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. 2021 Dec 28;7(1):240–258. doi: 10.1021/acsomega.1c04549

Outer Membrane Vesicles Secreted by Helicobacter pylori Transmitting Gastric Pathogenic Virulence Factors

Sisi Wei †, Xiaoya Li †, Jingjing Wang †, Yaojie Wang †, Cong Zhang †, Suli Dai †, Xian Wang ‡, Xiaoqing Deng †, Lianmei Zhao †,*, Baoen Shan †,*
PMCID: PMC8756444  PMID: 35036696

Abstract

graphic file with name ao1c04549_0009.jpg

Helicobacter pylori (H. pylori) is known to be a major pathogen causing gastric diseases through its direct localization in gastric epithelium cells. H. pylori releases outer membrane vesicles (OMVs) throughout the growth process. The content, function, and mechanism of H. pylori OMVs in gastric epithelial cells remain unclear. In this study, we extracted and characterized H. pylori OMVs of two strains (standard strain NCTC11637 and clinical strain Hp-400) and analyzed the specific content by proteomic technology. We identified more than 400 proteins in H. pylori OMVs. In addition, we investigated the impact of H. pylori OMVs on cellular functions by detecting proteomic changes in GES1 cells. GES1 cells cocultured with increasing concentrations of H. pylori OMVs were subjected to quantitative proteomic analyses using label-free methods for relative quantitation. The results showed that a total of 4261 proteins were verified, 153 of which were significantly altered in abundance when cocultured with NCTC11637 OMVs, and a total of 4234 proteins in Hp-400 OMVs, 390 of which were significantly altered. Gene ontology analysis and Kyoto encyclopedia of genes and genomes pathway mapping identified significantly altered inflammatory and cancer signaling pathways, including metabolic pathways and the PI3K-Akt signaling pathway. Furthermore, we explored the proteomic changes in GES1 cells induced by H. pylori. Bioinformatics analysis showed that changes in multiple pathways coincided with OMV-mediated proteomic changes. Based on these results, H. pylori induced pathogenicity in epithelial cells at least partially by secreting OMVs that mediated dramatic and specific proteomic changes in host cells. Data are available via ProteomeXchange with identifiers PXD025216, PXD025259, and PXD025281.

Introduction

Helicobacter pylori (H. pylori) is a spiral, microaerophilic, and Gram-negative bacterium that primarily colonizes the human stomach.1H. pylori persists in the human stomach lifelong and is predicted to have infected approximately half of the global population to cause multiple diseases, such as chronic gastritis, peptic ulcer, gastric mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. H. pylori was also identified as a type I carcinogen by the WHO (World Health Organization) and contributes to a higher occurrence of gastric carcinoma.

Outer membrane vesicles (OMVs) are nanosized particles derived from the outer membrane of Gram-negative bacteria and play central roles in initiating and regulating pathogenesis in the host. OMVs generally have a diameter of 20–250 nm and are secreted under all environmental conditions and during all growth phases.2,3 Originally considered as artifacts of the cell wall, OMVs are now accepted as a general secretion system.4 OMVs carry a large amount of cargo from their parent bacterium, including virulence factors and toxins, such as outer membrane proteins, adhesins, invasions, proteases, and lipopolysaccharide (LPS),5,6 illustrating that OMV secretion is an additional virulence mechanism of pathogens. The cargo may either be located in the vesicle lumen or integrated into the vesicle membrane.7,8 Compared to other secretion systems, OMVs protect their contents from the external environment and transport their cargo over a long distance.3,9

Similar to other Gram-negative bacteria, H. pylori spontaneously secretes OMVs that play important roles in the pathogen–host interaction mechanism.10 Several studies showed that the secreted H. pylori OMVs are internalized by gastric epithelial cells.11−13 After internalization, OMVs regulate gastric epithelial cell proliferation, facilitate the secretion of inflammatory factors, and induce apoptosis.12,14 In addition, H. pylori OMVs cause genomic instability in epithelial cells, as assessed using the cytokinesis-block micronuclei assay.15 Furthermore, H. pylori OMVs induce human eosinophil degranulation.16 Based on these results, we speculated that OMVs derived from H. pylori contributed to the H. pylori-induced pathogenic effects on the stomach.

In this study, we purified and identified proteins in H. pylori-derived OMVs. We detected the protein contents of OMVs, including cagA, vacA, ureB, outer membrane proteins, and other virulence factors. We also found that H. pylori OMVs promoted the secretion of inflammatory cytokines, consistent with their parental bacteria. Furthermore, we identified proteomic changes in GES1 cells in response to OMVs or their parental bacteria. The bioinformatics analysis showed that multiple pathways overlapped, suggesting that OMVs contain most of the contents from their parental bacteria. Therefore, we highlight that H. pylori secretes and delivers gastric pathogenic virulence factors mostly via outer membrane vesicles.

Results

Purification and Characterization of the H. pylori OMVs

H. pylori continuously secretes OMVs into the extracellular environment during growth. We collected a conditioned medium from NCTC11637 or Hp-400 and isolated OMVs after culturing for 72 h. Then, the H. pylori OMVs were characterized using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). TEM images revealed that the vesicles showed a spherical, bilayered morphology and a typical cup-shaped structure (Figure 1A,B). Additionally, NTA results showed that the size distribution of the OMVs ranged from 50 to 250 nm in diameter (Figure 1C,D), which is the typical size of OMVs produced by Gram-negative bacteria. Taken together, we successfully purified H. pylori OMVs.

Figure 1.

Figure 1

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Purification and characterization of the H. pylori OMVs. (A) Representative TEM images of OMVs secreted by NCTC11637. (B) Representative TEM images of OMVs secreted by Hp-400. (C) NTA analysis of the size distributions and numbers of OMVs derived from NCTC11637. (D) NTA analysis of the size distributions and numbers of OMVs derived from Hp-400.

Subsequently, in order to detect the proteomic changes in GES1 cells cocultured with gradually increasing concentrations of OMVs and H. pylori as well as to reveal the influence of OMVs and H. pylori on host gastric epithelial cells, an experimental scheme focused on the HPLC-MS/MS method was adopted and its workflow is shown in Figure 2.

Figure 2.

Figure 2

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Schematic experimental workflow: First, the contents of purified OMVs were examined using HPLC-MS/MS. Then, GES1 cells cocultured with gradually increasing concentrations of OMVs and H. pylori were subjected to quantitative proteomic analysis. Finally, bioinformatics analysis was performed on the above data, on which basis, the exact pathways and proteins that changed in the infected GES1 cell proteome were characterized. The MS analysis of each sample was performed in triplicate, and data analysis was performed with the software PD2.2.

