Uptake of Helicobacter pylori outer membrane vesicles by ... · 46 (32), leading to speculation...
Transcript of Uptake of Helicobacter pylori outer membrane vesicles by ... · 46 (32), leading to speculation...
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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
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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
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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
531
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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
647
648
649
650
651
652
653
654
655
656
657
658
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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
Chlorpromazine ( g/ml)
OM
Vup
take
(%
of c
ontr
ol)
60190:v1
5 10 150
50
100
150
*
C
Chlorpromazine ( g/ml)
OM
V u
ptak
e (%
of c
ont
rol)
LDL (graph)
5 10 150
50
100
150
*
D
Chlorpromazine ( g/ml)
LDL
upta
ke (
% o
f con
trol
)
661
662
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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