uptake of helicobacter pylori outer membrane vesicles by ... · 46 (32), leading to speculation...

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1

Uptake of Helicobacter pylori outer membrane vesicles 2

by gastric epithelial cells 3

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Heather Parker1, Kenny Chitcholtan

1, Mark B. Hampton

2, Jacqueline I. Keenan

1* 8

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Departments of 1Surgery & 2Pathology, University of Otago, Christchurch, New Zealand. 10

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Running title: UPTAKE OF H. PYLORI OMV 13

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*Corresponding Author: Dr Jacqueline Keenan, University of Otago, PO Box 4345, 15

Christchurch, New Zealand. Phone: 64 3 3640 570. Fax: 64 3 3641 427. E-mail: 16

[email protected] 17

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Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.00299-10 IAI Accepts, published online ahead of print on 27 September 2010

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ABSTRACT 19

Helicobacter pylori colonize the human stomach where they stimulate a persistent 20

inflammatory response. H. pylori is considered non-invasive, however LPS-enriched outer 21

membrane vesicles (OMV), continuously shed from the surface of this bacterium, are 22

observed within gastric epithelial cells. The mechanism of vesicle uptake is poorly 23

understood, and this study was undertaken to examine the roles of bacterial VacA cytotoxin 24

and LPS in OMV binding, and cholesterol and clathrin-mediated endocytosis in vesicle 25

uptake by gastric epithelial cells. OMV association was examined using a fluorescent 26

membrane dye to label OMV and comparison was made between the association of vesicles 27

from a VacA+ strain and OMV from a VacA

- isogenic mutant strain. Within 20 minutes 28

essentially all associated OMV were intracellular and vesicle binding appeared to be 29

facilitated by the presence of VacA cytotoxin. Uptake of vesicles from the VacA+ strain was 30

inhibited by H. pylori LPS (58% inhibition with 50 たg/ml LPS) while uptake of OMV from 31

the VacA- mutant strain was less affected (25% inhibition with 50 たg/ml LPS). Vesicle 32

uptake did not require cholesterol. However, uptake of OMV from the VacA- mutant strain 33

was inhibited by a reduction in clathrin-mediated endocytosis (42% with 15 たg/ml 34

chlopromazine) while uptake of OMV from the VacA+ strain was less affected (~25% 35

inhibition with 15 たg/ml chlopromazine). We conclude that VacA toxin enhances the 36

association of H. pylori OMV with cells and that the presence of the toxin may allow vesicles 37

to exploit more than one pathway of internalization. 38

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INTRODUCTION 39

Infection with the gastric pathogen Helicobacter pylori results in chronic gastritis (13) and 40

is associated with increased risk for the development of peptic ulcer disease (35), gastric 41

carcinoma (41, 57) and gastric lymphoma (5, 60). H. pylori persistence, in an environment 42

where peristalsis and sloughing of cells are continually occurring, is mediated by a variety of 43

adhesins present on the bacterial surface (14, 21, 36, 40). However, despite the ability to 44

adhere to the gastric epithelium, the majority of organisms remain unattached to surface cells 45

(32), leading to speculation that lipopolysaccharide (LPS)-enriched outer membrane vesicles 46

(OMV) shed by these bacteria (15, 19, 26) contribute to H. pylori pathogenesis via the 47

persistent delivery of bacterial virulence factors (including the vacuolating cytotoxin VacA) 48

and antigens to the gastric mucosa (26, 27). Observations that H. pylori OMV modulate 49

gastric epithelial cell proliferation (22), induce apoptosis (3), stimulate secretion of the pro-50

inflammatory cytokine interleukin-8 (22), increase micronuclei formation (8), and are 51

observed at the luminal surface (15, 26) and within cells of the gastric epithelium (15) 52

support this hypothesis. 53

OMV shedding by gram-negative bacteria is well described in the literature (reviewed by 54

Kuehn and Kesty (33)), yet little is known of the mechanisms of vesicle adherence to and 55

internalization within mammalian host cells. The adherence of enterotoxigenic 56

Escherichia coli (ETEC) OMV to host cells is mediated via a heat-labile enterotoxin LT 57

associated with these OMV (31), whereas leukotoxin, associated with OMV shed by 58

Actinobacillus actinomyctemcomitans, is not involved in vesicle binding (12). The 59

internalization of ETEC, Porphyromonas gingivalis and Pseudomonas aeruginosa OMV has 60

been shown to involve cholesterol-rich lipid rafts (6, 16, 31) and recently, Kaparakis & 61

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colleagues (25) reported that uptake of H. pylori OMV is also lipid raft-dependent. This is in 62

contrast to the uptake of Shigella flexneri OMV which occurs via phagocytosis, with 63

proposed subsequent fusion of OMV with the phagosomal membrane and release of vesicle 64

contents into the cell cytoplasm (24). 65

In this study, we sought to examine whether VacA cytotoxin associated with H. pylori 66