Identification of the Protein Contents of H. pylori OMVs

Next, we determined the protein contents of H. pylori OMVs to evaluate the mechanisms by which these components confer their immunomodulatory and cytotoxic activities to host cells, as these disease-associated activities are also transferred by the bacterium from which the vesicles are derived. We detected the protein contents of OMVs using HPLC-MS/MS analysis, and 436 proteins were found in NCTC11637 OMVs (Table 1) and 372 proteins in Hp-400 OMVs (Table 2). Although a significant overlap in the proteins identified between NCTC11637 and Hp-400 was observed, not all proteins appeared to be shared, suggesting that these two H. pylori strains have different genotypes (Figure 3A). The proteomic analysis illustrated the enrichment of membrane proteins, adhesins, porins, and several proteins known to regulate cell proliferation, cytokine secretion, and other host cellular processes in H. pylori OMVs. In addition, the OMVs contained the previously documented toxins cagA, vacA, and several OMV components possessing immunological activity, including urease, HpaA, OMP18, peptidyl-prolyl-cis-trans-isomerase, and gamma-glutamyl transpeptidase (Tables 1 and 2). Furthermore, the GO analysis revealed similar cellular components for the OMV contents in the two strains, mainly including the cytoplasmic part, such as the cytoplasm and cytomembrane (Figure 3B,C). Taken together, the H. pylori OMVs are equipped with the molecules required to interact with host cells in a manner similar to the intact pathogen.

Table 1. Protein Contents in NCTC11637 OMVs.

