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Incorporation of Heterologous Outer Membrane and Periplasmic Proteins into Escherichiacoli Outer Membrane Vesicles*

N.C. Kesty and M.J. Kuehn‡

Duke University Medical Center, Department of Biochemistry, Durham, NC 27710

Running Title: Heterologous Proteins in Bacterial Vesicles

* This work was supported by a Burroughs Wellcome Fund Investigator in Pathogenesis of Infectious DiseaseAward and an American Lung Association Research Grant.‡ To whom correspondence should be addressed: Duke University Medical Center, Box 3711, Durham, NC 27710.Tel.: 919-684-2545; Fax: 919-684-8885; E. mail: meta.kuehn@duke.edu.

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on October 24, 2003 as Manuscript M307628200 by guest on D

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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 if 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 entercolitica,

was detected in purified outer membrane and in vesicles from E. coli strains DH5a, 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 if 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, DH5a, and the pathogenic enterotoxigenic E. coli (ETEC) 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 by electron microscopy.

These studies are the first to demonstrate the incorporation of heterologously expressed outer

membrane and periplasmic proteins into bacterial vesicles.

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Gram-negative bacteria secrete proteins solubly associated 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 which 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 intoxifies 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

1 The abbreviations used are: LT, heat-labile enterotoxin; ETEC, enterotoxigenic E. coli; GFP, green fluorescentprotein; IPTG, isopropyl-1-thio-b-D-galactopyranoside; MBP, maltose binding protein; RFU, relative fluorescenceunits; Tat, twin arginine transporter; OMP, outer membrane protein.

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treatment of cells with gentamicin, but they differed from native vesicles in their composition

and size (15-17). Since 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 if 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 entercolitica 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 DH5a (Statagene Subcloning), MC4100 and

ETEC (ATCC 43886) were grown in LB or CFA broth (1% casamino acids, 0.15% yeast extract,

0.005% MgSO4, 0.005% MnCl2), respectively. HB101/pVM102 (Ail) was kindly provided by

Dr. Virginia Miller (18). Strains were grown in the presence of kanamycin (10 mg/mL) and/or

ampicillin (100 mg/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

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

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solution (Sigma Cell Culture) and maintained in a humidified atmosphere containing 5% CO2 at

37ºC. 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 Pharmacia) 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 37ºC 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 previously described (2)

with the following modifications. Bacteria were pelleted (10 min, 10,000 x g) and vesicles were

then pelleted from the supernatant (1 h, 40,000 x g), filter sterilized (0.45 mm; 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 determined using Coomassie Plus

(Pierce). Vesicles were fluorescently labeled by diluting 1:1 with fluorescein isothiocyanate

(FITC) (1 mg/ml in 50 mM Na2CO3, 100 mM NaCl; pH 9.2) and incubated for 1 h at 25ºC.

Vesicles were pelleted (52,000 x 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.

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Ail Confirmation—To confirm that the 17 kDa band detected in DH5a/Ail vesicles by

Coomassie stained SDS-PAGE was Ail, purified DH5a/Ail vesicles (15 mg) were loaded on a

15% SDS-PAGE, transferred to PVDF 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; 4ºC; 30 min), the

proteins pelleted (10 min; 16000 x 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). Results were quantified using NIH Image.

Periplasm and Outer Membrane Preparations—To obtain periplasm from DH5a, 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 lysosyme (600 mg/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

previously described (2).

Outer membranes were purified using the sarkosyl method as previously described (24).

To purify outer membrane from DH5a strains with and without Ail, spheroplasts collected from

the periplasm preparation were resuspended in ice-cold 10 mM Tris, pH 8.0, sonicated and the

cells pelleted (5 min; 8000 g). Whole membranes were pelleted from the supernatants (60 min;

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40000 g) and washed in 10 mM Tris, pH 8.0 before resuspending 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; 40000 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 previously described (25). To detect lumenal GFP, 3 mg 43886/Tat-

GFP/TatABCE vesicles were incubated (37ºC; 30 min) with 0.1 mg/ml Pronase (Boehringer

Mannheim) followed by Complete protease inhibitor (Roche; 37ºC; 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 PVDF (BioRad) and blocked in PBS 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)

and autoradiography.