OMV was involved in vesicle binding. We also examined the rate of OMV internalization 67

and the involvement of LPS, cholesterol and clathrin-mediated endocytosis in vesicle uptake 68

by AGS gastric epithelial cells. We report that within 20 min essentially all VacA+ OMV 69

associated with AGS cells are intracellular and that uptake is enhanced by the presence of 70

vesicle-associated cytotoxin. Excess H. pylori LPS reduced vesicle uptake having a more 71

significant effect on VacA+ OMV than VacA

- vesicle uptake. Uptake of both VacA

+ and 72

VacA- OMV did not require cholesterol. However, a reduction in clathrin-mediated 73

endocytosis inhibited VacA- OMV uptake and to a lesser extent VacA

+ OMV internalization. 74

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MATERIALS AND METHODS 81

Bacterial strains. The well characterized H. pylori clinical isolate 60190 (ATCC 49503) 82

was used in this study. H. pylori 60190 is a vacA s1m1 strain (2) producing a cytotoxin that 83

induces extensive vacuolation in cultured epithelial cells (9). H. pylori strain 60190:v1, in 84

which the VacA gene has been disrupted by insertional mutagenesis resulting in both the 85

absence of the 87-kDa protein and vacuolating cytotoxin production (11), was also used. 86

Harvesting and labeling of OMV. H. pylori were grown in 2.8% (wt/vol) Brucella broth 87

(Becton Dickinson and Company, Sparks, MD, USA) supplemented with 5% fetal bovine 88

serum (Invitrogen, Auckland, NZ) at 37oC under micro-aerobic conditions with constant 89

rotation (120 rpm). At 72 h incubation, bacteria were removed by two centrifugations (12,000 × 90

g, 15 min, 4oC) and the final supernatants ultracentrifuged (200,000 × g, 2 h, 4

oC) to recover 91

OMV. After three washes in phosphate-buffered saline (PBS) OMV were stored at &20°C 92

until required. Vesicles to be labeled were washed once after recovery then, to standardize 93

labeling, were suspended in 1 ml PBS/50 mg OMV pellet. OMV were labeled with 1% 94

(vol/vol) 3,3’-dioctadecyloxacarbocyanine perchlorate or 1,1’-dioctadecyl-3,3,3’,3’-95

tetramethylindodicarbocyanine perchlorate (Vybrant™

DiO & Vybrant™

DiD, respectively; 96

Molecular Probes, Eugene, OR, USA) by incubation for 20 min at 37oC. Free dye was 97

removed by two washes with PBS and labeled OMV were stored at 4oC for up to six weeks. 98

The protein concentration of OMV was determined (37) and preparations were examined by 99

transmission electron microscopy (TEM) to confirm the absence of whole bacteria and flagella 100

by overlaying aliquots of OMV suspension onto Formvar-coated 200 mesh copper grids and 101

negatively staining with 1% ammonium molybdate (pH 7.4). 102

Cell culture. The AGS human gastric adenocarcinoma cell line (ATCC CRL-1739) was 103

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cultured in F-12 Nutrient Mixture (HAM) (+ L-glutamine) (Invitrogen, Auckland, NZ) and 104

supplemented with 10% (vol/vol) fetal bovine serum and 1% (vol/vol) penicillin-105

streptomycin-glutamine supplement. Cells were cultured at 37oC with 5% CO2. 106

Cell proliferation . Cell proliferation was determined using a colorimetric assay that 107

measures the amount of 5-bromo-2’-deoxyuridine (BrdU) incorporated into cellular DNA 108

(Roche Diagnostics, Manneheim, Germany). AGS cells (1 × 104) were cultured overnight in 109

96-well plates then incubated for a further 24 h with labeled or unlabeled OMV from strain 110

60190 (0.05 – 20 µg). The BrdU assay was performed as per kit instructions. Briefly, 111

individual wells were incubated with BrdU labeling solution (10 µM BrdU) for 2 h. After 112

removal of the medium, a FixDenat solution was added at room temperature for 30 min then 113

replaced with anti-BrdU-POD (diluted 1:100) for 2 h. After washing, substrate solution was 114

applied and absorbance read at 370 nm every minute for 30 min with a SpectraMax plate 115

reader (Molecular Devices, USA). The level of BrdU incorporation was calculated from the 116

slope of the linear portion of the graph. 117

Flow cytometry. Cells (3 x 105) were cultured overnight then washed once with PBS and 118

the media replaced prior to addition of labeled OMV (200 µg OMV protein) for up to 6 h. 119

After incubation, cells were washed to remove unbound OMV and lifted with trypsin/EDTA 120

(Invitrogen). Fluorescence measurements were made using a FACS vantage flow cytometer 121

(Beckman Coulter Cytomics FC 500 MPL, AUS) and CXP software (Beckman Coulter 122

2005). A total of ten thousand events were collected for each sample. Mean fluorescence 123

intensity (MFI) values of cells incubated in the absence of OMV were subtracted from the 124

values of OMV-treated cells. 125

To determine the proportion of internalized OMV, extracellular vesicle fluorescence was 126