number accession gene symbol number accession gene symbol
1 P42383 groL 2 P56003 tuf
3 P69996 ureB 4 P0A0S1 flaA
5 P77872 katA 6 O26107 HP_1588
7 P56002 fusA 8 P55980 cagA
9 O25905 HP_1350 10 P55994 dnaK
11 P55987 atpA 12 P56418 acnB
13 O25242 dnaN 14 P56063 icd
15 P56112 HP_0175 16 P56185 rnj
17 P55975 tsf 18 P94845 glnA
19 P55988 atpD 20 P55969 hpaA
21 O25806 rpoBC 22 O25743 Ggt
23 O25294 pepA 24 O25017 HP_0231
25 P56116 htpG 26 P55981 vacA
27 O25284 HP_0558 28 P21762 ahpC
29 P52093 ftnA 30 P14916 ureA
31 P71404 clpB 32 O25011 msrAB
33 P56149 aspA 34 P0A0V0 lpp20
35 O25556 Omp19 36 P56088 guaB
37 P56008 rpsA 38 G2J5T2 hp1018/19
39 O25286 FabG 40 P50610 flgE
41 O25948 Ald 42 Q07911 flaB
43 O25750 Omp18 44 O06913 frdA
45 O25325 hemE 46 O25318 HP_0596
47 P43313 dps 48 O25751 tolB
49 O25349 HydB 50 O25739 HP_1111
51 P56420 tig 52 O26102 pdxJ
53 O25883 fumC 54 O25786 GlnH
55 O25656 PqqE 56 O25997 HP_1461
57 P56030 rplB 58 P56036 rplJ
59 O25749 HP_1124 60 O25546 HP_0879
61 O25321 HylB 62 O25371 YmxG
63 O24854 ribH 64 P56126 lysS
65 O24897 PutA 66 O25570 Omp20
67 O25015 Omp6 68 O24993 plsX
69 P56070 ppsA 70 O25326 HP_0605
71 O25135 HP_0371 72 O25216 PepF
73 O06914 frdB 74 O25736 HP_1108
75 P56001 rpoA 76 O25668 CbpA
77 A0A0M3KL20 C694_06140 78 O26082 CeuE
79 P56033 rplE 80 O25311 HP_0589
81 P56062 gltA 82 O24923 HP_0097
83 O24944 HP_0130 84 O25225 typA
85 O25534 pgbB 86 O25254 hslU
87 O25995 HP_1457 88 P96786 fliD
89 O26084 HP_1564 90 O25993 HP_1454
91 O25158 HP_0397 92 P56114 gatA
93 P56109 fba 94 O25825 HP_1227
95 O25856 NQO3 96 O34523 Omp29
97 O25927 lpxA 98 O25134 HP_0370
99 O25399 HP_0690 100 P56029 rplA
101 O25738 HP_1110 102 O25414 HP_0710
103 O25423 HP_0721 104 O25347 HP_0630
105 O25715 HP_1083 106 P56456 ileS
107 P56145 pheT 108 P56031 rplC
109 O24914 HP_0087 110 O25732 Cad
111 O25116 pyrG 112 O25383 HP_0672
113 O25787 HP_1173 114 O25166 HP_0410
115 O25052 AddB 116 O25465 HP_0773
117 O25008 iscS 118 O25067 amiE
119 P56060 kdsA 120 O25658 HdhA
121 O25936 fbp 122 P55982 nrdA
123 O25089 HP_0322 124 P66928 trxA
125 P56047 rplV 126 O24990 fabI
127 P0A0R3 groS 128 O26104 FlgG
129 O25560 hypB 130 O25571 Omp21
131 O25873 HP_1286 132 P56078 rplY
133 O25608 rdxA 134 O25410 Omp15
135 O24913 mqo 136 O25312 HP_0590
137 P56004 efp 138 O25925 mreB
139 P56431 trxB 140 O25327 MtrC
141 P25177 glmM 142 O25147 HP_0385
143 P56111 edd 144 P56146 pheS
145 O25731 glk 146 O25820 Dld
147 O25372 gatB 148 P56034 rplF
149 P42445 recA 150 P56460 metK
151 P56071 thrS 152 O25088 tatA
153 P56154 pgk 154 P56458 serS
155 O24922 HP_0096 156 O25009 HP_0221
157 P56032 rplD 158 O24925 TlpA
159 O24911 TlpC 160 O26004 ilvE
161 O25373 HP_0659 162 O24870 Omp2
163 Q09066 ureG 164 O25776 fldA
165 O25720 TktA 166 O25079 HP_0309
167 O24924 thrC 168 P56082 atpG
169 O25503 speE 170 P55972 infB
171 P56457 leuS 172 O25313 HP_0591
173 O25034 Omp7 174 P56155 pyrF
175 P64655 HP_0135 176 P55995 lon
177 O25872 HP_1285 178 O26075 yajC
179 P66609 rpsG 180 P94851 HP_1488
181 O25744 HAP1 182 O25140 DsbC
183 O24947 HP_0134 184 Q48248 cdh
185 O25151 tpx 186 O25779 TrxB
187 O25046 HP_0267 188 P71408 ftsH
189 P55834 rplL 190 O25018 HP_0232
191 P56046 rplU 192 O25341 AspB
193 P66328 rpsJ 194 P56035 rplI
195 O25597 dadA 196 O25369 bamA
197 O25389 HP_0678 198 P96551 gltX1
199 O25684 HP_1043 200 O24886 fcl
201 O25671 fur 202 P66572 rpsE
203 P56069 metB 204 O25607 HP_0953
205 O25029 rhpA 206 O25756 AtpH
207 O25530 RfaD 208 P48285 eno
209 P66052 rplK 210 O25625 HP_0973
211 O25728 hcpC 212 P56089 glyA
213 O25729 HP_1099 214 O24976 HP_0170
215 P56007 scoB 216 O25249 pgbA
217 O25762 HP_1143 218 P56106 pyrH
219 O25998 HP_1462 220 P56459 aspS
221 O25068 Fla 222 O24951 HP_0139
223 P66637 rpsI 224 O25036 Omp8
225 P56191 ddl 226 P56052 rpmC
227 O25087 hugZ 228 P48370 gyrA
229 O25080 pgdA 230 O25276 Cag22
231 O25157 HP_0396 232 O25773 proC
233 O25996 HP_1458 234 O25424 ansA
235 P56020 rpsM 236 O25283 accA
237 O25342 ispG 238 O25442 HP_0746
239 P56038 rplM 240 P56009 rpsB
241 O25771 Omp25 242 P56011 rpsD
243 O25001 hcpA 244 P56041 rplP
245 O24996 HP_0204 246 P66449 rpsQ
247 O25164 HP_0408 248 O25681 HP_1037
249 Q48255 aroQ 250 P56018 rpsK
251 P0A0X4 rpsL 252 O25791 Omp27
253 P56417 tyrS 254 O25999 HP_1463
255 P56010 rpsC 256 O25250 GlcD
257 O25572 HP_0914 258 P56006 scoA
259 O25176 HP_0422 260 O24999 mrp
261 P56156 clpP 262 O25360 gltX2
263 O26037 HP_1507 264 O25413 HP_0709
265 O25229 HP_0485 266 O25781 pgi
267 O25564 HP_0906 268 P56039 rplN
269 P56084 atpC 270 O25673 HP_1029
271 O24949 HP_0137 272 O25452 HP_0757
273 O25553 HP_0893 274 O24865 HP_0020
275 O26035 RibG 276 O25949 HP_1399
277 O25255 HP_0518 278 O26083 CeuE
279 O25213 HP_0466 280 O25253 hslV
281 O25006 HP_0218 282 P55992 gyrB
283 O24950 HP_0138 284 O25280 HP_0554
285 P56141 trpA 286 P56110 zwf
287 O25076 HP_0305 288 O25926 clpX
289 O25930 bamD 290 P56045 rplT
291 P56097 ftsZ 292 O25899 tonB
293 P56128 argS 294 O25489 HP_0809
295 O25664 ispDF 296 O25234 HP_0492
297 O25511 pseB 298 O25737 HP_1109
299 P64653 HP_0122 300 O25516 thiM
301 O25990 HP_1451 302 O25801 asd
303 O25310 HP_0588 304 O24934 HP_0112
305 O25030 HP_0248 306 O24943 HP_0129
307 O24941 Omp4 308 P56086 atpF
309 P56455 hisS 310 P55970 grpE
311 O25566 HP_0908 312 P56044 rplS
313 O25524 YheS 314 O25257 Cag1
315 O25982 ppiA 316 O25470 HP_0781
317 O25992 HP_1453 318 O25931 TyrA
319 P66185 rpmE 320 P56162 pyrE
321 O25853 nuoD 322 P56067 cysM
323 O24884 HP_0043 324 O25510 OmpP1
325 O25421 HP_0719 326 P56075 ndk
327 P55976 nusG 328 O24991 lpxD
329 P56396 trpS 330 O25019 HP_0233
331 O25529 hldE 332 O25614 gpsA
333 P43312 sodB 334 O26067 HP_1542
335 P56021 rpsZ 336 O25565 FlgD
337 O25858 nuoI 338 O25759 Soj
339 O25584 surE 340 P66119 rplW
341 P56000 valS 342 P55971 gapA
343 O25362 Slt 344 P55985 truD
345 O25758 parB 346 O25782 HP_1167
347 O25343 dapD 348 O25032 OppD
349 O25956 bioB 350 O26094 RibC
351 O25686 acsA 352 O25748 slyD
353 O25953 HsdM 354 P66621 rpsH
355 Q59465 cadA 356 O25277 Cag24
357 O25171 Cfa 358 P56124 proS
359 P56040 rplO 360 P55979 bcp
361 P56195 deoB 362 O25913 HP_1359
363 O25521 HsdM 364 O25132 HP_0368
365 O25757 AtpF′ 366 O25896 HP_1338
367 O25525 guaC 368 O25121 dxs
369 O25549 ruvA 370 P56104 adk
371 O25293 ychF 372 O25595 alr
373 P56737 trpD 374 O26103 pdxA
375 P56137 purA 376 P56452 alaS
377 P94842 ybgC 378 O24885 gmd
379 O24864 CheV 380 P56184 prs
381 O25475 secA 382 O25477 HP_0788
383 O24973 OmpR 384 O26096 metN
385 P56157 dnaE 386 O24890 HP_0049
387 O26064 HP_1539 388 O25533 coaX
389 P56115 hemL 390 O25577 carB
391 P56176 nnr 392 O25335 HP_0614
393 P56153 ppa 394 O25469 HP_0780
395 O25624 HP_0971 396 O25376 hemN
397 O25929 fliW2 398 O25281 HP_0555
399 O25233 HP_0490 400 O25398 HP_0689
401 O25435 Gpt 402 O24994 fabH
403 O25136 dcd 404 P56022 rpsO
405 O26074 secD 406 P56074 hemB
407 O25382 Omp14 408 O25991 mnmE
409 O25945 Omp30 410 P55990 gdhA
411 P56028 rpsU 412 O25430 HP_0730
413 O25696 HP_1056 414 O25122 lepA
415 O25390 WbpB 416 P56127 metG
417 O25348 HydA 418 P56142 trpB
419 O25902 Gap 420 P56122 aroC
421 O25849 cobB 422 O24956 FixO
423 O25308 HP_0586 424 O25484 ribB
425 O25594 YckK 426 O25817 purD
427 O25500 Lex2B 428 P56131 miaB
429 O25055 GppA 430 O25195 HP_0447
431 O25912 HP_1358 432 O25142 HP_0379
433 O25363 HP_0646 434 O25082 HP_0312
435 O25509 HP_0838 436 O25278 Cag25
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Table 2. Protein Contents in Hp-400 OMVs.