Negative Staining EM—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 x 104

cells/well and incubated overnight (37ºC, 5% CO2) to allow cell adherence. Cells were washed

with Hanks buffer (2x) and incubated in (100 ml) with FITC-labeled vesicles (1 mg) in serum-

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and antibiotic-free media. Following incubation, wells were washed with Hanks (2x) to remove

unbound vesicles and 100 mL Hanks containing 1% Triton X-100 was added. Fluorescence was

quantitated using a 96-well plate FLUOstar Galaxy fluorometer (BMG Labtechnologies) with

excitation at 485nm and emission at 520nm. Relative fluorescence units (RFU) were converted

to micrograms of vesicle protein using separate standard curves that established RFU/mg of

FITC-DH5a vesicles and FITC-DH5a Ail vesicles (r = 0.99), which were present on each

microtitre plate tested.

Fluorescence and Immunofluorescence Microscopy—CHO or HT29 (1.6 x 105 cells/well)

were plated on permanox microwell chamber slides (Nunc Inc.) and incubated (37ºC) with

vesicles (1-5 mg/well) for various times in serum- and antibiotic-free media. Unbound vesicles

were then removed and the cells 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% BSA. Cells were then incubated 1 h with rabbit anti-GFP antibody

(2.5 mg/ml) and visualized with rhodamine red-X-labeled goat anti-rabbit antibody (2.5 mg/ml;

Jackson Immunoresearch). Slides were mounted with a cover slip and ProLong Antifade kit

(Molecular Probes, Inc.) and the cells observed using a Nikon Eclipse TE200 laser scanning

confocal microscope.

RESULTS

Ail is present in E. coli vesicles—To determine if 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. entercolitica outer membrane protein Ail under the

control of its native promoter (18). Ail was detected in purified outer membrane preparations

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and in vesicles produced by DH5a/Ail (Fig. 1A, B), MC4100/Ail and HB101/Ail (data not

shown) and its presence in vesicles confirmed by N-terminal sequencing. Cofractionation in

Optiprep density gradients of Ail with OmpA and OmpF/C, outer membrane components of

native vesicles (Fig. 2A, B), showed that Ail was shed into the culture supernatant in association

with other outer membrane vesicle components. EM examination of vesicles in low density

Optiprep fractions revealed that more than 90% were closed vesicles, not membrane whirls or

fragments of lysed vesicles. These data demonstrated that heterologous outer membrane proteins

expressed in E. coli are present in native-like vesicles.

In order to investigate the effect of a heterologous protein on yield, we compared vesicle

production as a function of protein concentration per CFU. After overnight growth, DH5a/Ail

produced 2.3-fold more vesicles per CFU than DH5a. A difference in vesicle density was not

detected, since vesicles with Ail migrated to the same density fraction as DH5a vesicles (see

peak vesicle fractions 4 and 5 Fig. 2A, B). In addition, no distinguishable difference was

detected in the vesicles by negative staining electron microscopy: DH5a vesicles ranged from

22 nm - 90 nm (Fig. 2C) and DH5a/Ail vesicles ranged from 22 nm - 77 nm (Fig. 2D) in

diameter. Therefore, outer membrane vesicle density and size was unaltered due to the

incorporation of a heterologous protein into the membrane, but vesicle yield increased.

To determine if 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%

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:Ail ratio appeared constant in each

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fraction (0.5 + 0.03 SEM; 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 due to its association with LPS,

induces the association and internalization of vesicles by eukaryotic cells (2,28).2 Since Ail is a

known adhesin/invasin which can confer an invasive phenotype to HB101 (18), we wanted to

determine if Ail is able to catalyze the internalization of DH5a vesicles. We used fluorescently

labeled vesicles to study cell association. Wild type DH5a 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 DH5a vesicle-cell association 10-fold over the background (Fig. 4C),

and that Ail-dependent vesicle-cell association was vesicle-concentration dependent (data not

shown). Interestingly, the unbound DH5a/Ail vesicles removed from the eukaryotic cells

contained Ail as shown by Coomasie staining (data not shown). This further supported the

finding that Ail was present in every vesicle, since 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 TatA,B,C and E—Next

we wanted to determine if 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

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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 (30,31).

DH5a 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 previously shown 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

if 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 DH5a and in 43886 after induction of Tat-GFP and

TatABCE (Fig. 5B, 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 DH5a/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

prep (Fig. 5B, C). These results demonstrated periplasmic localization of fluorescent GFP in

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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—Since Tat-GFP was transported and

properly folded in the periplasm, we wanted to determine if 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 DH5a- and 43886-derived vesicles

when TatABCE was expressed (Fig. 6A, 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 Omps F/C, OmpA and GFP in the same fractions (Fig. 7A, fractions 4-6). In

contrast, soluble GFP and MBP (Fig. 7B, fraction 8-9) 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.

