Title

探討幽門螺旋桿菌GroES之羧基端對人類PBMC及KATO-Ⅲ細胞IL-8表現之影響

Translated Titles

Effect of carboxyl terminus of Helicobacter pylori GroES on IL-8 expression in human PBMC and KATO-Ⅲ cell

DOI

10.6342/NTU.2008.02865

Authors

游元鼎

Key Words

幽門螺旋桿菌 ; 致病因子 ; 免疫反應 ; GroES ; 羧基端 ; IL-8 ; Helicobacter pylori ; virulenece factor ; GroES ; PBMC ; IL-8

PublicationName

臺灣大學生物化學暨分子生物學研究所學位論文

Volume or Term/Year and Month of Publication

2008年

Academic Degree Category

碩士

Advisor

周綠蘋

Content Language

繁體中文

Chinese Abstract

幽門螺旋桿菌被證實會造成胃部之慢性發炎並且與許多胃部之疾病(例如胃炎、胃潰瘍、十二指腸潰瘍與胃癌)有很大之關聯,至目前為止雖然有很多致病因子被研究出來,但是幽門螺旋桿菌的致病機制至今還尚未完全釐清。 在實驗室先前的研究中,發現了幽門螺旋桿菌新的致病因子GroES,而且GroES對胃癌病人血清有很高的抗原性,同時也會使得周邊血液單核球細胞釋放前發炎細胞激素,並對胃上皮細胞產生免疫反應及細胞增生之現象,而此造成之現象可能與胃癌的形成有關。GroES是一個熱休克蛋白,並且在細胞中與GroEL形成一個co-chaperone。經由文獻搜尋後得知,Escherichia coli或是其他微生物之 GroES並不會造成細胞之免疫反應,而幽門螺旋桿菌之GroES所造成細胞之免疫反應是否與其蛋白質序列有關,因此,我們分析了不同物種之GroES的蛋白質序列,發現幽門螺旋桿菌之GroES在羧基端有28個胺基酸片段延伸,而這片段是在其他物種中從未發現。 因此,我們想進一步了解此胺基酸片端是否會造成細胞產生免疫反應,所以我們將全長GroES中的後28個胺基酸片段刪除 (deletion),並藉由分析周邊血液單核球細胞以及胃上皮細胞所釋放之IL-8,發現這樣的刪除會使得原本GroES的喪失誘導細胞釋放IL-8之能力,並且使得GroES無法與胃上皮細胞表面進行結合。再進一步的分析後,我們發現這一段28個胺基酸之片段,會引發周邊血液單核球細胞之IL-8的釋放,似乎這一段胺基酸片段是GroES活性的重要來源,並進一步造成宿主細胞之免疫反應。

English Abstract

Helicobacter pylori (H. pylori) which can cause chronic inflammation is now emerging as a risk factor in the development of chronic gastritis, duodenal ulcer, gastric ulcer, and gastric cancer (1-2%). Although many virulence factors of H. pylori have been reported, but the pathogenic mechanism of H. pylori still remains unclear. In the previous study, we have found a novel GC-associated virulence factor which is GroES of H. pylori and this protein may contribute to gastric carcinogenesis via induction of inflammation and promotion of cell proliferation. GroES, a heat shock protein family is well known as a co-chaperone with GroEL. In the previous study of Escherichia coli GroES, it showed significant activity only on the expression and release of IL-6 in keratinocytes and E-selectin in monocytes and endothelial cells. So It sound like that there is no immunogencity in Escherichia coli GroES-treat cells. H. pylori GroES contains 118 amino acids. After comparing the amino acid sequences, the difference among GroES of different species was that there are extra 28-amino-acid in the carboxyl terminus of Helicobacter pylori GroES. This region was not present in any of the other bacterial GroES homologues characterized. We were interested in the contribution of the 28-amino-acid carboxyl terminus of H. pylori GroES in the effect of inflammation. We deleted the 28-amino-acid carboxyl terminus of H. pylori GroES and purified the protein that contains 1-90 amino acids of H. pylori GroES〈△rGroES(1-90)〉. We investigated the IL-8 expression in △rGroES(1-90)-treated PBMC (peripheral blood mononuclear cell) and gastric epithelial cell (KATO-Ⅲ). By comparing with wild type H. pylori and E. coli GroES, we found that deletion of C-terminal amino acid (91-118) of H. pylori GroES reduce IL-8 expression in both cells. It can be observed that △rGroES(1-90) doesn’t bind to surface of KATO-Ⅲ cell but H. Pylori rGroES(1-118) does. The synthesized peptide of H. pylori C-terminal amino acid (91-118) can induce IL-8 expression in PBMC. This peptide may contribute to the activity of H. pylori GroES whereas △rGroES(1-90) has no effect on IL-8 expression. Therefore, GroES seems to be involved in the inflammatory response of H. Pylori infection through its carboxyl terminus of 28-amino-acid.