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quenched with trypan blue (0.025% final concentration). To confirm trypan blue quenched 127

DiO fluorescence, cells were incubated with labeled OMV for 4 h then fixed and 128

permeabilized using a Fix & Perm®

Cell Permeabilization Kit (Caltag Laboratories, 129

Burlingame, CA, USA) as per manufacturer’s instructions. Fluorescence was measured 130

before and after the addition of trypan blue. 131

Fluorescence microscopy. AGS cells (3 × 105), cultured overnight on glass coverslips, 132

were incubated with 200 µg DiO-labeled OMV for 5 h. Following fixation (4% 133

paraformaldehyde, 45 min), the cells were washed extensively in PIPES buffer, and 134

coverslips mounted in SlowFade® without glycerol (Molecular Probes, Eugene, OR, USA). 135

Where immunofluorescence microscopy was used to visualise intracellular OMV, the cells 136

were blocked with 5% (wt/vol) BSA for 60 min at room temperature. After washing, the 137

cells were permeabilised with 0.5% (vol/vol) Triton X-100 for 20 min and washed again 138

before being incubated overnight at 4°C with a murine IgG1 subclass monoclonal antibody to 139

H. pylori Lpp20 (27). Primary antibody binding was detected using a fluorescien 140

isothiocyante (FITC)-conjugated goat anti-mouse secondary antibody. Cells were examined 141

and imaged using a Leitz Aristoplan microscope (Germany) fitted with a Photometrics 142

KAF1400 CCD camera and QUIPS Smartcapture software, version 1.3 (Vysis Inc, IL, USA). 143

Images were formatted in Adobe Photoshop (CS2). 144

Cell treatments. To investigate the role of LPS in OMV association, DiO-labeled OMV 145

were added to cells at the same time as H. pylori LPS then cells were incubated for 4 h before 146

fluorescence measurements. The effect of several pharmacological agents (cycloheximide, 147

nystatin, chlorpromazine & MくCD; Sigma, St Louis, MO, USA) on OMV uptake was 148

examined by pre-treating cells with each drug for the following times: nystatin and MくCD, 149

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1 h; chlorpromazine, 30 min; cycloheximide, 15 min. Cells were then incubated with 150

labeled-OMV for 2 - 4 h. Control cells were incubated without inhibitor for the same period 151

of time. To assess drug cytotoxicity, AGS cells were incubated in media alone or with the 152

highest concentration of each inhibitor for 4 h then stained with 0.75% propidium iodide for 153

10 min and fluorescence measured by flow cytometry. Cytotoxicity was expressed as the 154

percentage of propidium-iodide positive cells. To assess inhibition of clathrin-mediated 155

endocytosis, cells were incubated with DiO-labeled LDL (15 µg) for 4 h. 156

Preparation of H. pylori LPS. Extraction of H. pylori LPS was performed using 157

conventional hot phenol-water treatment (23). Subsequent purification of this crude LPS was 158

performed by using enzymatic treatments with RNase A, DNase II and proteinase K (all from 159

Sigma) as described previously (38). 160

Cholesterol quantification. AGS cells (3 x 105) were cultured overnight, washed once 161

with PBS prior to incubation for 1 h at 37oC in F12 media containing 5 mg/ml of MくCD. 162

The cholesterol content of cells was measured using the cholesterol CHOD-PAP reagent 163

(Roche Diagnostics, Indianapolis, IN). 164

Neutral red assay. Cells (3 x 104) were cultured overnight in 96-well micro-titre plates 165

then incubated with OMV (10 – 50 µg OMV protein) for 6 h. Untreated AGS cells were 166

used as controls for background vacuolation. Vacuolation was assessed by staining with 167

neutral red as previously described (10). 168

Statistical analysis. Results are the means ± standard errors of the means (SE). Data 169

were analyzed by t-test, Pearson’s correlation, One Way Analysis of Variance (ANOVA) or 170

Two Way ANOVA. If the ANOVA p value was < 0.05, this was followed by Dunnett’s 171

post-hoc test. 172

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RESULTS 173

Assessment of the rate of H. pylori OMV internalizaiton by AGS cells. We and others 174

have previously shown that H. pylori OMV are internalized by gastric epithelial cells (8, 15, 175

44, 45). In this study, we sought to examine the rate and mechanism of OMV internalization 176

using the lipophilic fluorescent membrane dye DiO to monitor OMV localization. The 177

addition of labeled OMV to AGS cells for 24 h induced a 73% decrease in proliferation (27 ± 178