43886 vesicles containing GFP were slightly more dense than those without GFP since

GFP and Omps F/C and A 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 DH5a vesicles containing GFP (data not shown). Although the

difference in density due to the presence of lumenal GFP in vesicles suggested that the lipid-

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. 7C, D). 43886 vesicles

ranged from 43 nm - 165 nm and 43886/Tat-GFP/TatABCE ranged from 20 nm - 165 nm in

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diameter, while DH5a vesicles ranged from 22 nm - 90 nm and DH5a/Tat-GFP/TatABCE

ranged from 25 nm - 90 nm in diameter. Note that native DH5a vesicles are approximately one-

half the size of 43886 vesicles (Fig. 7C, 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 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, maltose binding protein (MBP;

Fig. 8A).

To determine if 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 (fractions 3-6; Fig.

7A) all contained the same ratio of Omps:GFP. Therefore, periplasmic GFP is neither selectively

enriched nor selectively excluded from vesicles and GFP appears to be included in every vesicle.

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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 if 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 DH5a/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.

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 since 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

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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. entercolitica 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 DH5a vesicle association with

CHO cells, and the DH5a/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 SufI Tat substrate to the periplasm was

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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 lab strains and pathogenic strains of

E. coli. We further observed that DH5a expressing Tat-GFP without TatABCE overexpression

appeared filamentous, much like the DTatC 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 DH5a/Tat-GFP/TatABCE spheroplasts did not have any detectable

fluorescence over background, very little cytoplasmic Tat-GFP was detectable by

immunoblotting, and at endogenous (low) levels of TatABCE, bacteria contained little to no

cytoplasmic GFP (data not shown). However, we did detect fluorescence and GFP in

spheroplasts of 43886/Tat-GFP/TatABCE. Since 43886 is more difficult to lyse than DH5a, 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 if 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 since 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

solubized with SDS. These results proved that GFP was lumenally packaged in vesicles.

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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 DH5a/Ail vesicles. Both DH5a and 43886 package outer membrane

proteins into vesicles to the same extent. Of total cellular OmpF/C, DH5a and 43886 package

0.23% and 0.17% in vesicles, respectively, while 0.14% of OmpA is packaged by DH5a 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 lipid-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

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through the inner and outer membrane, such as by the Tat and the general secretory pathway,

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, while 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 (28,39,40).

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 there tools provide valuable methods to study

the nature of vesicle-mediated transport.

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FIGURE LEGENDSFIG. 1. Ail is detected in the outer membrane and in vesicles produced by DH5a/Ail.

Coomassie-stained SDS-PAGE of 1 mg outer membrane fraction (A) and purified vesicles (B)

from DH5a (1.5 mg) and DH5a/Ail (2 mg). Arrows 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.

Coomassie-stained SDS-PAGE of fractions of increasing density (left to right) of DH5a (A) and

DH5a/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 DH5a (C) and DH5a/Ail (D) vesicles. Size bar applies to both panels.

FIG. 3. Ail and endogenous OMPs are packaged equally into vesicles. Coomassie-stained

SDS-PAGE of DH5a/Ail cell lysate (C) and DH5a/Ail vesicles (V). Positions of OmpF/C,

OmpA and Ail are indicated.

FIG. 4. FITC-labeled DH5a/Ail vesicles associate with CHO cells. Confocal microscopy of

CHO cells incubated for 3 h with FITC- DH5a (A) and FITC- DH5a/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 vesicles/well)

following a 3 h incubation of CHO cells with 5 mg FITC- DH5a/Ail (gray) and FITC- DH5a

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(black) vesicles. Error bars are SEM. Significant difference (*) was determined by the Student t

test (p<0.001, n > 2). Data representative of two trials done in duplicate.

FIG. 5. Tat-GFP is transported to the periplasm in DH5a and 43886. A, TatABCE

increases Tat-GFP transport to the periplasm of DH5a. Data represented as fold-increase of

fluorescence in DH5a/Tat-GFP periplasm + SEM with induction of TatABCE using 0.1 mM

IPTG for 6 h. Significant difference (*) as determined by the Student t test (p<0.002, n = 6). B,

Anti-GFP (upper) and anti-MutL (lower) immunoblot analysis of periplasm and spheroplasts for

DH5a/Tat-GFP /TatABCE and spheroplasts for DH5a 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 part B. Positions of mature GFP (27 kDa) and MutL (68 kDa) are

indicated.