Topic Category 醫藥衛生 > 基礎醫學
醫學院 > 生物化學暨分子生物學研究所
Reference
  1. 3. Houghton, J., Wang, T. C. (2005) Gastroenterology 128, 1567-1578
    連結:
  2. 4. Crew, K. D., and Neugut, A. I. (2006) World J Gastroenterol 12, 354-362
    連結:
  3. 7. Robinson, K., Argent, R. H., and Atherton, J. C. (2007) Best Pract Res Clin Gastroenterol 21, 237-259
    連結:
  4. 9. Gewirtz, A. T., Yu, Y., Krishna, U. S., Israel, D. A., Lyons, S. L., and Peek, Jr R. M. (2004) J Infect Dis 189, 1914-1920
    連結:
  5. 14. Ernst, P. B. a. G., B. D. (2000) Annu Rev Microbiol 54, 615-640
    連結:
  6. 15. Blaser, M. J., and Atherton, J. C. (2004) J Clin Invest 113, 321-333
    連結:
  7. 16. Lai, L. H., and Sung, J. J. Y. (2007) Best Pract Res Clin Gastroenterol 21, 261-279
    連結:
  8. 17. Parkin, D. M. Oncogene 23, 6329-6340
    連結:
  9. 19. Correa, P. (1996) Cancer Epidemiol Biomarkers Prev 5, 477-481
    連結:
  10. 20. Correa, P., and Houghton, J. (2007) Gastroenterology 133, 659-672
    連結:
  11. 22. Andersen, L. (2007) Helicobacter 12 Suppl 2, 12-15
    連結:
  12. 23. Montecucco, C., and Rappuoli, R. (2001) Nat Rev Mol Cell Biol 2, 457-466
    連結:
  13. 36. Amieva, M. R., and El-Omar, E. M. (2008) Gastroenterology 134, 306-323
    連結:
  14. 40. Tammer, I., Brandt, S., Hartig, R., Kig, W., and Backert, S. (2007) Gastroenterology 132, 1309-1319
    連結:
  15. 41. Amieva, M. R., Vogelmann, R., Covacci, A., Tompkins, L. S., Nelson, W. J., and Falkow, S. (2003) Science 300, 1430-1434
    連結:
  16. (2007) Oncogene 26, 4617-4626
    連結:
  17. 43. Creighton, T. E. (1991) Nature 352, 17-18
    連結:
  18. 51. Fan, T. C., Chang, H. T., Chen, I. W., Wang, H. Y., and Chang, M. D. T. (2007) Traffic 8, 1778-1795
    連結:
  19. 52. Xu, Q. B. (2003) Curr Opin Cardiol 18, 245-252
    連結:
  20. 1. Marshall, B. J., and Warren, J. R. (1984) Lancet 1, 1311-1315
  21. 2. Kandulski, A., Selgrad, M., and Malfertheiner, P. (2008) Dig Liver Dis 40, 619-626
  22. 5. Lehours, P., and Yilmaz, O. (2007) Helicobacter 12 Suppl 1, 1-3
  23. 6. Portal-Celhay, C., and Perez-Perez, GI. (2006) Clin Sci 110, 305-314
  24. 8. Ding, S. Z., Torok, A. M., Smith, M. F., and Goldberg, J. B. (2005) Helicobacter 10, 193-204
  25. 10. Ishihara, S., Rumi, M. A. K., Kadowaki, Y., Ortega-Cava, C. F., Yuki, T., Yoshino, N., Miyaoka, Y., Kazumori, H., Ishimura, N., Amano, Y., and Kinoshita, Y. (2004) J Immunol 173, 1406-1416
  26. 11. Chang, Y. J., Wu, M. S., Lin, J. T., Sheu, B. S., Muta, T., Inoue, H., and Chen, C. C. (2004) Mol Pharmacol 66, 1465-1477
  27. 12. Smith, M. F., Jr., Mitchell, A., Li, G., Ding, S., Fitzmaurice, A. M., Ryan, K., Crowe, S., and Goldberg, J. B. (2003) J Biol Chem 278, 32552-32560
  28. 13. Tummala, S., Keates, S., and Kelly, C. P. (2004) Curr Opin Gastroenterol 20, 592-597