4% of control) similar to that induced by unlabeled OMV (30 ± 2% of control) (means ± SE, 179

n = 3) indicating that labeling did not interfere with the interaction of vesicles with AGS 180

cells. 181

Internalization of OMV was assessed quantitatively by flow cytometry using the cell 182

impermeant dye trypan blue to quench extracellular DiO-vesicle fluorescence. The 183

effectiveness of trypan blue at quenching DiO-OMV fluorescence was confirmed by 184

incubating cells with labeled OMV and comparing the fluorescence of permeabilized and 185

non-permeabilized cells treated with this dye. Complete loss of DiO fluorescence was 186

observed in permeabilized cells upon addition of trypan blue while non-permeabilized cells 187

showed no change in fluorescence (Fig. 1A & B). To examine the rate of OMV 188

internalization, AGS cells were incubated with DiO-labeled OMV for up to 4 h and 189

fluorescence measured before (total associated OMV) and after (intracellular OMV) the 190

addition of trypan blue. A linear increase in total associated OMV was observed over the 191

4 h, with Pearson’s coefficient correlation r value = 0.997 (p = 0.003; Fig. 1C). At each time 192

point assessed the majority of the OMV associated with the cells were intracellular (Fig. 1C). 193

Examination of earlier time points showed that internalization of OMV peaked at 20 min and 194

then plateaued (Fig. 1D). 195

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De novo protein synthesis is not required for vesicle binding. The steady increase in 196

OMV association with cells over time could be explained by specific vesicle binding that is 197

dependent on upregulation or recycling of receptors after internalization of OMV. To 198

examine whether vesicle binding stimulates de novo synthesis of OMV receptors, AGS cells 199

were pretreated with increasing concentrations of the protein synthesis inhibitor 200

cycloheximide. Inhibition of protein synthesis had no effect on the association of OMV with 201

AGS cells (ANOVA p = 0.878; Fig. 1E). 202

VacA enhances OMV association with AGS cells. We have previously shown that 203

VacA is localized on the surface of H. pylori OMV (26) and that OMV-associated cytotoxin 204

is biologically active (22). These findings, plus evidence that heat-labile enterotoxin has a 205

role in the internalization of OMV shed from enterotoxigenic E. coli (31), led us to consider 206

that vesicle-associated VacA may also play a role in the uptake of H. pylori OMV. Uptake 207

of OMV from the vacuolating strain 60190 was compared to its isogenic mutant 60190:v1 208

that does not produce a toxin (11). The vacA gene has been disrupted in this mutant by 209

insertion of a kanamycin cassette preventing formation of the cytotoxin (11). Although the 210

proteome of H. pylori OMVs has not been defined, OMV from this mutant strain are 211

assumed to have the same phenotype as wild-type OMV, except for the absence of the VacA 212

protein. Examination by fluorescence microscopy after 5 h confirmed a previous observation 213

(8) that vesicles shed from both wild type and VacA- mutant strains are internalized by AGS 214

cells (Fig. 2A & B). To confirm that the observed fluorescence was OMV and not free dye, 215

we labeled vesicles with DiO then, after incubation with AGS cells, used a polyclonal 216

antibody directed against H. pylori OMV to detect vesicles. Immuno-labeled OMV co-217

localized with DiO-labeled OMV (Fig. 2C). 218

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While the data obtained by fluorescence microscopy demonstrated that uptake of VacA- 219

mutant OMV can occur, there was no indication of the rate of association of these OMV. To 220

investigate this, AGS cells were incubated with labeled OMV from either strain for up to 6 h 221

and examined at regular time points by flow cytometry. As shown in Figure 3A, wild type 222

OMV associated with AGS cells at a faster rate than OMV from the VacA- strain (p < 0.01 at 223

6 h). 224

To further examine the role of VacA in OMV binding, vesicles from strain 60190 were 225

labeled with DiO (green) and vesicles from strain 60190:v1 with DiD (red). Cells were 226

incubated with OMV from either strain or a 50:50 mix of both. Co-incubation of wild type 227

OMV with VacA- OMV had no effect on the association of wild type vesicles with cells (Fig. 228

3B). In contrast, the association of OMV from the VacA mutant strain was significantly 229

inhibited (p = 0.002, Fig. 3B). This observation, that incubation with wild type OMV 230

inhibited the association of VacA- OMV, suggests the presence of VacA enhances vesicle 231

binding. 232

OMV uptake is inhibited by LPS. LPS has been implicated as a potential H. pylori 233

adhesin (14). AGS cells were co-incubated with wild type or VacA- OMV and increasing 234

concentrations of purified H. pylori LPS. OMV uptake was inhibited in a dose-dependent 235

manner by H. pylori LPS, with the effect most evident with VacA+ OMV (58% ± 2.5 236

inhibition of wild type OMV uptake compared with 25% inhibition ± 2.4 of VacA- OMV 237

uptake with 50 µg/ml LPS) (Fig. 4) (Dunnett’s post-hoc test p < 0.05). In combination with 238

Fig. 3, this suggests that VacA is important in facilitating the uptake of OMV by an LPS-239

inhibitable mechanism, while in the absence of VacA LPS plays a less significant role. 240

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Cholesterol is not required for OMV uptake. Depletion of plasma membrane 241

cholesterol, which disrupts lipid rafts (34), has been reported to inhibit entry of purified 242

VacA into host cells (42, 53). To examine whether depletion of plasma membrane 243

cholesterol could likewise reduce the uptake of VacA+ OMV, AGS cells were pretreated with 244