number accession gene symbol number accession gene symbol
1 P42383 groL 2 P56003 tuf
3 P69996 ureB 4 P77872 katA
5 O25806 rpoBC 6 G2J5T2 hp1018/19
7 O26107 HP_1588 8 O24870 Omp2
9 P55987 atpA 10 O25743 HP_1118
11 P56418 acnB 12 P55975 tsf
13 P56063 icd 14 O25011 msrAB
15 P0A0V0 lpp20 16 P71404 clpB
17 P55969 hpaA 18 P55994 dnaK
19 O25905 HP_1350 20 O25286 HP_0561
21 O25751 tolB 22 P94845 glnA
23 O25242 dnaN 24 P55988 atpD
25 O25791 Omp27 26 A0A0M3KL20 C694_06140
27 P56002 fusA 28 O25017 HP_0231
29 O25311 HP_0589 30 O25825 HP_1227
31 O25321 HP_0599 32 O25294 pepA
33 O26083 HP_1562 34 O25423 HP_0721
35 P14916 ureA 36 O06913 frdA
37 O25732 HP_1104 38 O25284 HP_0558
39 O25052 AddB 40 P56008 rpsA
41 O25015 Omp6 42 O25786 GlnH
43 O24925 TlpA 44 P56112 HP_0175
45 O25749 HP_1124 46 O26084 HP_1564
47 P56456 ileS 48 P50610 flgE
49 O25840 Omp28 50 P21762 ahpC
51 P55981 vacA 52 P52093 ftnA
53 P56036 rplJ 54 O25993 HP_1454
55 P56149 aspA 56 O25312 HP_0590
57 O25883 fumC 58 O25216 PepF
59 O25147 HP_0385 60 O25656 PqqE
61 O25997 HP_1461 62 P56062 gltA
63 O25738 HP_1110 64 O25414 HP_0710
65 P56185 rnj 66 O25736 HP_1108
67 P56145 pheT 68 O26082 CeuE
69 P56155 pyrF 70 O24968 pyrF
71 O26102 pdxJ 72 O25995 HP_1457
73 O25927 lpxA 74 O25750 Omp18
75 O24944 HP_0130 76 O25157 HP_0396
77 O24923 HP_0097 78 O25872 HP_1285
79 O25046 HP_0267 80 P56116 htpG
81 O24922 HP_0096 82 O25729 Eda
83 O25992 HP_1453 84 O25158 HP_0397
85 O25597 dadA 86 O24993 plsX
87 O25055 GppA 88 O25510 OmpP1
89 O25229 HP_0485 90 P55982 nrdA
91 O25556 Omp19 92 P66928 trxA
93 P55993 rpoD 94 O25402 HyuA
95 O25135 HP_0371 96 O25349 HydB
97 O25728 hcpC 98 P0A0R3 groS
99 P43313 dps 100 P56070 ppsA
101 O25757 AtpF′ 102 O26042 FrpB
103 O25739 HP_1111 104 O25326 HP_0605
105 O25371 YmxG 106 O25225 typA
107 O06914 frdB 108 P56030 rplB
109 O25088 tatA 110 P56060 kdsA
111 P56047 rplV 112 P55980 cagA
113 P56111 edd 114 O25313 HP_0591
115 O25045 pyrC′ 116 P56420 tig
117 O25776 fldA 118 O25257 Cag1
119 P56088 guaB 120 O25771 Omp25
121 O25936 fbp 122 O25176 HP_0422
123 P56007 scoB 124 O25948 Ald
125 P56078 rplY 126 P56431 trxB
127 O25570 Omp20 128 O25756 AtpH
129 O25399 FadA 130 P25177 glmM
131 O25715 HP_1083 132 O25787 HP_1173
133 P56034 rplF 134 O25658 HdhA
135 O25607 HP_0953 136 O25369 bamA
137 P56110 zwf 138 O25318 HP_0596
139 O25562 HP_0902 140 O25076 HP_0305
141 O26071 HP_1546 142 O25625 HP_0973
143 O24996 HP_0204 144 P56001 rpoA
145 O25410 Omp15 146 O25325 hemE
147 O25781 pgi 148 O25153 CheA
149 O26067 HP_1542 150 O24913 mqo
151 O24947 HP_0134 152 O25403 HP_0696
153 P56154 pgk 154 O25465 HP_0773
155 P42445 recA 156 O24914 HP_0087
157 O24911 TlpC 158 O24897 PutA
159 O25327 MtrC 160 O25534 pgbB
161 P56146 pheS 162 O25442 HP_0746
163 O25773 proC 164 O25742 HP_1117
165 O25503 speE 166 O24881 HP_0040
167 P56458 serS 168 O25372 gatB
169 O24950 HP_0138 170 O25373 HP_0659
171 O25998 HP_1462 172 P56114 gatA
173 O25546 HP_0879 174 O25668 CbpA
175 P56067 cysM 176 O25469 HP_0780
177 O25249 pgbA 178 O25069 DppA
179 O25873 HP_1286 180 O25230 HP_0486
181 O24909 HP_0080 182 P56106 pyrH
183 O25470 HP_0781 184 P56046 rplU
185 P56126 lysS 186 O25458 ftsY
187 P56082 atpG 188 P56075 ndk
189 O25273 Cag19 190 P56006 scoA
191 O25140 DsbC 192 O25926 clpX
193 P56109 fba 194 P56460 metK
195 O25134 HP_0370 196 O25213 HP_0466
197 Q48248 cdh 198 P56127 metG
199 P64655 HP_0135 200 P56052 rpmC
201 O26091 rlpA 202 P56031 rplC
203 Q09066 ureG 204 O25018 HP_0232
205 O25902 Gap 206 P56457 leuS
207 O24929 TlpB 208 O25009 NifU
209 O25424 ansA 210 O26031 Omp32
211 O25564 HP_0906 212 P56104 adk
213 O25477 HP_0788 214 O25475 secA
215 P56035 rplI 216 O25474 lolA
217 O25165 guaA 218 O25283 accA
219 P56468 purB 220 O25574 FrpB
221 O24924 thrC 222 O25572 HP_0914
223 P56004 efp 224 P56032 rplD
225 O25452 HP_0757 226 P56455 hisS
227 O25508 HP_0837 228 P56137 purA
229 O25036 Omp8 230 O25925 mreB
231 O25594 YckK 232 P56459 aspS
233 O24949 HP_0137 234 O25999 HP_1463
235 P56018 rpsK 236 O25820 Dld
237 P56029 rplA 238 O25255 HP_0518
239 O24863 HP_0018 240 O25087 hugZ
241 O26037 HP_1507 242 P56084 atpC
243 O24930 CpdB 244 P56020 rpsM
245 O25368 mqnE 246 P55970 grpE
247 O25218 Omp11 248 P64653 HP_0122
249 O25234 HP_0492 250 O25383 HP_0672
251 O25762 HP_1143 252 P66637 HP_1143
253 O26039 plsY 254 O24864 CheV
255 O26052 HP_1524 256 O25073 DppF
257 O24999 mrp 258 O25684 HP_1043
259 P56041 rplP 260 O25288 HP_0564
261 P56039 rplN 262 O25930 BamD
263 O24951 HP_0139 264 O25089 HP_0322
265 O25426 HP_0726 266 O25116 pyrG
267 O25256 HP_0519 268 P94844 dapB
269 P66052 rplK 270 O25573 FrpB
271 O25856 NQO3 272 O25713 HP_1081
273 O25276 Cag22 274 O25362 Slt
275 O25355 Omp13 276 O25090 Nuc
277 O25472 HP_0783 278 P56089 glyA
279 P64649 HP_0031 280 P56033 rplE
281 O24854 ribH 282 O24941 Omp4
283 O34523 Omp29 284 O25770 murG
285 O25595 alr 286 O25336 ligA
287 O25489 HP_0809 288 P56044 rplS
289 O26004 ilvE 290 P55971 gapA
291 P56069 metB 292 O25289 HP_0565
293 P56086 atpF 294 P48285 eno
295 O25152 CheW 296 O24946 SdaC
297 P56197 aroA 298 O25297 HP_0573
299 O25397 HP_0688 300 O25990 HP_1451
301 O24865 HP_0020 302 O25343 dapD
303 O87326 trl 304 O25507 HP_0836
305 O25171 Cfa 306 O25681 HP_1037
307 O24871 HP_0028 308 O25161 HP_0405
309 P94851 HP_1488 310 O25072 DppD
311 O25029 rhpA 312 O25571 Omp21
313 O25530 RfaD 314 O25696 HP_1056
315 O25671 fur 316 O25144 YJR117W
317 P55995 lon 318 O25079 HP_0309
319 O25560 hypB 320 O26096 metN
321 O25509 HP_0838 322 O25612 HP_0958
323 P66119 rplW 324 O25512 CoaBC
325 O25296 apt 326 O25032 OppD
327 P56061 panC 328 O25281 HP_0555
329 O25748 slyD 330 O25484 ribB
331 P66328 rpsJ 332 O25250 GlcD
333 O25337 CheV 334 O25584 surE
335 P56467 folD 336 O24886 fcl
337 O26075 yajC 338 P56072 sdaA
339 P56011 rpsD 340 O25772 Omp26
341 O24991 lpxD 342 O25166 HP_0410
343 O25039 xseA 344 P55972 infB
345 O25382 Omp14 346 O25348 HydA
347 P56191 ddl 348 P56153 ppa
349 O25413 HP_0709 350 O25714 MsbA
351 O25673 HP_1029 352 O25991 mnmE
353 P66572 rpsE 354 P55834 rplL
355 P56000 valS 356 P55992 gyrB
357 P56040 rplO 358 O25945 Omp30
359 O25535 HP_0864 360 O25008 iscS
361 P55976 nusG 362 P55986 HP_1459
363 O25347 Mda66 364 P56038 rplM
365 O25274 Cag20 366 O25151 tpx
367 O25852 HP_1262 368 P56022 rpsO
369 P56156 clpP 370 O25931 TyrA
371 O25614 gpsA 372 P56097 ftsZ
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Figure 3.

