are ubiquitously shed by Gram-negative bacteria and have been shown to be vehicles for intercellular transport. They are particularly significant to the study of pathogenic bacteria because they contain and deliver virulence factors to host cells. To understand vesicle formation and vesicle-mediated transport and trafficking inside the host cell, we need to manipulate both vesicle adhesins and vesicle content. Here we show that both membrane and lumenal protein contents of vesicles produced by laboratory and pathogenic strains of E. coli can be manipulated such that native vesicles contain active heterologous factors.

Ail is a protein from Y. enterocolitica that is abundantly expressed in the outer membrane of E. coli. We demonstrate that Ail is also packaged in E. coli vesicles without altering the size or shape of the vesicles. Ail expression allows HB101, a normally non-invasive E. coli strain, to invade CHO cells (18). Similarly, we found that Ail increased DH5α vesicle association with CHO cells and that the DH5α/Ail vesicles appeared by microscopy z-sectioning to be internalized. Thus, engineered vesicles can be used to investigate host cell association and trafficking mediated by specific outer membrane protein adhesins that are active and presented in their native membrane environment. Although whole bacteria pathogens may activate potentially interesting regulatory effects, vesicles from engineered strains can provide a valuable, novel tool in studying a specific outer membrane protein in its native, membrane context. Vesicles provide a “constant expression” level of an Omp, which can be particularly useful in its initial characterization, whereas changes in the bacterial outer membrane composition induced in response to host cells could confuse results. Thus, we propose that the initial interaction and subsequent cellular response due to a specific surface component can be studied using heterologous proteins incorporated in vesicles.

In addition to modifying vesicle surface components, we were interested in changing the soluble, lumenal cargo of vesicles to contain a fluorescent marker, GFP. Previously, GFP fused to the Tat signal sequence has been shown to be transported to the periplasm by the Tat pathway in the E. coli laboratory strain MC4100 (19, 31). We demonstrate here that Tat-GFP can also be used in pathogenic E. coli strains. The transport of the SufI Tat substrate to the periplasm was shown to depend on the level of Tat expression in the cell (21, 32). Likewise, we show that the Tat transporter is limiting for the Tat-GFP substrate in both the lab strains and the pathogenic strains of E. coli. We further observed that DH5α expressing Tat-GFP without TatABCE overexpression appeared filamentous, much like the ΔTatC mutants (33), perhaps because overloading the Tat transporter with the fusion protein resulted in the lack of a functional Tat transporter for natural Tat substrates.

Interestingly, the DH5α/Tat-GFP/TatABCE spheroplasts did not have any detectable fluorescence over that of background, very little cytoplasmic Tat-GFP was detectable by immunoblotting, and, at endogenous (low) levels of TatABCE, spheroplasts contained little to no cytoplasmic GFP (data not shown). However, we did detect fluorescence and GFP in spheroplasts of 43886/Tat-GFP/TatABCE. Because 43886 is more difficult to lyse than DH5α, we used polymyxin B to purify periplasm. Thus, whereas the spheroplasts’ content did not leak into the periplasmic preparation (as determined by MutL immunoblotting), it was not determined whether the spheroplast fraction contained unlysed cells that could explain the GFP detected in this fraction.

To determine whether periplasmic GFP entered vesicles, we first showed that crude and gradient-purified vesicle preparations from TatABCE-overexpressing bacteria contained GFP. However, it was possible that this associated GFP cofractionated with vesicles because it was bound to their exterior. This was of particular concern, because bacteria expressing cytosolic GFP were highly osmotically sensitive (20). We found that periplasmic GFP did not decrease outer membrane integrity and that vesicle GFP was protected from Pronase unless the vesicles were solubilized with SDS. These results proved that GFP was lumenally packaged in vesicles.