5 mg/ml methyl-く-cyclodextrin (MくCD), a cholesterol sequestering agent (47). MくCD-245

treatment resulted in no difference in cholesterol levels per mg of total cell protein (37.74 µg 246

± 1.87 for untreated cells, 33.16 µg ± 2.21 for MくCD-treated cells) (means ± SE, n=3; p = 247

0.133). This was unexpected as a reduction in cholesterol of up to ~50% has been reported 248

with similar concentrations of MくCD (42, 53). However, pretreatment of AGS cells with as 249

little as 1 mg/ml MくCD was sufficient to significantly inhibit vacuole formation in response 250

to 60190 OMV (Fig. 5A), while vesicle uptake was increased by MくCD at all concentrations 251

tested (Fig. 5B) (Dunnett’s post-hoc test p < 0.001 and p < 0.05, respectively). Higher 252

concentrations of MくCD were not used as, in addition to disrupting lipid rafts, these have 253

been reported to inhibit clathrin-mediated endocytosis (47, 55) and to induce marked changes 254

in cellular morphology in HeLa cells (53). Instead, cells were treated with nystatin, a 255

cholesterol-binding agent (50), that disrupts lipid rafts but does not affect clathrin-mediated 256

endocytosis (49). Pretreatment with nystatin (25 たg/ml) inhibited wild-type OMV-mediated 257

cell vacuolation (Fig. 5C) (Dunnett’s post-hoc test p < 0.05) but had no effect on wild-type or 258

VacA- mutant OMV uptake (Fig. 5D) ((ANOVA p = 0.114 and p = 0.811, resepctively). 259

Inhibition of clathrin-mediated endocytosis reduces OMV uptake. Clathrin-mediated 260

endocytosis was explored as a potential pathway for vesicle internalization. This pathway 261

can mediate the constitutive uptake of ligands such as transferrin or low-density lipoprotein 262

as well as ligand-triggered receptor uptake of proteins (17). Chlorpromazine, a known 263

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inhibitor of clathrin-mediated endocytosis (59), significantly inhibited vacuole formation in 264

AGS cells treated with 60190 OMV when added at a concentration of 15 µg/ml (Dunnett’s 265

post-hoc test p < 0.001; Fig. 6A) and had a dose-response effect on wild-type OMV uptake 266

(ANOVA p = 0.011; Fig. 6B). Chlorpromazine (15 µg/ml) reduced the uptake of VacA- 267

OMV to a greater extent (42% ± 8.9) (Dunnett’s post-hoc test p < 0.05; Fig. 6C). A 268

reduction in clathrin-mediated endocytosis was confirmed by measurement of LDL uptake 269

(Dunnett’s post-hoc test p < 0.05; Fig. 6D). We were not able to completely inhibit clathrin-270

mediated endocytosis with chlorpromazine as concentrations of this drug greater than 271

15 たg/ml were cytotoxic, as assessed by propidium iodide staining (data not shown). 272

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DISCUSSION 274

The H. pylori vacuolating cytotoxin is secreted in a soluble form and is also associated 275

with OMV (45). Vesicle-associated VacA has been observed at the surface (26) and within 276

the gastric mucosa (15), however, little is known of the interactions of OMV-associated 277

cytotoxin with host cells. The data presented in this study indicates that VacA is not 278

essential for H. pylori OMV uptake but the presence of the cytotoxin increases the rate of 279

vesicle association with cells. 280

Purified VacA is reported to bind to multiple cell surface components, including 281

sphingomyelin (18), glycosphinglipids (46), glycosylphosphatidylinositol (GPI)-anchored 282

protein(s) (43) and receptor-like protein tyrosine phosphatases ß (39), with subsequent 283

clustering of the receptor-bound toxin in cholesterol-rich lipid rafts (39, 42, 53). Horstman et 284

al. speculated that such binding of vesicle-bound bacterial toxins to multiple binding sites 285

acts to bring vesicles into closer proximity with host cells, thus allowing secondary adhesions 286

to increase the “intimacy” between the two (20). In support of this, VacA+ OMV associated 287

with cells quicker than VacA- OMV and inhibited the association of VacA

- vesicles 288

suggesting the toxin increases the avidity of vesicle binding. In addition, H. pylori LPS 289

inhibited OMV uptake to a greater extent with VacA+ OMV. We speculate that binding of 290

vesicle-associated VacA to cells increases the interaction of OMV LPS with cells, increasing 291

the avidity of the overall vesicle binding reaction. In studies of other bacteria, the 292

aminopeptidase PaAP, one of the major protein constituents of OMV from P. aeruginosa 293

cystic fibrous isolates, was recently shown to increase the rate of association of 294

P. aeruginosa OMV with lung epithelial cells (4). In addition, a heat-labile enterotoxin (LT) 295

associated with ETEC OMV increases the association of E. coli OMV with cells (31). In the 296

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absence of LT, E. coli OMV binding is significantly reduced (31). 297