Figure 3

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Identification of protein contents of H. pylori OMVs. (A) Proteins in NCTC11637 and Hp-400 detected by HPLC-MS/MS. (B) Cellular components of NCTC11637 OMV contents revealed by GO analysis. (C) Cellular components of Hp-400 OMV contents revealed by GO analysis.

H. pylori OMVs Promoted the Secretion of Inflammatory Factors of GES1 Cells

H. pylori colonizes the gastric mucosa and causes acute and chronic gastritis accompanied by a chronic pro-inflammatory environment, and thus the inflammatory response is the main characteristic of H. pylori infection. As shown above, OMVs contain a variety of virulence factors; therefore, we hypothesized that OMVs induce inflammation similar to the bacterium from which they are derived. Inflammatory factors were detected in the cultured supernatant of GES1 cells cocultured with OMVs (40 μg) or H. pylori (50:1). The levels of secreted IL-5, IL-6, IFN-γ, IL-8, IL-12P70, and TNF-α were significantly increased when cells were cocultured with either OMVs or H. pylori (Figure 4A). In particular, IL-6, IL-8, and TNF-α play an important role in the activation of neutrophils and lymphocytes and the induction of T cell activation, proliferation, and differentiation. The levels of other inflammatory factors, IL-2, IL-10, and IL-17, were also slightly increased (Figure 4B), although the difference was not significant. Additionally, we did not observe a difference in cytokine levels between H. pylori-treated cells and OMV-treated cells, suggesting that H. pylori induced an inflammatory response mainly through OMVs.

Figure 4.

Figure 4

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H. pylori and OMVs induced secretion of inflammatory factors. (A) Inflammatory factors, IL-5, IL-6, IFN-γ, IL-8, IL-12P70, and TNF-α, in the cultural supernatant were detected by flow cytometry. (B) Inflammatory factors, IL-2, IL-10, IL-17, IFN-α, and IL-4, the in cultural supernatant were detected by flow cytometry. *p < 0.05, **p < 0.01.

The Proteomic Changes in GES1 Cells Cocultured with NCTC11637 OMVs Were Consistent with Those of GES1 Cells Cocultured with the NCTC11637 Strain

GES1 cells were cocultured with increasing concentrations of OMVs (0, 10, 20, or 40 μg) or bacteria (control, 1:1, 10:1, or 50:1) and subjected to quantitative proteomic analyses using label-free methods for relative and absolute quantitation to further define the effects of NCTC11637 OMVs and the parental strain on gastric epithelial cells. A total of 4261 proteins were quantified in GES1 cells cocultured with OMVs, 79, 128, and 153 of which were markedly changed (|fold change| > 2) in abundance after treatment with 10, 20, and 40 μg of OMVs, respectively, compared to control samples (Figure 5A and Supporting information Table S1); we described the difference in the proteome of cells treated with OMVs (40 μg). KEGG and GO analyses were next used to find biologically relevant canonical signaling pathways that were significantly altered by OMVs. In the KEGG pathway analysis, RNA transport and degradation, oxidative phosphorylation, metabolism, tight junctions, cytoskeleton, and extracellular matrix signaling were significantly altered (Figure 5B). In the GO analysis, including biological processes, cellular components and molecular functions, IL-12 signaling pathways, VEGF receptor pathway, antioxidant activity, apoptosis, and other terms were dramatically altered (Figure 5C). These pathways are related to immune regulation and carcinogenesis.

Figure 5.

Figure 5

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Proteomic changes of GES1 infected by NCTC11637 OMVs and bacteria. (A) Heat map showing differentially expressed proteins in different groups of GES1 cocultured with increasing NCTC11637 OMVs (0, 10, 20, and 40 μg/well). (B) KEGG analysis of differentially expressed proteins in GES1 control and GES1 cocultured with 40 μg of NCTC11637 OMVs. (C) GO analysis of differentially expressed proteins in GES1 control and GES1 cocultured with 40 μg of NCTC11637 OMVs. (D) Heat map showing differentially expressed proteins in different groups of GES1 cocultured with increasing NCTC11637 (0, 1:1, 10:1, and 50:1). (E) KEGG analysis of differentially expressed proteins in GES1 control and GES1 cocultured with 50:1 NCTC11637. (F) GO analysis of differentially expressed proteins in GES1 control and GES1 cocultured with 50:1 NCTC11637. (G) Venn diagram revealed the overlapped proteins between differentially expressed proteins in GES1 infected by NCTC11637 and OMVs. (H) Top 10 hub genes of the overlapped proteins in panel G.