GFP-containing vesicles were denser; however, the average vesicle size did not change. This result suggested that the protein-lipid ratio increased, presumably as a result of the inclusion of heterologous soluble cargo. Unlike the GFP-containing vesicles, there did not appear to be a density difference in DH5α/Ail vesicles. Both DH5α and 43886 package outer membrane proteins into vesicles to the same extent. Of total cellular OmpF/C, DH5α and 43886 package 0.23% and 0.17% in vesicles, respectively, whereas 0.14% of OmpA is packaged by DH5α, and 0.15% by 43886. These data correspond to previously reported values (26, 27). Therefore, there may be an optimal protein-lipid ratio within the outer membrane, even when an abundant heterologous membrane protein is present. Although the lipid to protein ratio appeared unaltered, the vesicle yield was slightly increased with the incorporation of Ail. However, induction of GFP, which altered vesicle density, did not change vesicle yield. We conclude that heterologous lumenal and outer membrane cargo may affect vesiculation differently.

Unfortunately, the intrinsic fluorescence of vesicles containing GFP was barely above background fluorescence and could not be used to detect cell-associated vesicles. However, GFP inside 43886 vesicles could be detected in vesicle-treated HT29 cells by amplifying GFP detection with antibodies to GFP, showing that GFP can be used as a lumenal marker protein. More importantly, this result demonstrates that ETEC vesicles carry lumenal components into eukaryotic cells.

In eukaryotic cells, signal sequences or protein modifications are necessary signals for protein sorting into different vesicles and organelles. For example, the endoplasmic reticulum retention signal sequence, KDEL, signals for retrograde transport from the Golgi to the ER (34), whereas exosomes are enriched in ubiquitinated proteins (35, 36). Bacterial protein secretion through the inner membrane, such as by the Tat and the sec pathways, also utilizes signal sequences (19, 37, 38). Currently, little is known about protein sorting into outer membrane vesicles produced by bacteria. Previous studies have reported that certain proteins such as LT, leukotoxin, and ClyA are enriched in vesicles, whereas other periplasmic proteins, such as DsbA, are excluded, suggesting that vesiculation is an active and directed process (2, 6, 8). In this study, the percentage of Ail or GFP packaged in vesicles corresponded to the amount of endogenous outer membrane and periplasmic components packaged in vesicles as well as to previous reports of the percentage of protein packaged in E. coli vesicles (26, 27). Presumably, GFP would not contain a vesicle “sorting sequence,” since it is produced by the jellyfish Aequorea victoria and is not a native bacterial protein. These data suggest that a cargo tag is not necessary for protein sorting into bacterial vesicles. Instead, we propose that the previously described toxin enrichment in vesicles may be due to cell envelope “hot spots” for vesicle budding. These enriched areas may occur due to the location of protein secretion machinery, such as the type II secretion machinery necessary for LT secretion through the inner and outer membranes (28, 30, 39).

In conclusion, heterologous outer membrane and periplasmic proteins, such as Ail and periplasmic GFP, are packaged in vesicles and can be used to modify the characteristics of the vesicle. Outer membrane and external proteins such as Ail and LT can be used to direct the interactions of vesicles with eukaryotic cells, and GFP and other lumenal vesicle cargo can be used to track soluble transported cargo. Together, these tools provide valuable methods to study the nature of vesicle-mediated transport.

Acknowledgments

We thank S.J. Bauman for the electron microscopy, J. Qiu for help with transformations and vesicle purifications, V. Miller for HB101/pVM102, C. Robinson for plasmid pJDT1, B. Cormack for plasmid pEGFP2, T. Yahr for plasmid pTatABCE, M. Jobling and R. Holmes for plasmid pK187, A. Johnson for anti-OM antibody, P. Modrich for anti-MutL antibody, P. Silver for anti-GFP antibody, S. Abraham for confocal microscopy facilities, and M. Reedy and C. Lucaveche for electron microscopy training and facilities.

Footnotes

1

The 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.

2

N. C. Kesty, K. M. Mason, and M. J. Kuehn, submitted for publication.

*

This work was supported by a Burroughs Wellcome Fund Investigator in Pathogenesis of Infectious Disease Award (to M. J. K.), a National Institutes of Health Grant, and an American Lung Association Research Grant.