Uptake of vesicles from several bacterial genera has been shown to be cholesterol-298

dependent (6, 25, 31). In addition, Kaparakis and colleagues (25) recently reported that 299

H. pylori OMV uptake is cholesterol-rich lipid raft-dependent. However, in our study 300

binding of cholesterol with MくCD or nystatin, disruptors of lipid raft formation (25, 49), did 301

not significantly inhibit H. pylori vesicle uptake. The disparity between the Kaparakis study 302

and ours may be a reflection of the uptake of vesicles from different H. pylori strains and/or 303

the use of serum-free cell culture medium (25). 304

MくCD, although having no significant effect on AGS cell cholesterol levels, significantly 305

decreased vacuolation in cells treated with VacA+ OMV. Low concentrations of MくCD have 306

also been shown to inhibit vacuolation in HeLa cells treated with purified VacA (53), adding 307

weight to evidence that membrane cholesterol is essential for VacA toxigenesis (39, 42). A 308

reduction in vacuolation in the absence of an effect on OMV uptake may be explained by a 309

greater sensitivity of toxin-mediated vacuole biogenesis to membrane cholesterol levels than 310

vesicle uptake. 311

VacA translocation to lipid rafts following binding to receptor-like protein tyrosine 312

phosphatases ß receptors in non-lipid raft domains on the cell surface is reportedly inhibited 313

by treatment with the same concentration of MくCD (5 mg/ml) used in our study (39). Thus, 314

if lipid rafts are required for VacA+ OMV uptake we expected to see reduced vesicle 315

internalization. Instead we observed that OMV uptake in MくCD-treated cells was increased. 316

The reason for this is not immediately apparent and requires further investigation. 317

Disruption of clathrin-mediated endocytosis with chlorpromazine reduced the uptake of 318

OMV. To our knowledge this is the first report demonstrating OMV internalization via 319

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clathrin-mediated endocytosis. Internalization of ETEC and P. aeruginosa OMV is 320

unaffected by chlorpromazine and these OMV rarely colocalize with clathrin (4, 31). Indeed, 321

ETEC OMV frequently colocalize with caveloe (31). In addition, P. gingivalis OMV 322

internalize via a Rac1-regulated lipid raft-dependent pathway that is independent of caveolin 323

and clathrin (16). 324

The observation that VacA+ OMV uptake was less inhibited by chlorpromazine than 325

uptake of VacA- OMV suggests that toxin-containing H. pylori OMV may be taken up into 326

gastric epithelial cells via more than one pathway. That nystatin had no significant effect on 327

VacA+ OMV uptake also supports this. However, further investigation is required to confirm 328

this hypothesis. Of note, several bacterial toxins (including anthrax, cholera and Shiga 329

toxins) are reportedly endocytosed by both clathrin-dependent and clathrin-independent 330

mechanisms (1, 52, 56). 331

The increase in OMV association over time, in conjunction with the observation that the 332

majority of associated OMV were intracellular 20 min and thereafter suggests to us, that 333

OMV are taken up and that binding is the rate limiting step, possibly due to a limited 334

numbers of receptors. The steady increase in fluorescence over time also suggests that, once 335

internalized, OMV persist within AGS cells for some time. This is supported by the 336

observation of intact OMV within cells after >72 h co-incubation (54). The association of 337

ETEC and P. aeruginosa OMV with cells has also been shown to increase with time (4, 31). 338

In agreement with Bauman & Kuehn (4) we consider this is consistent with (i) vesicle 339

binding initiating upregulation of OMV receptors; or (ii) that receptors are recycled back to 340

the plasma membrane after internalization of their cargo (7). We found OMV association 341

was unaffected by cycloheximide indicating that if specific receptors are involved they are 342

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already translated. We have observed that pertubation of the actin cytoskeleton inhibits 343

vesicle association (H. Parker and J. Keenan, unpublished data) suggesting disruption of 344

receptor recycling. 345

The small size of OMV (50 – 300 nm) makes their enumeration difficult. In this study 346

OMV were quantified based on protein concentration. While this may vary between strains 347

it is one of the more commonly used methods for OMV enumeration (16, 24, 25, 48, 58). As 348

an alternative, OMV may be quantified based on the concentration of lipopolysaccharide (51) 349

or toxin (6) in vesicle preparations. However, the amount of LPS and VacA cytotoxin also 350

varies between H. pylori strains (26, 29), as well as within a strain grown under different 351

conditions (28, 30). In addition, use of a VacA- strain in this study precluded OMV 352

enumeration based on cytotoxin concentration. Immunolabelling of VacA+ OMV shows 353

relatively few (< 10) toxin molecules per OMV (15, 26, 54). Therefore, we considered that 354

its absence from OMV may not significantly change the overall vesicle protein concentration 355

thus allowing for reasonable comparison between OMV from wild type and VacA- OMV. 356

In summary, we have shown that VacA toxin enhances the association of H. pylori OMV 357

with cells and that VacA- OMV are internalized via clathrin-mediated endocytosis while 358