  29. 18. Parsonnet, J., Friedman, G. D., Vandersteen, D. P., Chang, Y., Vogelman, J. H., Orentreich, N., and Sibley, R. K. (1991) N Engl J Med 325, 1127-1131
  30. 21. Eaton, K., Brooks, C. L., Morgan, D. R., and Krakowka, S. (1991) Infect Immun 59, 2470-2475
  31. 24. Boren, T., Falk, P., Roth, K. A., Larson, G., and Normark, S. (1993) Science 262, 1892-1895
  32. 25. Ilver, D., Arnqvist, A., Ogren, J., Frick, I. M., Kersulyte, D., Incecik, E. T., Berg, D. E.,
  33. Covacci, A., Engstrand, L., and Boren, T. (1998) Science 279, 373-377
  34. 26. Evans, D. G., Karjalainen, T. K., Evans, D. J. Jr., Graham, D. Y., and Lee, C. H. (1993) J Bacteriol 175, 674-683
  35. 27. Jones, A. C., Logan, R. P., Foynes, S., Cockayne, A., Wren, B. W., and Penn, C. W. (1997) J Bacteriol 179, 5643-5647
  36. 28. Mahdavi, J., Sonden, B., Hurtig, M., Olfat, F. O., Forsberg, L., Roche, N., Angstrom, J., Larsson, T., Teneberg, S., Karlsson, K. A., Altraja, S., Wadstrom, T., Kersulyte, D., Berg, D. E., Dubois, A., Petersson, C., Magnusson, K. E., Norberg, T., Lindh, F., Lundskog, B. B., Arnqvist, A., Hammarstrom, L., and Boren, T. (2002) Science 297, 573-578
  37. 29. Valkonen, K. H., Wadstrom, T., and Moran, A. P. (1997) Infect Immun 65, 916-923
  38. 30. Megraud, F., Neman-Simha, V., and Brugmann, D. (1992) Infect Immun 60, 1858-1863
  39. 31. Suzuki, M., Miura, S., Suematsu, M., Fukumura, D., Kurose, I., Suzuki, H., Kai, A., Kudoh, Y., Ohashi, M., and Tsuchiya, M. (1992) Am J Physiol 263, G719-725
  40. 32. Jabri, E., Carr, M. B., Hausinger, R. P., and Karplus, P. A. (1995) Science 268, 998-1004
  41. 33. Satin, B., Del Giudice, G., Della Bianca, V., Dusi, S., Laudanna, C., Tonello, F., Kelleher, D., Rappuoli, R., Montecucco, C., and Rossi, F. (2000) J Exp Med 191, 1467-1476
  42. 34. D'Elios, M. M., Amedeo, A., Cappon, A., Del Prete, G., and de Bernard, M. (2007) FEMS Immunol Med Microbiol. 50, 157-164
  43. 35. McClain, M. S., Schraw, W., Ricci, V., Boquet, P., and Cover, T. L. (2000) Mol Microbiol 37, 433-442
  44. 37. Dhar, S. K., Soni, R. K., Das, B. K., and Mukhopadhyay, G. (2003) Mol Cell Biochem 253, 207-215
  45. 38. Ogura, K., Maeda, S., Nakao, M., Watanabe, T., Tada, M., Kyutoku, T., Yoshida, H., Shiratori, Y., and Omata, M. (2000) J Exp Med 192, 1601-1610
  46. 39. Stein, M., Bagnoli, F., Halenbeck, R., Rappuoli, R., Fantl, W. J., and Covacci, A. (2002) Mol Microbiol 43, 971-980
  47. 42. Murata-Kamiya, N., Kurashima, Y., Teishikata, Y., Yamahashi, Y., Saito, Y., Higashi, H., Aburatani, H., Akiyama, T., Peek, R. M., Jr. Azuma, T., and Hatakeyama, M.
  48. 44. Macchia, G., Massone, A., Burroni, D., Covacci, A., Censini, S., and Rappuoli, R. (1993) Mol Microbiol 9, 645-652