Similarly, we identified 4360 proteins in GES1 cells cocultured with the NCTC11637 strain; 53, 165, and 367 proteins displayed significantly altered abundance (|fold change| > 2) after infection with the bacteria at multiplicities of infection (MOIs) of 1:1, 10:1, and 50:1, respectively, compared to uninfected samples (Figure 5D and Supporting information Table S2). Therefore, we analyzed the proteome of infected GES1 cells in the 50:1 group. The KEGG analysis showed significant changes in amino acid metabolism, p53 signaling pathway, ECM-receptor interaction, and epithelial cell signaling in response to the H. pylori infection (Figure 5E). The GO analysis revealed that T cell-mediated immunity, integrin binding, mitotic cell cycle, antioxidant activity, and chromosome organization were dramatically altered (Figure 5F) in response to NCTC11637 infection.

In addition, 35 proteins overlapped between the proteomes of GES1 cells infected with NCTC11637 and OMVs (Figure 5G). Furthermore, the top 10 hub genes were screened (Figure 5H). Taken together, these results revealed that NCTC11637 OMVs led to changes in the GES1 cell proteome and the altered pathways mapped to the donor bacteria.

The Proteomic Changes in GES1 Cells Cocultured with Hp-400 OMVs Were in Accordance with Those of GES1 Cells Cocultured with Hp-400

Another H. pylori strain, Hp-400, was used to detect proteomic changes in cells cocultured with OMVs or the H. pylori strain and to further confirm our hypothesis that OMVs play vital roles in H. pylori-treated GES1 cells. Hp-400 is a clinical strain isolated from northern China, where the incidence of gastric cancer is high. Consistent with our hypothesis, the quantitative proteomic analysis verified a total of 4234 proteins in GES1 cells cocultured with Hp-400 OMVs, 303, 236, and 390 of which exhibited significantly altered (|fold change| > 1.5) abundance following infection with 10, 20, and 40 μg of Hp-400 OMVs, respectively, compared to uninfected samples (Figure 6A and Supporting information Table S3). Consistent with the aforementioned findings, we described the GES1 proteomic change induced by OMVs (40 μg). The KEGG analysis revealed marked changes in several pathways, including amino acid metabolism, spliceosome, RNA process, and protein exporting (Figure 6B). The GO analysis showed significant changes in cadherin binding, endocytosis, mitochondrial matrix, and ubiquitin binding pathways in GES1 cells cultured with 40 μg of OMVs (Figure 6C).

Figure 6.

Figure 6

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Proteomic changes of GES1 infected by Hp-400 OMVs and bacteria. (A) Heat map showing differentially expressed proteins in different groups of GES1 cocultured with increasing Hp-400 OMVs (0, 10, 20, and 40 μg/well). (B) KEGG analysis of differentially expressed proteins in GES1 control and GES1 cocultured with 40 μg of Hp-400 OMVs. (C) GO analysis of differentially expressed proteins in GES1 control and GES1 cocultured with 40 μg of Hp-400 OMVs. (D) Heat map showing differentially expressed proteins in different groups of GES1 cocultured with increasing Hp-400 (0, 1:1, 10:1, and 50:1). (E) KEGG analysis of differentially expressed proteins in GES1 control and GES1 cocultured with 50:1 Hp-400. (F) GO analysis of differentially expressed proteins in GES1 control and GES1 cocultured with 50:1 Hp-400. (G) Venn diagram revealed the overlapped proteins between differentially expressed proteins in GES1 infected by Hp-400 and OMVs. (H) Top 10 hub genes of the overlapped proteins in panel G.

We also detected proteomic changes in GES1 cells cocultured with Hp-400 cells. A total of 4406 proteins were identified, and 243, 307, and 405 proteins were significantly changed (|fold change| > 2) in abundance after infection with Hp-400 at MOIs of 1:1, 10:1, and 50:1, respectively, compared to uninfected samples (Figure 6D and Supporting information Table S4). We characterized changes in the GES1 cell proteome after infection with 50:1 Hp-400. The KEGG analysis showed significant changes in oxidative phosphorylation, phagosome, pyrimidine metabolism, and p53 signaling pathways (Figure 6E). In addition, the GO analysis showed that T cell-mediated immunity, apoptosis, protein–DNA complex assembly, and the cell cycle were altered (Figure 6F).

Certain pathways altered by Hp-400 OMVs were also changed in response to Hp-400, including adhesion molecules, RNA polymerase, protein processing in the endoplasmic reticulum, and RNA processing. Forty-three proteins overlapped between Hp-400 OMV- and Hp-400-infected GES1 cells (Figure 6G), of which the top 10 hub genes were screened (Figure 6H). These results further indicated that H. pylori affected the proteomes of gastric epithelial cells partially by secreting OMVs.

OMVs and H. pylori Mediated the Upregulation of VTN and C3 in GES1 Cells

We integrated the top 10 hub genes shown in Figures 5H and 6H to confirm the common markers of H. pylori and OMV infection. Furthermore, we screened the expression abundance of these proteins and found that the levels of most proteins increased progressively with the increase in the concentrations of OMVs or H. pylori (Figure 7A,B). Among these proteins, VTN and C3 were both elevated in response to treatments with OMVs and H. pylori strains. Hence, we detected the expression of VTN and C3 using laser scanning confocal microscopy (LSCM). Both OMVs and H. pylori promoted VTN and C3 expressions (Figure 7C). Taken together, we revealed that VTN and C3 were the pathogenic targets of H. pylori on gastric epithelium cells by secreting OMVs.

Figure 7.

Figure 7

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Screening and verification of the hub genes altered both by H. pylori and OMVs. (A) Actual expression of hub genes in NCTC11637 and OMV proteomic data. (B) Actual expression of hub genes in Hp-400 and OMV proteomic data. (C) Immunofluorescence analysis of VTN and C3 in GES1 infected by NCTC11637 or Hp-400 or their OMVs. *p < 0.05, **p < 0.01, ***p < 0.001.

Discussion

In the study, we aimed to demonstrate the pathogenicity of H. pylori primarily through secretion of OMVs. OMVs are released by kinds of Gram-negative bacteria and contain proteins, DNA, toxins, peptidoglycan, and lipids, which play roles in the infection process, including helping to build a colonization niche22 and the delivery of virulence factors and toxins to host cells.23 We isolated OMVs using size exclusion chromatography (SEC) and identified 436 proteins in NCTC11637 OMVs and 372 proteins in Hp-400 OMVs. The global proteomic analysis of H. pylori OMVs illustrated that there were a variety of proteins in OMVs, including well-known toxin proteins of H. pylori, which further emphasized the crucial contribution of OMVs to mediate pathogenesis in the host. Several main toxin factors were detected in H. pylori OMVs, such as vacA and cagA. The vacA gene is conserved among all H. pylori strains, which has the ability to induce cell vacuolation. CagA is a strain-specific H. pylori gene that is considered a marker for strains that lead to a high risk of gastric cancer. The delivery of Cag A protein is mainly through the bacterial type four secretion system, which causes a direct effect on epithelial cells, including disrupting cell signaling pathways and cell polarity.24−26

H. pylori is reported to have a high degree of genomic diversity because of high frequencies of mutation and recombination.27−30 Recently, Furuta et al. reported multi-locus sequence typing and whole genome sequence analyses of very closely related H. pylori strains from the same family members consisting of parents and children in Japan, suggesting adaptation to a new host through mutations in virulence-related genes, restriction-modification genes, and OMP genes.31,32 In our study, NCTC11637 was the standard strain, and Hp-400 was isolated from gastric tissues from patients in Hebei Province, an area with a high incidence of gastric cancer. These strains induced similar but not identical proteomic changes in GES1 cells, indicating that different strains have different pathogenic mechanisms. The results suggested that precise individualized treatment is necessary in clinical applications.

