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Abstract
Helicobacter pylori is a spiral, slow growing gram-negative microaerophilic bacterium. It has been shown to be the etiological agent of gastroduodenal diseases, such as chronic gastritis, gastric and duodenal ulcers, and gastric cancer. General culture condition of H. pylori is 5% O2, 10% CO2 and 100% humid atmosphere. We have compared proliferation protein expression profile of H. pylori incubated under normal microaerophilic (10% CO2) and environment stress (4% CO2, 18% CO2) conditions. H. pylori cultured under environment stress displayed coccoid morphology and time-dependent decrease in proliferation. We have further compared the protein expression profiles of H. pylori under normal growing and environment stress conditions by a global proteomic analysis, which includes high-resolution 2-DE followed by matrix-assisted laser desorption/ionization time of flight and nanoelectrospray/tandem mass spectrometry. In total, 42 protein spots were found to be up- or down-regulated by more than 2-fold under environment stress conditions. Of the 42 protein spots processed, 27 spots were identified; they represented 19 genes, including 2 kinds of hypothetical proteins.
References
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Figure 1.
Morphology change of H. pylori. Original culture grown under 5% O2 and 10% CO2 (A) were transferred to the new medium and cultured 3 more days under 4% CO2 (B), 10% CO2 (C) and 18% CO2 (D).
Figure 2.
Two-DE of whole cell protein extracts (200 μg) of H. pylori. The proteins were separated on an IPG strip of pH 5.0∼8.0 and subsequently on a 12.5% SDS-PAGE gel and then detected by silver staining. The original gel size is 17×20×0.12 cm. The numbers on the left indicate molecular mass markers and pI values at the top of the panel. (A) 10% CO2, 5% O2 (B) 5% CO2, 5% O2 (C) 18% CO2, 5% O2
Figure 3.
Comparison of expression intensity in over two fold increased or decreased protein spots in CO2 4% and/or 18%. X axis and Y axis represent spot number and spot intensity, respectively. SSP No is standard spot number in PDQUEST program.
Table 1.
Identified up or down-regulated proteins under various CO2 concentration of H. pylori strain 26695 by using tandem ms/ms
| SSP No | Proteins name | Accession no | MW | pI | Amino acid seq | Score | HP No |
|---|---|---|---|---|---|---|---|
| 0506e | Translation elongation factor EF-Tu (TufB) | gi:2314366 | 43647.7 | 4.93 | R.ELLSAYEFPGDDTPIVAGSALR.A | 66 | 1205 |
| K.TFLMPVEDVFSIAGR.G | 19 | ||||||
| 1011c | 3-dehydroquinase type II (AroQ) | gi:2314182 | 18483.25 | 4.93 | K.ILVIQGPNLNMLGHR.D | 65 | 1038 |
| K.QGNLDVELEFFQTNFEGEIIDK.I | 72 | ||||||
| R.GLSLAGNQVLTR.T | 63 | ||||||
| 2104b | Conserved hypothetical protein | gi:2314773 | 28417.54 | 5.24 | R.QALSAATLTLFK.M | 46 | 1588 |
| K.TETTLIEQNMLSK.I | 73 | ||||||
| R.AQDFEILTSR.L | 32 | ||||||
| 2901a | Polynucleotide phosphorylase (Pnp) | gi:2314375 | 76894.54 | 5.34 | K.NQYFDEIK.G | 45 | 1213 |
| K.ESLNMIEMR.S | 41 | ||||||