References

  • 1.Beveridge TJ. J Bacteriol. 1999;181:4725– 4733. doi: 10.1128/jb.181.16.4725-4733.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Horstman AL, Kuehn MJ. J Biol Chem. 2000;275:12489–12496. doi: 10.1074/jbc.275.17.12489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Devoe IW, Gilchrist JE. J Exp Med. 1973;138:1156–1167. doi: 10.1084/jem.138.5.1156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Keenan J, Day T, Neal S, Cook B, Perez-Perez G, Allardyce R, Bagshaw P. FEMS Microbiol Lett. 2000;182:259–264. doi: 10.1111/j.1574-6968.2000.tb08905.x. [DOI] [PubMed] [Google Scholar]
  • 5.Fiocca R, Necchi V, Sommi P, Ricci V, Telford J, Cover TL, Solcia E. J Pathol. 1999;188:220–226. doi: 10.1002/(SICI)1096-9896(199906)188:2<220::AID-PATH307>3.0.CO;2-C. [DOI] [PubMed] [Google Scholar]
  • 6.Kato S, Kowashi Y, Demuth DR. Microb Pathog. 2002;32:1–13. doi: 10.1006/mpat.2001.0474. [DOI] [PubMed] [Google Scholar]
  • 7.Wai SN, Takade A, Amako K. Microbiol Immunol. 1995;39:451– 456. doi: 10.1111/j.1348-0421.1995.tb02228.x. [DOI] [PubMed] [Google Scholar]
  • 8.Wai SN, Lindmark B, Soderblom T, Takade A, Westermark M, Oscarsson J, Jass J, Richter-Dahlfors A, Mizunoe Y, Uhlin BE. Cell. 2003;115:25–35. doi: 10.1016/s0092-8674(03)00754-2. [DOI] [PubMed] [Google Scholar]
  • 9.Yokoyama K, Horii T, Yamashino T, Hashikawa S, Barua S, Hasegawa T, Watanabe H, Ohta M. FEMS Microbiol Lett. 2000;192:139–144. doi: 10.1111/j.1574-6968.2000.tb09372.x. [DOI] [PubMed] [Google Scholar]
  • 10.Kolling GL, Matthews KR. Appl Environ Microbiol. 1999;65:1843–1848. doi: 10.1128/aem.65.5.1843-1848.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Demuth DR, James D, Kowashi Y, Kato S. Cell Microbiol. 2003;5:111–121. doi: 10.1046/j.1462-5822.2003.00259.x. [DOI] [PubMed] [Google Scholar]
  • 12.Li Z, Clarke AJ, Beveridge TJ. J Bacteriol. 1998;180:5478–5483. doi: 10.1128/jb.180.20.5478-5483.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Beermann C, Wunderli-Allenspach H, Groscurth P, Filgueira L. Cell Immunol. 2000;201:124–131. doi: 10.1006/cimm.2000.1640. [DOI] [PubMed] [Google Scholar]
  • 14.Shoberg RJ, Thomas DD. Infect Immun. 1993;61:3892–3900. doi: 10.1128/iai.61.9.3892-3900.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Kadurugamuwa JL, Beveridge TJ. J Bacteriol. 1996;178:2767–2774. doi: 10.1128/jb.178.10.2767-2774.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Kadurugamuwa JL, Beveridge TJ. Antimicrob Agents Chemother. 1998;42:1476–1483. doi: 10.1128/aac.42.6.1476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Kadurugamuwa JL, Beveridge TJ. J Bacteriol. 1995;177:3998– 4008. doi: 10.1128/jb.177.14.3998-4008.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Miller VL, Falkow S. Infect Immun. 1988;56:1242–1248. doi: 10.1128/iai.56.5.1242-1248.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Thomas JD, Daniel RA, Errington J, Robinson C. Mol Microbiol. 2001;39:47–53. doi: 10.1046/j.1365-2958.2001.02253.x. [DOI] [PubMed] [Google Scholar]