OMVs serve as vehicles for toxin delivery into host cells to promote bacterial pathogenicity and induce an inflammatory response. In this study, we mimicked the in vivo interaction between H. pylori or OMVs and the gastric mucosa through the coculture of GES1 cells and H. pylori or OMVs. Inflammatory factors were detected using flow cytometry; we demonstrated that OMVs contribute, at least in part, to driving a robust inflammatory response in gastric epithelial cells. A number of cytokines are elevated when infected by OMVs and H. pylori. For example, IL-8 is a potential neutrophil chemoattractant and activating factor that mediates strong pro-inflammatory responses. IL-8 levels are increased by H. pylori infection in a cag-dependent manner,33 and polymorphisms in IL-8 are associated with increased risks of chronic atrophic gastritis and gastric cancer.34,35 IL-6 is a significant mediator of inflammation that promotes a Th17-mediated inflammatory response. IL-6 expression is associated with the disease status among patients with H. pylori-associated gastritis36 and gastric cancer.37 TNF-α is a cytokine involved in systemic inflammation and the Th1 response, and TNF levels are increased in patients with H. pylori-associated gastritis.36

In addition to altering inflammatory signaling pathways, H. pylori has also been shown to disrupt cellular junctional complexes38 and induce cytoskeletal rearrangements that are suggestive of the uncontrolled growth induced by growth factors.39H. pylori has also been shown to disrupt the balance between gastric epithelial cell proliferation and apoptosis.40 However, the molecular mechanism of virulence factor delivery via OMVs has been unclear. We speculated that the main function of OMVs is to mimic parental pathogens and induce pathological damage. In addition to the well-established secretion systems, OMVs have been recently considered a new independent secretion system. Many “well-known” virulence factors and toxins have been identified that use OMVs as an alternative secretory pathway. OMVs provide unique advantages compare to other secretion systems by transporting high concentrations of proteins and delivering them to target destinations over long distances. Transmission of bacterial proteins by OMVs into host cells appears to be an important aspect in pathogens. In our study, disease pathways and networks induced by OMVs are directly related to gastrointestinal injury, disease, and development of cancer. These described pathways and networks will allow future functional analyses of specific proteomic targets that have been previously uncharacterized with response to either H. pylori infection or gastric carcinogenesis but now may play an important role in the development of gastric injury and cancer. A more thorough understanding of these networks will enable the exploitation of targetable pathways and effectors for clinical benefits and disease prevention.

VTN and complement C3 are two proteins that were detected through label-free mapping and were upregulated upon treatments with H. pylori and OMVs from both NCTC11637 and Hp-400. These targets were validated by LSCM, and the data were consistent with the HPLC-MS/MS results. VTN has not been previously identified to be associated with H. pylori infection. However, it has been previously shown to promote gastric cancer cell growth and motility in vitro and in vivo. In addition, VTN was also identified as a factor contributing to a poor prognosis of gastric cancer.41 In contrast, complement C3 has been reported to be activated directly by H. pylori,(42) and overexpression of complement C3 correlates with gastric cancer progression by activating the JAK2/STAT3 pathway.43

In conclusion, by utilizing proteomic approaches and pathway analyses, we were able to define proteomic changes in GES1 cells in response to H. pylori or OMVs infection. These data mirrored alterations observed among humans infected with H. pylori, further validating our conjecture that H. pylori delivers pathogenic factors by secreting OMVs. Importantly, this technique and approach facilitated the identification and validation of novel protein targets that play important roles in H. pylori-induced gastric diseases in individuals at a high risk of infection. Indeed, this technique and approach prospectively accelerates the identification of novel biomarkers that arise in the early inflammatory and carcinogenic cascade and are conductive to therapeutic intervention and disease prevention.

Materials and Methods

H. pylori Culture

Two kinds of H. pylori were used in the article. The standard strain NCTC11637 was donated by the Shijiazhuang Center for Disease Control and Prevention. The well-characterized clinically isolated H. pylori 400 strain was separated from gastric tissues obtained from patients in Hebei Province, which has a high incidence of gastric cancer, and was preserved in the China General Microbiological Culture Collection Center (CGMCC 15126). H. pylori was cultured for 72 h in a Columbia blood plate medium under microaerobic conditions. H. pylori used for OMV isolation was cultured in brain heart infusion broth (BHI, Oxoid) supplemented with 10% fetal bovine serum (BI) and 2% antibiotics for 72 h at 37 °C under microaerobic conditions and with constant rotation (150 rpm).

OMV Preparation and Purification

OMVs were isolated using size exclusion chromatography (SEC).17 Briefly, after 72 h of incubation, the broth cultures were centrifugated (3000g, 15 min) to remove bacteria. The culture supernatants were then filtered via a 0.22 μm filter (Millipore, USA) to eliminate contaminating particles. The filtered supernatant was condensed to 1 mL using Amicon Ultra-15 centrifugal filter units (Millipore, USA) for use in Exosupur columns in accordance with the manufacturer’s instructions (Echo Biotech, China). OMVs were collected and condensed to an appropriate volume by centrifugation through Amicon Ultra-4 centrifugal filter units (Millipore, USA). The morphology was characterized using transmission electron microscopy (TEM, JEOL2100F). The particle size distribution and concentration of the OMVs were measured using nanoparticle tracking analysis (NTA).

Cell Culture

The immortalized gastric epithelial cell line, GES1, was obtained from Procell Life Science & Technology (Wuhan, China), which was cultured in RPMI 1640 (Gibco, UA), supplemented with 10% fetal calf serum (BI, Israel), penicillin, and streptomycin (Invitrogen, UA), and incubated at 37 °C with 5% CO2.

Cytokine Detection

GES1 cells (1 × 105) were seeded in 6-well plates and cultured for 24 h before OMVs and H. pylori were added. Forty micrograms of total OMVs or 5 × 106H. pylori were added to each well. After 48 h of coculture, the cellular supernatant was collected for cytokine detection, including IL-2, IL-5, IFN-α, IL-10, IL-6, IFN-γ, IL-8, IL-17, IL-4, IL-12P70, and TNF-α, using flow cytometry in accordance with the manufacturer’s instructions (RAISE CARE).

Mass Spectrometry-Based Proteome Profiling

Protein Extraction and Digestion

RIPA buffer was added into the H. pylori or OMVs cocultured GES1 cells and purified OMVs for protein extraction and then sonicated for 5 s on and 5 s off with a total of six cycles. The proteins were then denatured at 95 °C for 2 min. The insoluble fragment was removed by centrifugation at 12,000g for 10 min, and the supernatant was used for the proteomic experiment. The protein concentration was measured using a BCA kit (Thermo).