FIG. 6. GFP is packaged into vesicles from bacteria overexpressing TatABCE. A, Anti-

OM (upper) and anti-GFP (lower) immunoblot of 1 mg vesicles from DH5a/Tat-GFP,

DH5a/Tat-GFP /TatABCE, and DH5a. Hours of induction with 0.1 mM IPTG are indicated. B,

Coomassie-stained SDS-PAGE (upper) and anti-GFP immunoblot (lower) of 10 mg 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.

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FIG. 7. GFP cofractionates with vesicle OMPs, and GFP-containing vesicles are slightly

more dense 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, 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 DH5a (B) and

43886 (C) strains without (left) and with (right) TatGFP/TatABCE. Size bars are shown for each

pair.

FIG. 8. GFP is a lumenal component of vesicles. A, Anti-GFP immunoblot analysis (upper

panel), anti-MBP immunoblot analysis (middle panel) and Coomassie-stained SDS-PAGE

(middle panel) of 10 mg 43886/Tat-GFP/TatABCE vesicles incubated with and without pronase

for 30 minutes. 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). Arrows indicate OmpF/C, GFP and MBP (42

kDa).

FIG. 9. GFP-containing 43886 vesicles associate with eukaryotic cells. Confocal microscopy

of HT29 cells incubated for 8 h with 5 mg GFP-containing 43886 (A) and DH5a (B) vesicles

visualized using anti-GFP and a rhodamine-labeled secondary antibody. Selected cells from

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upper panels (boxed) are magnified in bottom panels. Magnification is indicated for pairs of

upper and lower panels.

Acknowledgements—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.

REFERENCES

1. Beveridge, T. J. (1999) J Bacteriol 181, 4725-47332. Horstman, A. L., and Kuehn, M. J. (2000) J Biol Chem 275, 12489-12496.3. Devoe, I. W., and Gilchrist, J. E. (1973) J Exp Med 138, 1156-11674. Keenan, J., Day, T., Neal, S., Cook, B., Perez-Perez, G., Allardyce, R., and Bagshaw, P. (2000) FEMS

Microbiol Lett 182, 259-264.5. Fiocca, R., Necchi, V., Sommi, P., Ricci, V., Telford, J., Cover, T. L., and Solcia, E. (1999) J Pathol 188,

220-226.6. Kato, S., Kowashi, Y., and Demuth, D. R. (2002) Microb Pathog 32, 1-13.7. Wai, S. N., Takade, A., and Amako, K. (1995) Microbiol Immunol 39, 451-4568. Wai, S. N., Lindmark, B., Soderblom, T., Takade, A., Westermark, M., Oscarsson, J., Jass, J., Richter-

Dahlfors, A., Mizunoe, Y., and Uhlin, B. E. (2003) Cell 115, 25-359. Yokoyama, K., Horii, T., Yamashino, T., Hashikawa, S., Barua, S., Hasegawa, T., Watanabe, H., and Ohta,

M. (2000) FEMS Microbiol Lett 192, 139-144.10. Kolling, G. L., and Matthews, K. R. (1999) Appl Environ Microbiol 65, 1843-184811. Demuth, D. R., James, D., Kowashi, Y., and Kato, S. (2003) Cell Microbiol 5, 111-12112. Li, Z., Clarke, A. J., and Beveridge, T. J. (1998) J Bacteriol 180, 5478-548313. Beermann, C., Wunderli-Allenspach, H., Groscurth, P., and Filgueira, L. (2000) Cell Immunol 201, 124-

131.14. Shoberg, R. J., and Thomas, D. D. (1993) Infect Immun 61, 3892-390015. Kadurugamuwa, J. L., and Beveridge, T. J. (1996) J Bacteriol 178, 2767-277416. Kadurugamuwa, J. L., and Beveridge, T. J. (1998) Antimicrob Agents Chemother 42, 1476-148317. Kadurugamuwa, J. L., and Beveridge, T. J. (1995) J Bacteriol 177, 3998-400818. Miller, V. L., and Falkow, S. (1988) Infect Immun 56, 1242-124819. Thomas, J. D., Daniel, R. A., Errington, J., and Robinson, C. (2001) Mol Microbiol 39, 47-53.20. Cormack, B. P., Valdivia, R. H., and Falkow, S. (1996) Gene 173, 33-3821. Yahr, T. L., and Wickner, W. T. (2001) Embo J 20, 2472-247922. Jobling, M. G., and Holmes, R. K. (1990) Nucleic Acids Res 18, 5315-5316

by guest on Decem

ber 6, 2020http://w

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.jbc.org/D

ownloaded from

23

23. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2 Ed.(Ford, N., Ed.), 1. 3 vols., Cold Spring Harbor Press, Cold Spring Harbor