| 3204b | Thioredoxin reductase (TrxB) | gi:2313959 | 33538.22 | 6.25 | R.ELVVPGFFIFVGYDVNNAVLK.Q | 53 | 0825 |
| R.LVQVVDIEK.M | 48 | ||||||
| 4205b | Chemotaxis protein (CheV) | gi:2313092 | 36587.05 | 6.19 | K.DLYAVNVFK.I | 19 | 0019 |
| R.ELTIPLIDMK.K | 23 | ||||||
| 5801b | Threonyl-tRNA synthetase (ThrS) | gi:2313207 | 70239.64 | 6.36 | R.IYGIAFATK.E | 30 | 0123 |
| K.LAGAYLGGDENNEMLI.R | 74 | ||||||
| 5901a | ATP-dependent protease binding subunit (ClpB) | gi:2314182 | 96683.36 | 6.27 | K.ALAQFLFDSDKNLIR.I | 27 | 0264 |
| 5911c | ATP-dependent protease binding subunit (ClpB) | gi:2314182 | 96683.36 | 6.27 | K.ALAQFLFDSDKNLIR.I | 25 | 0264 |
| 6302b | ATP-binding protein (Mpr) | gi:2313292 | 45197.63 | 8.58 | K.VGLLDADVYGPNIPR.M | 74 | 0207 |
| 6801e | Hydantoin utilization protein A (HyuA) | gi:2313818 | 78532.79 | 6.94 | K.LLPGNEVIGPAIVESDATTFVIPK.G | 33 | 0695 |
| 7001d | Modulator of drug activity (Mda66) | gi:2313748 | 21604.61 | 7.16 | K.NPQVEQYLNSLTTHLR.Q | 24 | 0630 |
| R.TIVSGPIGGVIGSK.L | 39 | ||||||
| 7702e | Hydantoin utilization protein A (HyuA) | gi:2313818 | 78532.79 | 6.94 | R.LVLSLPLVAMDSVGAGAGSFVR.I | 22 | 0695 |
| K.IIQDAWDELTLK.V | 50 | ||||||
| R.GVVATQKPVIPVEK.E | 24 | ||||||
| 7705e | Hydantoin utilization protein A (HyuA) | gi:2313818 | 78532.79 | 6.94 | R.TIVSGPIGGVIGSK.L | 59 | 0695 |
| K.YDDPLIPLKR.I | 44 | ||||||
| R.TIVSGPIGGVIGSK.L | 31 | ||||||
| 7801e | Hydantoin utilization protein A (HyuA) | gi:2313818 | 78532.79 | 6.94 | R.LVLSLPLVAMDSVGAGAGSFVR.I | 26 | 0695 |
| K.IIQDAWDELTLK.V | 24 | ||||||
| 8102b | Uease alpha subunit (UreA) | gi:2313154 | 26539.49 | 8.96 | R.IFGFNALVDR.Q | 71 | 0073 |
| K.LNYVEAVALISAHIMEEAR.A | 18 | ||||||
| K.LLAPILPLVK.E | 26 | ||||||
| 8603f | Conserved hypothetical iron-sulfur protein | gi:2313222 | 54236.31 | 7.04 | K.DGDEANEIIYNLAK.E | 53 | 0138 |
| R.LDEYLELFEK.N | 48, | ||||||
| 8702a,e | Hydantoin utilization protein A (HyuA) | gi:2313818 | 78532.79 | 6.94 | R.GVVATQKPVIPVEK.E | 25, | −0695 |
| R.TIVSGPIGGVIGSK.L | 68, | ||||||
| 8801a | Glucose inhibited divistion protein (GidA) | gi:2313303 | 69683.72 | 7.13 | R.LGLMEEDFYK.E | 38, | −0213 |
| K.GIPGLSLEAVEK.L | 42, | ||||||
| 8903a,e | N-methylhydantoinase | gi:2313819 | 86511.66 | 6.95 | R.TVLLVDEK. | 31, | −0696 |
| K.GGPGFGDPIER.D | 45, |
Table 2.
Identified up or down-regulated proteins under various CO2 concentration of H. pylori strain 26695 by using MALDI-TOF MS
| SSP No | Proteins name | Accession no | MW | pI | HP no |
|---|---|---|---|---|---|
| 5001b | Co-chaperone (GroES) | gi:2313085 | 12990.87 | 6.59 | 0011 |
| 2906a | NADH-ubiquinone oxidoreductase, NQO3 subunit (NQO3) | gi:2314431 | 94229.31 | 5.12 | 1266 |
| 3903a | Protein translocase subunit (SecA) | gi:2313910 | 99083.31 | 5.65 | 0786 |
| 3909a | Protein translocase subunit (SecA) | gi:2313910 | 99083.31 | 5.65 | 0786 |
| 4107c | Alkyl hydroperoxide reductase (AhpC) | gi:2314747 | 22235.57 | 6.25 | 1563 |
Table 3.
Identified proteins classification by functional annotation of using COGs
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