  49. 45. Engstrand, L., Scheynius, A., and Pahlson, C. (1991) Am J Gastroenterol 86, 976-980
  50. 46. Yamaguchi, H., Osaki, T., Kurihara, N., Kitajima, M., Kai, M., Takahashi, M., Taguchi, H., and Kamiya, S. (1999) J Med Microbiol 48, 927-933
  51. 47. Kawahara, Y., Yokota, K., Mizuno, M., Yunoki, N., Uesu, T., Okada, H., Kobayashi, K., Hirai, Y., Oguma, K., and Tsuji, T. (1999) Gut 45, 20-23
  52. 48. Zhao, Y., Yokota, K., Ayada, K., Yamamoto, Y., Okada, T., Shen, L. H., and Oguma, K. (2007) J Med Microbiol 56, 154-164
  53. 49. Takenaka, R., Yokota, K., Ayada, K., Mizuno, M., Zhao, Y., Fujinami, Y., Lin, S. N., Toyokawa, T., Okada, H., Shiratori, Y., and Oguma, K. (2004) Microbiology-Sgm 150, 3913-3922
  54. 50. Lin, Y. F. W., M. S. Chang, C. C. Lin, S. W. Lin, J. T. Sun, Y. J. Chen, D. S., and Chow, L. P. (2006) Mol Cell Proteomics 5, 1484-1496
  55. 53. Yokota, S., Tsubaki, K., Kuriyama, T., Shimizu, H., Ibe, M., Mitsuda, T., Aihara, Y., Kosuge, K., and Nomaguchi, H. (1993) Clin Immunol Immunopathol 67, 163-170
  56. 54. Hinode, D., Yoshioka, M., Tanabe, S. I., Miki, O., Masuda, K., and Nakamura, R. (1998) FEMS Microbiol Lett 167, 1-6
  57. 55. Launois, P., Ndiaye, M. N., Cartel, J. L., Mane, I., Drowart, A., Vanvooren, J. P., Sarthou, J. L., and Huygen, K. (1995) Infect Immun 63, 88-93
  58. 56. Marcatili, A., Cipollaro de l'Ero, G., Galdiero, M., Folgore, A., and Petrillo, G. (1997) Microbiology 143, 45-53
  59. 57. Galdiero, M., de l'Ero, G. C., and Marcatili, A. (1997) Infect Immun 65, 699-707
  60. 58. Ferrero, R. L., Thiberge, J. M., Kansau, I., Wuscher, N., Huerre, M., and Labigne, A. (1995) Proc Natl Acad Sci U S A 92, 6499-6503
  61. 59. Fattorini, L., Creti, R., Nisini, R., Pietrobono, R., Fan, Y., Stringaro, A., Arancia, G., Serlupi-Crescenzi, O., Iona, E., and Orefici, G. (2002) J Med Microbiol 51, 1071-1079
  62. 60. Kansau, I., Guillain, F., Thiberge, J. M., and Labigne, A. (1996) Mol Microbiol 22, 1013-1023
  63. 61. El-Omar, E. M., Carrington, M., Chow, W. H., McColl, K. E. L., Bream, J. H., Young, H. A., Herrera, J., Lissowska, J., Yuan, C. C., Rothman, N., Lanyon, G., Martin, M.,
  64. Fraumeni, J. F., and Rabkin, C. S. (2000) Nature 404, 398-402
  65. 62. Kai, H., Kitadai, Y., Kodama, M., Cho, S., Kuroda, T., Ito, M., Tanaka, S., Ohmoto, Y., and Chayama, K. (2005) Anticancer Res 25, 709-713
  66. 63. Suerbaum, S., Thiberge, J. M., Kansau, I., Ferrero, R. L., and Labigne, A. (1994) Mol Microbiol 14, 959-974
  67. 64. Cun, S., Li, H., Ge, R., Lin, M. C. M., and Sun, H. (2008) J Biol Chem 283, 15142-15151
  68. 65. Fukata, M., and Abreu, M. T. (2008) Oncogene 27, 234-243
  69. 66. Smith, M. F. J., Mitchell, A., Li, G., Ding, S., Fitzmaurice, A. M., Ryan, K., Crowe, S., and Goldberg, J. B. (2003) J Biol Chem 278, 32552-32560