VacA+ OMV may be able exploit more than one pathway of internalization. We propose that 359

constitutive release (26) and uptake (15) of OMV from the surface of these predominantly 360

non-invasive bacteria contributes to H. pylori pathogenesis via the persistent delivery of 361

virulence factors and antigens to gastric epithelial cells. 362

363

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ACKNOWLEDGEMENTS 364

We thank Tim Cover (Division of Infectious Diseases, Vanderbilt University School of 365

Medicine, USA) for the kind gift of the VacA mutant. 366

This work was supported by a Bright Futures Doctoral Scholarship to HAP and funding 367

from the Cancer Society of New Zealand (to JIK and MBH). 368

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530

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533

A 534

535

536

537

538

539

540

541

B 542

543

544

545

546

547

548

549

550

1 2 3 40

50

100

150

200 Total associated OMV

Internalized OMV

C

Time (h)

OM

V a

sso

ciat

ion

(mea

n flu

oes

cenc

e in

ten

sity

)

10 15 20 25 30 35 40 45 50 55 600

50

100

150

200

D

Time (min)

Pro

port

ion

of in

tern

aliz

ed O

MV

(% o

f tot

al D

io fl

uo

resc

ence

)

0.1 1.0 10.00

20

40

60

80

100

120

E

Cycloheximide ( g/ml)

OM

V a

ssoc

iatio

n (%

of c

on

trol

)

551 552

100 101 102 103 104

DiO

Eve

nts

Before trypan blue

After trypan blue

100 101 102 103 104

DiO

Eve

nts

Before trypan blue

After trypan blue

Eve

nts

0 1

DiO

2 3

Before trypan blue

After trypan blue

10 10 10 10 104

Eve

nts

0 1

DiO

2 3

Before trypan blue

After trypan blue

10 10 10 10 104

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FIG. 1. Flow cytometry assessment of the association of DiO-labeled OMV (strain 60190) 553

with AGS cells. (A & B) To confirm that trypan blue quenches DiO-OMV fluorescence, 554

cells were incubated with labeled OMV for 4 h then (A) fixed and permeabilized or (B) not 555

permeablized. Fluorescence was measured before and after the addition of trypan blue. (C) 556

The proportion of intracellular OMV was monitored over time. Cells were incubated with 557

DiO-labeled 60190 OMV for 1 – 4 h then fluorescence measured before (total associated) 558

and after (intracellular) the addition of trypan blue. (D) The proportion of intracellular OMV 559

during the first hour incubation. (E) Inhibition of de novo protein synthesis does not inhibit 560

the association of OMV with AGS cells. Cells were pretreated with cycloheximide for 561

15 min prior to incubation with labeled 60190 OMV for 2 h then fluorescence measured by 562

flow cytometry. Results for (C - E) are ± SE for three independent experiments. 563

564

565

566

567

568

569

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570

571

572 573

574

FIG. 2. OMV internalization occurs in the presence and absence of VacA. OMV from (A) 575

strain 60190 and (B) its VacA- isogenic mutant 60190:v1 within AGS cells. Green – OMV; 576

red – actin cytoskeleton. Images are a side view of composite Z-stack series. (C) To confirm 577

DiO fluorescence was OMV-associated, AGS cells were incubated with DiO-labeled OMV 578

for 5 h, fixed and immunolabeled with an antibody to H. pylori OMV detected with a PE-579

conjugated secondary antibody. No fluorescence was observed when cells were incubated 580

without OMV (Control). Image shows two adjacent cells whose cytoplasm contains labeled 581

OMV (n – nucleus; c – cytoplasm). (A – C) images are a representative of three independent 582

experiments. 583

584

585

586

587

588

n

c n

c

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2 4 60.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5 60190 60190 v:1

*

Hours

OM

V a

sso

ciat

ion

(fo

ld in

crea

se)

A

100% 50:50 100% 50:500

20

40

60

80

100

120

140

60190 60190 v:1

**

B

OM

V a

ssoc

iatio

n (%

of c

ontr

ol)

589

590

FIG. 3. The presence of VacA increases vesicle association with AGS cells. (A) Cells were 591

incubated for up to 6 h with DiO-labeled OMV from strain 60190 or 60190:v1 and the 592

increase in cell associated vesicle fluorescence was measured by flow cytometry. Data are 593

presented as fold increase of the fluorescence measured at 2 h for each experiment. (B) AGS 594

cells incubated for 4 h with DiO-labeled wild type OMV (black bar) or DiD-labeled VacA- 595

mutant OMV (grey bar) or a 50:50 mix of labeled OMV (hatched bar) from both strains. 596

Measurement of DiO fluorescence is shown over the bar designated 60190 while 597

measurement of DiD fluorescence is shown over the bar designated 60190 v:1. Results are 598

± SE for three (A) or four (B) independent experiments. (A) *, results are statistically 599

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different at the 6 h time point (p < 0.01). (B) **, significantly fewer VacA-