24. Nikaido, H. (1994) Methods Enzymol 235, 225-23425. Bernadac, A., Gavioli, M., Lazzaroni, J. C., Raina, S., and Lloubes, R. (1998) J Bacteriol 180, 4872-487826. Mug-Opstelten, D., and Witholt, B. (1978) Biochim Biophys Acta 508, 287-29527. Hoekstra, D., van der Laan, J. W., de Leij, L., and Witholt, B. (1976) Biochim Biophys Acta 455, 889-89928. Horstman, A. L., and Kuehn, M. J. (2002) J Biol Chem 277, 32538-32545.29. Feilmeier, B. J., Iseminger, G., Schroeder, D., Webber, H., and Phillips, G. J. (2000) J Bacteriol 182, 4068-

4076.30. Thomas, J. D., Daniel, R. A., Errington, J., and Robinson, C. (2001) Mol Microbiol 39, 47-53.31. Santini, C. L., Bernadac, A., Zhang, M., Chanal, A., Ize, B., Blanco, C., and Wu, L. F. (2001) J Biol Chem

276, 8159-816432. Sargent, F., Gohlke, U., De Leeuw, E., Stanley, N. R., Palmer, T., Saibil, H. R., and Berks, B. C. (2001)

Eur J Biochem 268, 3361-336733. Stanley, N. R., Findlay, K., Berks, B. C., and Palmer, T. (2001) J Bacteriol 183, 139-14434. Pelham, H. R. (1996) Cell Struct Funct 21, 413-41935. Stoorvogel, W., Kleijmeer, M. J., Geuze, H. J., and Raposo, G. (2002) Traffic 3, 321-33036. Thery, C., Zitvogel, L., and Amigorena, S. (2002) Nat Rev Immunol 2, 569-57937. Pugsley, A. P. (1993) Microbiol Rev 57, 50-10838. Lory, S. (1998) Curr Opin Microbiol 1, 27-3539. Scott, M. E., Dossani, Z. Y., and Sandkvist, M. (2001) Proc Natl Acad Sci U S A 98, 13978-1398340. Tauschek, M., Gorrell, R. J., Strugnell, R. A., and Robins-Browne, R. M. (2002) Proc Natl Acad Sci U S A

99, 7066-7071.

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Figure 1

DH5a DH5a/AilOM

A

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Vesicles

OmpF/COmpA

Ail

DH5a DH5a/Ail

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Ail

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1 2 3 4 5 6 7 8 9

Figure 2

DH5a

OmpF/COmpA

Ail

DH5a DH5a/Ail

A B

1 2 3 4 5 6 7 8 9

1421

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C DDH5a DH5a/Ail

25 nm

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C V

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DH5a/Ail DH5a0

100

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* C

ell A

ssoc

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Figure 4

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DH5a/AilDH5a

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Figure 5Fo

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Time (h)

B DH5a/Tat-GFP/TatABCE

0 2 4 6 0 2 4 6 6

DH5aPeriplasm Spheroplast

GFP

MutL

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25

Time(h)

GFP

0 2 4 6 0 2 4 6 6

C 43886/Tat-GFP/TatABCE 43886Periplasm Spheroplast

MutL

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Figure 6

43886/Tat-GFP/TatABCE

43886 43886/Tat-GFP

Time (h) 6 12 6 12 6 12

DH5a/Tat-GFP/TatABCE

GFP

A DH5a/Tat-GFP

DH5a

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OM Ag

Time (h) 6 12 6 12 6 12

GFP

OmpAOmpF/C

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Figure 7

Omp F/COmp A

1 2 3 4 5 6 7 8 943886/

Tat-GFP/TatABCE

43886

GFP

A

43886/Tat-GFP/TatABCE

Omp F/COmp A

DH5a/Tat-GFP/TatABCEC

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DH5a

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B 1 2 3 4 5 6 7 8 9

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Figure 8

Pronase - + +SDS - - +

GFP

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Figure 9

25 mm

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A BDH5a/TatGFP/TatABCE43886/TatGFP/TatABCE

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Nicole C. Kesty and Meta J. KuehnEscherichia coli outer membrane vesicles

Incorporation of heterologous outer membrane and periplasmic proteins into

published online October 24, 2003J. Biol. Chem. 

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