A filter-aided sample preparation (FASP) procedure was used for protein digestion. Briefly, proteins were loaded in 10 kDa centrifugal filter tubes (Thermo, 88513), the disulfide bond was cleaved with 50 mM DTT in 300 μL UA buffer (8 M urea in 0.1 M Tris–HCl, pH 8.5) for 30 min in 37 °C, alkylated with 50 mM IAA in 300 μL of UA buffer for 30 min in the dark, washed thrice with 300 μL of UA buffer, and then washed twice with 300 μL of 50 mM NH4HCO3. All the above steps were centrifuged at 12,000g at 25 °C. Proteins were digested at 37 °C for 18 h with trypsin (Promega) at a concentration of 1:100 (w/w) in 50 mM NH4HCO3. After digestion, peptides were eluted by centrifugation. Subsequently, peptides were purified and extracted using homemade C18 tips (Empore) in 80% ACN and 2% TFA. Peptides were lyophilized and acidified in 0.1% FA. The peptide concentration was determined by the BCA peptide quantification kit (Thermo).

Proteomic Analysis

For proteomic analysis, the peptides (∼1 μg of each sample) were loaded on a nanoflow HPLC Easy-nLC1200 system (Thermo Fisher Scientific), using a 90 min LC gradient at 300 nL/min. Buffer A consisted of 0.1% (v/v) FA in H2O and buffer B consisted of 0.1% (v/v) FA in 80% ACN. The gradient was set as follows: 2–8% B in 1 min, 8–28% B in 60 min, 28–37% B in 14 min, 37–100% B in 5 min, and 100% B in 10 min. Proteomic analyses were performed on a Q Exactive HF mass spectrometer (Thermo Fisher Scientific). The spray voltage was set at 2100 V in a positive ion mode, and the ion transfer tube temperature was set at 320 °C. Data-dependent acquisition was performed using Xcalibur software in a profile spectrum data type. The MS1 full scan was set at a resolution of 60,000 at m/z 200, AGC target 3e6 and maximum IT 20 ms by an orbitrap mass analyzer (350–1500 m/z), followed by “top 20” MS2 scans generated by higher energy collisional dissociation (HCD) fragmentation at a resolution of 15,000 at m/z 200, AGC target 1e5 and maximum IT 45 ms. The fixed first mass of the MS2 spectrum was set 110.0 m/z. An isolation window was set at 1.6 m/z. The normalized collision energy (NCE) was set at NCE 27%, and the dynamic exclusion time was 45 s. Precursors with charges 1, 8, and >8 were excluded for MS2 analysis.

Database Searching of MS Data

All preliminary data processing was performed in Proteome Discoverer 2.2 using an ion currently-based label-free quantification method or basic protein identification similar to that previously described.18 Identification of peptides was performed with Sequest HT using a maximum 10 ppm mass tolerance for the parent ion and a 0.02 Da fragment tolerance for tandem mass spectrometry. All data were searched against the UniProtSwissProt Human canonical database (downloaded on Uniprot, 2019) or UniProtSwissProt H. Pylori database (downloaded on Uniprot, 2019). Carbamido methylation of cysteines was considered as a static modification; acetylation of the protein N-termini and oxidation of methionine were applied as potential variable modification. Multiple testing corrections were performed using false discovery rate calculations, as previously described.19 A 1% false discovery rate cutoff was applied to both the peptide spectral matches (calculated using Percolator20) and peptide group levels. Quantification ratios for each peptide were determined via pairwise analysis of individual peptides and then averaged for peptide group and protein levels. Significance was then determined by analysis of variance based on the peptide background at both the peptide group and protein levels.21

The criterion for differentially expressed proteins was |fold change| > 2. For enrichment analyses, gene ontology (GO) was analyzed using ClueGo of Cytoscape, and the enrichment terms with a p value less than 0.05 was reported. Kyoto Encyclopedia of Genes and Genomes (KEGG) was analyzed online (http://enrich.shbio.com) and the top 30 of enriched pathways were presented in the figures along with the p-value.

Immunofluorescence Assay

GES1 cells were seeded on the glass placed in the 24-well plate in advance, treated with OMVs and H. pylori for 24 h. Then, they were fixed with methanol for 6 h at 4 °C and permeabilized by 0.1% Triton X-100. The cells were blocked with sheep serum and incubated with primary antibodies overnight at 4 °C, VTN (A1667, ABclonal) and C3 (A13283, ABclonal). The protein signals were detected by anti-rabbit IgG Fab2 conjugated with Alexa Fluor 488 (Cell Signaling Technology, USA). Finally, the cells were incubated with DAPI for 15 min and visualized by a laser confocal microscope (Nikon).

Statistical Analysis

All statistical analyses were performed using SPSS version 13.0 software. All data are presented as the mean ± standard deviation from three independent experiments that were each measured in triplicate. One-way analysis of variance and the student’s t test were performed for comparison as described. A chi-square test was used to analyze categorical variables. A p value of less than 0.05 was considered statistically significant (*p value < 0.05), and all statistical tests were two-tailed.

Acknowledgments

This study was supported by the Natural Science Foundation of China (nos. 81772550, 81673642, 81502032, 81973520, and 81902798), the Outstanding Youth Foundation of Hebei Province, China (H2019206697), and the Natural Science Foundation of Hebei Province (H2020206131).

Glossary

Abbreviations

H. pylori

Helicobacter pylori

OMVs

outer membrane vesicles

HPLC-MS/MS

high-performance liquid chromatography–tandem mass spectrometry

GO

gene ontology

KEGG

Kyoto encyclopedia of genes and genomes

MALT

mucosa-associated lymphoid tissue

LPS

lipopolysaccharide

BHI

brain heart infusion

CGMCC

China General Microbiological Culture Collection Center

FBS

fetal bovine serum

SEC

size exclusion chromatography

FASP

filter-aided sample preparation

IAA

indole acetic acid

DTT

dithiothreitol

UA

urea

TFA

trifluoroacetic acid

FA

formic acid

ACN

acetonitrile

NCE

normalized collision energy

PD

proteome discoverer

TEM

transmission electron microscopy

NTA

nanoparticle tracking analysis

C3

complement C3

VTN

vitronectin

LSCM

laser scanning confocal microscope

VEGF

vascular endothelial growth factor

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c04549.

  • Detailed data of differentially expressed proteins in GES1 cocultured with increasing concentrations of OMVs (0, 10, 20, or 40 μg) and H. pylori (1:1, 10:1, or 50:1) in 11,637 and 400 (PDF)

Author Contributions

# S.W. and X.L. contributed equally to this study.

The authors declare no competing financial interest.

Notes

Mass spectrometry experimental data have been deposited on ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD025216, PXD025259, and PXD025281.

Supplementary Material

ao1c04549_si_001.pdf (1.1MB, pdf)

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