OMV are 600

internalized when mixed 50:50 with wild-type OMV. (p = 0.002). 601

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602

603

3.125

6.25

12.5 25 50

3.125

6.25

12.5 25 50

0

20

40

60

80

100

60190 60190:v1

*

*

*

*

* * * *

H. pylori LPS ( g/ml)

OM

V u

pta

ke (

% o

f co

ntr

ol)

604 605

606

FIG. 4. H. pylori LPS competitively inhibits OMV uptake by AGS cells. LPS (3.125 –607

50 µg/ml) was added to cells at the same time as labeled OMV from either wild type or 608

VacA- mutant strains. Cells were then incubated for 4 h and fluorescence measured after the 609

addition of trypan blue. Results are ±SE for three independent experiments performed in 610

triplicate. Overall, purified LPS had a significant effect on wild type and VacA- mutant 611

OMV uptake (p < 0.0001 and p = 0.0002, respectively). *, results are statistically significant 612

from controls not treated with purified LPS (p < 0.05). 613

614

615

616

617

618

619

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1 3 50

20

40

60

80

100

Methyl-beta-cyclodextran (mg/ml)

***

A

******

Vac

uola

tion

(% o

f con

trol

)

1 3 50

50

100

150

B

*****

*

Methyl-beta-cyclodextran (mg/ml)

OM

V u

ptak

e (%

of c

ontr

ol)

5 25 500

20

40

60

80

100

*

C

Nystatin (ug/ml)

Vac

uola

tion

(% o

f con

trol

)

*

5 25 50 5 25 500

50

100

150 60190 60190:v1

D

Nystatin (ug/ml)

OM

V u

ptak

e (%

of c

ontr

ol)

620 621

622

FIG. 5. Effect of MくCD and nystatin on vacuole formation and vesicle uptake in AGS cells. 623

(A) Cells were pre-treated with MくCD (1 – 5 mg/ml) for 1 h then incubated with 60190 624

OMV for 6 h. Vacuolation was measured by neutral red assay. (B) Treatment with MくCD 625

increased the uptake of 60190 OMV with AGS cells. Cells were pre-treated with MくCD for 626

1 h then incubated with DiO-labeled OMV for 4 h. Fluorescence was measured after the 627

addition of trypan blue. (C) Cells were pre-treated with nystatin (5 – 50 mg/ml) for 1 h then 628

incubated with OMV for 6 h. Vacuolation was measured by neutral red assay. (D) Cells 629

were pre-treated with nystatin for 1 h then incubated with DiO-labeled 60190 OMV or 630

labeled 60190:v1 OMV for 4 h. Fluorescence was measured after the addition of trypan 631

blue. Results for (A & C) are ±SE for three separate experiments performed in triplicate; (B 632

& D) are ±SE for three separate experiments. Overall, MくCD and nystatin had a significant 633

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effect on vacuole formation (p < 0.0001 and p = 0.027, respectively). *, **, ***, results are 634

statistically significant from controls not treated with MくCD or nystatin (p < 0.05, p < 0.01, 635

and p < 0.001, respectively). 636

637

638

639

640

641

642

643

644

645

646

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648

649

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651

652

653

654

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656

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659

660

5 10 150

50

100

150

Chlorpromazine ( g/ml)

***

60190A

Inhi

bitio

n (%

of c

ontr

ol

60190 (graph)

5 10 150

50

100

150

B


OM

Vup

take

(%

of c

ontr

ol)

60190:v1

5 10 150

50

100

150

*

C


OM

V u

ptak

e (%

of c

ont

rol)

LDL (graph)

5 10 150

50

100

150

*

D


LDL

upta

ke (

% o

f con

trol

)

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FIG. 6. Effect of chlorpromazine on vacuole formation and vesicle uptake in AGS cells. 663

(A) Cells were pre-treated with chlorpromazine (5 – 15 たg/ml) for 30 min then incubated 664

with 60190 OMV for 6 h. Vacuolation was measured by neutral red assay. (B & C) 665

Chlorpromazine reduces OMV uptake. Cells were pretreated with chlorpromazine for 666

30 min prior to 4 h incubation with labeled-OMV from either strain 60190 (B) or 60190v:1 667

(C) or with fluorescently labeled LDL (D). Fluorescence was measured after the addition of 668

trypan blue. Results for (A) are ±SE for three independent experiments performed in 669

triplicate. Overall, vacuolation decreased in 60190 OMV treated cells in response to a dose-670

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34

dependent increase in chlorpromazine (p < 0.0001). Results for (B) and (C) are ±SE for six 671

independent experiments for OMV and three separate experiments for LDL (D). Overall, 672

there was a significant decrease in 60190 (p = 0.011), 60190v:1 (p = 0.003) and LDL (p = 673

0.002) uptake in response to increasing concentration of chlorpromazine. *, ***, results are 674

statistically significant from controls not treated with chlorpromazine (p < 0.05 and < 0.001, 675

respectively). 676

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uptake of helicobacter pylori outer membrane vesicles by ... · 46 (32), leading to speculation...

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