  • 20.Cormack BP, Valdivia RH, Falkow S. Gene. 1996;173:33–38. doi: 10.1016/0378-1119(95)00685-0. [DOI] [PubMed] [Google Scholar]
  • 21.Yahr TL, Wickner WT. EMBO J. 2001;20:2472–2479. doi: 10.1093/emboj/20.10.2472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Jobling MG, Holmes RK. Nucleic Acids Res. 1990;18:5315–5316. doi: 10.1093/nar/18.17.5315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2. Cold Spring Harbor Press; Cold Spring Harbor, NY: 1989. pp. 1.82–1.84. [Google Scholar]
  • 24.Nikaido H. Methods Enzymol. 1994;235:225–234. doi: 10.1016/0076-6879(94)35143-0. [DOI] [PubMed] [Google Scholar]
  • 25.Bernadac A, Gavioli M, Lazzaroni JC, Raina S, Lloubes R. J Bacteriol. 1998;180:4872– 4878. doi: 10.1128/jb.180.18.4872-4878.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Mug-Opstelten D, Witholt B. Biochim Biophys Acta. 1978;508:287–295. doi: 10.1016/0005-2736(78)90331-0. [DOI] [PubMed] [Google Scholar]
  • 27.Hoekstra D, van der Laan JW, de Leij L, Witholt B. Biochim Biophys Acta. 1976;455:889– 899. doi: 10.1016/0005-2736(76)90058-4. [DOI] [PubMed] [Google Scholar]
  • 28.Horstman AL, Kuehn MJ. J Biol Chem. 2002;277:32538–32545. doi: 10.1074/jbc.M203740200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Feilmeier BJ, Iseminger G, Schroeder D, Webber H, Phillips GJ. J Bacteriol. 2000;182:4068– 4076. doi: 10.1128/jb.182.14.4068-4076.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Tauschek M, Gorrell RJ, Strugnell RA, Robins-Browne RM. Proc Natl Acad Sci U S A. 2002;99:7066–7071. doi: 10.1073/pnas.092152899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Santini CL, Bernadac A, Zhang M, Chanal A, Ize B, Blanco C, Wu LF. J Biol Chem. 2001;276:8159– 8164. doi: 10.1074/jbc.C000833200. [DOI] [PubMed] [Google Scholar]
  • 32.Sargent F, Gohlke U, De Leeuw E, Stanley NR, Palmer T, Saibil HR, Berks BC. Eur J Biochem. 2001;268:3361–3367. doi: 10.1046/j.1432-1327.2001.02263.x. [DOI] [PubMed] [Google Scholar]
  • 33.Stanley NR, Findlay K, Berks BC, Palmer T. J Bacteriol. 2001;183:139–144. doi: 10.1128/JB.183.1.139-144.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Pelham HR. Cell Struct Funct. 1996;21:413– 419. doi: 10.1247/csf.21.413. [DOI] [PubMed] [Google Scholar]
  • 35.Stoorvogel W, Kleijmeer MJ, Geuze HJ, Raposo G. Traffic. 2002;3:321–330. doi: 10.1034/j.1600-0854.2002.30502.x. [DOI] [PubMed] [Google Scholar]
  • 36.Thery C, Zitvogel L, Amigorena S. Nat Rev Immunol. 2002;2:569–579. doi: 10.1038/nri855. [DOI] [PubMed] [Google Scholar]
  • 37.Pugsley AP. Microbiol Rev. 1993;57:50–108. doi: 10.1128/mr.57.1.50-108.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Lory S. Curr Opin Microbiol. 1998;1:27–35. doi: 10.1016/s1369-5274(98)80139-2. [DOI] [PubMed] [Google Scholar]
  • 39.Scott ME, Dossani ZY, Sandkvist M. Proc Natl Acad Sci U S A. 2001;98:13978–13983. doi: 10.1073/pnas.241411198. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES