胃幽門螺旋桿菌（Helicobacter pylori）是一種螺旋狀的革蘭氏陰性菌，在全世界超過50％以上的人口的胃黏膜中都可以發現它的存在。慢性胃炎，消化性潰瘍，胃及十二指腸潰瘍，胃腺癌和粘膜相關淋巴組織（MALT）淋巴瘤等的胃部疾病皆發現與胃幽門桿菌的感染有關，由於胃幽門螺旋桿菌為如此惡名昭彰的致病菌以及感染人類的廣泛性，而若要找出有效對抗它的藥物，深入地瞭解這種致病菌就顯得極為重要。脂多醣 （lipopolysaccharide）是胃幽門螺旋桿菌的毒力因子之一，其由脂質A (lipid A)，核心寡糖 (core oligosaccharide) 和O-抗原 (O-antigen) 所構成。由於脂多醣會誘發宿主細胞產生強烈的免疫反應，因此它對宿主來說是有毒性的。本篇論文主要討論的對象為HP0044和HP1275基因，它們分別被預測為經表現後可產生GDP-D-甘露糖脫水酶（RfbD）和磷酸甘露糖苷酶 （AlgC）兩種蛋白質，而這兩種酶在岩藻糖合成中扮演了了關鍵的角色。 我們建構了HP0044基因缺失的菌株和HP1275基因缺失的菌株，由這兩種基因缺失的菌株產生的脂多醣均呈現截短的結構，這樣的結果表明這兩種基因的產物確實都有參與脂多醣的生合成。另外，這兩種基因缺失的菌株表現出了較低的的生長速率，在感染能力，貼附能力及進到宿主細胞內的能力也都較為低落，並且對界面活性劑以及抗生素的敏感性增加。我們還發現在此二基因缺失的菌株中胃幽門螺旋桿菌表面的疏水性及自我凝集的程度皆大幅提高。我們也發現似乎fucose有參與到醣基化作用當中，但這還需要更多相關的實驗去證實。我們目前正在嘗試進行大量提取脂多醣的試驗，以獲得足夠的脂多醣去進行結構分析。經分析所獲得的發現將有助於釐清LPS的結構及功能和胃幽門螺旋桿菌感染的關係。

Helicobacter pylori is a spiral-shaped gram-negative bacteria and is recognized as a human pathongen which infects more than 50% human population worldwide. Infection by this bacterium is associated with chronic gastritis, peptic ulcer, gastroduodenal ulcer, gastric adenocarcinoma and mucosa-associated lymphatic tissue ( MALT ) lymphoma. Because H. pylori is such a notorious pathogen and causes a wide range of infection, it is extremely important to have an thoroughly understanding of this pathogen if we want to develop a drug that is effective against it. Lipopolysaccharide ( LPS ), one of the virulence factors of H. pylori, is composed of lipidA, core oligosaccharide and O-antigen polysaccharide. This structure is toxic to the hosts, because of its potent immunomodulating and immunostimulating properties. This study was focused on the HP0044 and HP1275 genes, which are predicted to encode GDP-D-mannose dehydratase ( RfbD ) and phosphomannomutase ( AlgC ), respectively. These two enzymes play key roles in fucose biosynthesis. We constructed the HP0044 knockout mutant and the HP1275 knockout mutant, and LPS produced by these two knockout mutants all showed a truncated structure, suggesting that both of them are indeed involved in LPS biosynthesis. In addition, these two knockout mutants also exhibited a reduced growth rate, weaker ability of infection, adhesion and internalization, and were more sensitive to detergents and antibiotics. We also found that the surface hydrophobicity of the H. pylori and the degree of autoaggregation were significantly increased in the knockout mutants. Furthermore, we also found that fucose seems to be involved in adhesion glycosylation, but this hypothesis requires more relevant experiments to confirm it. We are currently trying to conduct a large-scale LPS extraction procedure to obtain enough sample for LPS structural analysis. The obtained findings are expected to shed light on the structure and function of LPS in H. pylori infection.

中文摘要……………………………………………………………………………………… i
Abstract………………………………………………………………………………………. ii
Table of contents…………………………………………………………………………... ...iii
List of tables…………………………………………………………………………………. vi
List of figures………………………………………………………………………………vii
Abbreviations……………………………………………………………………………….viii
Introduction…………………………………………………………………………………..1
1-1 The history and characteristics of Helicobacter pylori……………………….………….1
1-2 The infection of H.pylori………………………………………………………………….2
1-3 Lipopolysaccharide (LPS) ………………………………………………………...………3
1-4 The biosynthesis of fucose ………………………………………………………………..6
1-5 Target genes: HP0044 and HP1275……………………………………………………….6
1-6 Outer membrane vesicles (OMVs) ……………………………………………………….8
1-7 The motivation of this study……………………………………………………………....9
Materials and methods………………………………………………………………….…..10
2-1 Materials ………………………………………………………………………………...10
2-2 Bioinformatic analyses………………………………………………………………..….10
2-3 Bacterial strains, plasmids and cell culture………………………………………………11
2-4 Molecular cloning of HP0044 gene and HP1275 gene…………………………………12
2-5 Protein SDS-PAGE and immunoblotting analysis………………………………………14
2-6 Isolation of outer membrane vesicles (OMVs) …………………………………….……15
2-7 Isolation and phenotypic analysis of H. pylori LPS…………………………………..…16
2-8 Growth curve analysis of H. pylori………………………………………………………17
2-9 Detergent (SDS) and antibiotic (novobiocin) sensitivity assay……………………….…17
2-10 The analysis of morphological changes on H. pylori-infected AGS cells……………18
2-11 Adhesion assay………………………………………………………………………18
2-12 Internalization assay…………………………………………………………………19
2-13 Hydrophobicity and autoaggregation assay…………………………………………19
2-14 Subcellular fractionation………………………………………………………………20
2-15 Statistical analysis……………………………………………………………………21
Results………………………………………………………………………………………..22
3-1 Sequence analysis of HP0044 and HP1275 protein in H. pylori 26695………………22
3-2 Confirmation of HP0044 and HP1275 knockout mutants and the corresponding complementary mutants……………………………………………………………….23
3-3 Effects of HP0044 and HP1275 mutations on LPS expression…………………….…25
3-4 The growth curve analysis of wild-type H. pylori 26695 strain, HP0044 and HP1275 knockout mutants and the corresponding knockout complementary mutants………26
3-5 Detergent (SDS) and antibiotic (novobiocin) sensitivity of H. pylori wild-type, HP0044 and HP1275 knockout mutants and the corresponding knockout complementary mutants………………………………………………………………………………27
3-6 Effects of HP0044 and HP1275 mutations on AGS cells morphological change after infection………………………………………………………………………………27
3-7 Effects of HP0044 and HP1275 mutations on adhesion and internalization level of H. pylori in infection………………………………………………………………….28
3-8 Effects of HP044 and HP1275 mutations on the hydrophobicity and autoaggregation of H. pylori……………………………………………………………………………….30
3-9 Comparison of electrophoretic mobility of BabA adhesin in different subcellular fractions from wild-type strain and various mutants…………………………………30
Discussions…………………………………………………………………………………32
References…………………………………………………………………………………36

1. Marshall, B.J. and J.R. Warren, Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet, 1984. 1(8390): p. 1311-5.
2. Marshall, B.J., et al., Attempt to fulfil Koch's postulates for pyloric Campylobacter. Med J Aust, 1985. 142(8): p. 436-9.
3. Olson, J.W. and R.J. Maier, Molecular hydrogen as an energy source for Helicobacter pylori. Science, 2002. 298(5599): p. 1788-90.
4. Warren, J.R. and B. Marshall, Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet, 1983. 1(8336): p. 1273-5.
5. Peterson, W.L., Helicobacter pylori and peptic ulcer disease. N Engl J Med, 1991. 324(15): p. 1043-8.
6. Hejazi, R. and M. Amiji, Stomach-specific anti-H. pylori therapy. I: Preparation and characterization of tetracyline-loaded chitosan microspheres. Int J Pharm, 2002. 235(1-2): p. 87-94.
7. Taneike, I., et al., Helicobacter pylori Intrafamilial Infections: Change in Source of Infection of a Child from Father to Mother after Eradication Therapy. Clin Diagn Lab Immunol, 2001. 8(4): p. 731-9.
8. van der Hulst, R.W., et al., Treatment of Helicobacter pylori infection: a review of the world literature. Helicobacter, 1996. 1(1): p. 6-19.
9. Chey, W.D., et al., ACG Clinical Guideline: Treatment of Helicobacter pylori Infection. Am J Gastroenterol, 2017. 112(2): p. 212-239.
10. Tomb, J.F., et al., The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature, 1997. 388(6642): p. 539-47.
11. Alm, R.A., et al., Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature, 1999. 397(6715): p. 176-80.
12. Oh, J.D., et al., The complete genome sequence of a chronic atrophic gastritis Helicobacter pylori strain: evolution during disease progression. Proc Natl Acad Sci U S A, 2006. 103(26): p. 9999-10004.
13. Baltrus, D.A., et al., The complete genome sequence of Helicobacter pylori strain G27. J Bacteriol, 2009. 191(1): p. 447-8.
14. Atherton, J.C., et al., Clinical and pathological importance of heterogeneity in vacA, the vacuolating cytotoxin gene of Helicobacter pylori. Gastroenterology, 1997. 112(1): p. 92-9.
15. Blaser, M.J., Not all Helicobacter pylori strains are created equal: should all be eliminated? Lancet, 1997. 349(9057): p. 1020-2.
16. Kalali, B., et al., H. pylori Virulence Factors: Influence on Immune System and Pathology. Mediators of Inflammation, 2014. 2014: p. 9.
17. Hazell, S.L., et al., Campylobacter pyloridis and gastritis: association with intercellular spaces and adaptation to an environment of mucus as important factors in colonization of the gastric epithelium. J Infect Dis, 1986. 153(4): p. 658-63.
18. Roesler, B.M., E.M.A. Rabelo-Gonçalves, and J.M.R. Zeitune, Virulence Factors of Helicobacter pylori: A Review. Clin Med Insights Gastroenterol, 2014. 7: p. 9-17.
19. Schreiber, S., et al., The spatial orientation of Helicobacter pylori in the gastric mucus. Proc Natl Acad Sci U S A, 2004. 101(14): p. 5024-9.
20. Clyne, M., A. Labigne, and B. Drumm, Helicobacter pylori requires an acidic environment to survive in the presence of urea. Infect Immun, 1995. 63(5): p. 1669-73.
21. Scott, D.R., et al., The role of internal urease in acid resistance of Helicobacter pylori. Gastroenterology, 1998. 114(1): p. 58-70.
22. Bauerfeind, P., et al., Synthesis and activity of Helicobacter pylori urease and catalase at low pH. Gut, 1997. 40(1): p. 25-30.
23. Dunne, C., B. Dolan, and M. Clyne, Factors that mediate colonization of the human stomach by Helicobacter pylori. World J Gastroenterol, 2014. 20(19): p. 5610-24.
24. Censini, S., et al., cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors. Proc Natl Acad Sci U S A, 1996. 93(25): p. 14648-53.
25. Argent, R.H., et al., Functional association between the Helicobacter pylori virulence factors VacA and CagA. J Med Microbiol, 2008. 57(Pt 2): p. 145-50.
26. J., P., et al., Helicobacter pylori lipopolysaccharide induces gastric epithelial cells apoptosis. IUBMB Life, 1996. 40(3): p. 597-602.
27. Cover, T.L. and S.R. Blanke, Helicobacter pylori VacA, a paradigm for toxin multifunctionality. Nat Rev Microbiol, 2005. 3(4): p. 320-32.
28. Sharma, S.A., et al., Activation of IL-8 gene expression by Helicobacter pylori is regulated by transcription factor nuclear factor-kappa B in gastric epithelial cells. J Immunol, 1998. 160(5): p. 2401-7.
29. Gnauck, A., R.G. Lentle, and M.C. Kruger, The Characteristics and Function of Bacterial Lipopolysaccharides and Their Endotoxic Potential in Humans. Int Rev Immunol, 2016. 35(3): p. 189-218.
30. Ulich, T.R., et al., The intratracheal administration of endotoxin and cytokines. I. Characterization of LPS-induced IL-1 and TNF mRNA expression and the LPS-, IL-1-, and TNF-induced inflammatory infiltrate. Am J Pathol, 1991. 138(6): p. 1485-96.
31. Kulp, A. and M.J. Kuehn, Biological functions and biogenesis of secreted bacterial outer membrane vesicles. Annu Rev Microbiol, 2010. 64: p. 163-84.
32. Ranoa, D.R.E., S.L. Kelley, and R.I. Tapping, Human Lipopolysaccharide-binding Protein (LBP) and CD14 Independently Deliver Triacylated Lipoproteins to Toll-like Receptor 1 (TLR1) and TLR2 and Enhance Formation of the Ternary Signaling Complex. J Biol Chem, 2013. 288(14): p. 9729-41.
33. Wang, X. and P.J. Quinn, Lipopolysaccharide: Biosynthetic pathway and structure modification. Prog Lipid Res, 2010. 49(2): p. 97-107.
34. Hug, I., et al., Helicobacter pylori lipopolysaccharide is synthesized via a novel pathway with an evolutionary connection to protein N-glycosylation. PLoS Pathog, 2010. 6(3): p. e1000819.
35. Rittig, M.G., et al., Smooth and rough lipopolysaccharide phenotypes of Brucella induce different intracellular trafficking and cytokine/chemokine release in human monocytes. J Leukoc Biol, 2003. 74(6): p. 1045-55.
36. Raetz, C.R.H. and C. Whitfield, Lipopolysaccharide Endotoxins. Annu Rev Biochem, 2002. 71: p. 635-700.
37. Edwards, N.J., et al., Lewis X structures in the O antigen side-chain promote adhesion of Helicobacter pylori to the gastric epithelium. Mol Microbiol, 2000. 35(6): p. 1530-9.
38. Moran, A.P., et al., Chemical characterization of Campylobacter jejuni lipopolysaccharides containing N-acetylneuraminic acid and 2,3-diamino-2,3-dideoxy-D-glucose. J Bacteriol, 1991. 173(2): p. 618-26.
39. Hashimoto, M., et al., Structural study on lipid A and the O-specific polysaccharide of the lipopolysaccharide from a clinical isolate of Bacteroides vulgatus from a patient with Crohn's disease. Eur J Biochem, 2002. 269(15): p. 3715-21.
40. Moran, A.P., B. Lindner, and E.J. Walsh, Structural characterization of the lipid A component of Helicobacter pylori rough- and smooth-form lipopolysaccharides. J Bacteriol, 1997. 179(20): p. 6453-63.
41. Pacheco, A.R., et al., Fucose Sensing Regulates Bacterial Intestinal Colonization. Nature, 2012. 492(7427): p. 113-7.
42. Ren, Y., et al., Biochemical characterization of GDP-L-fucose de novo synthesis pathway in fungus Mortierella alpina. Biochem Biophys Res Commun, 2010. 391(4): p. 1663-9.
43. Webb, N.A., et al., Crystal structure of a tetrameric GDP-d-mannose 4,6-dehydratase from a bacterial GDP-d-rhamnose biosynthetic pathway. Protein Sci, 2004. 13(2): p. 529-39.
44. Kneidinger, B., et al., Identification of two GDP-6-deoxy-D-lyxo-4-hexulose reductases synthesizing GDP-D-rhamnose in Aneurinibacillus thermoaerophilus L420-91T. J Biol Chem, 2001. 276(8): p. 5577-83.
45. Ye, R.W., N.A. Zielinski, and A.M. Chakrabarty, Purification and characterization of phosphomannomutase/phosphoglucomutase from Pseudomonas aeruginosa involved in biosynthesis of both alginate and lipopolysaccharide. J Bacteriol, 1994. 176(16): p. 4851-7.
46. Deatherage, B.L., et al., Biogenesis of bacterial membrane vesicles. Mol Microbiol, 2009. 72(6): p. 1395-407.
47. Mashburn, L.M. and M. Whiteley, Membrane vesicles traffic signals and facilitate group activities in a prokaryote. Nature, 2005. 437(7057): p. 422-5.
48. Rakoff-Nahoum, S., M.J. Coyne, and L.E. Comstock, An ecological network of polysaccharide utilization among human intestinal symbionts. Curr Biol, 2014. 24(1): p. 40-49.
49. Renelli, M., et al., DNA-containing membrane vesicles of Pseudomonas aeruginosa PAO1 and their genetic transformation potential. Microbiology, 2004. 150(Pt 7): p. 2161-9.
50. Vanaja, S.K., et al., Bacterial Outer Membrane Vesicles Mediate Cytosolic Localization of LPS and Caspase-11 Activation. Cell, 2016. 165(5): p. 1106-1119.
51. Altman, E., et al., Effect of the HP0159 ORF mutation on the lipopolysaccharide structure and colonizing ability of Helicobacter pylori. FEMS Immunol Med Microbiol, 2008. 53(2): p. 204-13.
52. Altman, E., et al., Lipopolysaccharide structures of Helicobacter pylori wild-type strain 26695 and 26695 HP0826::Kan mutant devoid of the O-chain polysaccharide component. Carbohydr Res, 2011. 346(15): p. 2437-44.
53. Li, H., et al., The redefinition of Helicobacter pylori lipopolysaccharide O-antigen and core-oligosaccharide domains. PLoS Pathog, 2017. 13(3): p. e1006280.
54. Horton, R.M., In vitro recombination and mutagenesis of DNA. SOEing together tailor-made genes. Methods Mol Biol, 1997. 67: p. 141-9.
55. Lefebvre, B., P. Formstecher, and P. Lefebvre, Improvement of the gene splicing overlap (SOE) method. Biotechniques, 1995. 19(2): p. 186-8.
56. Horton, R.M., et al., Gene splicing by overlap extension. Methods Enzymol, 1993. 217: p. 270-9.
57. Haas, R., T.F. Meyer, and J.P. van Putten, Aflagellated mutants of Helicobacter pylori generated by genetic transformation of naturally competent strains using transposon shuttle mutagenesis. Mol Microbiol, 1993. 8(4): p. 753-60.
58. Gerrits, M.M., et al., Role of the rdxA and frxA genes in oxygen-dependent metronidazole resistance of Helicobacter pylori. J Med Microbiol, 2004. 53(Pt 11): p. 1123-8.
59. Wai, S.N., A. Takade, and K. Amako, The release of outer membrane vesicles from the strains of enterotoxigenic Escherichia coli. Microbiol Immunol, 1995. 39(7): p. 451-6.
60. Fomsgaard, A., M.A. Freudenberg, and C. Galanos, Modification of the silver staining technique to detect lipopolysaccharide in polyacrylamide gels. J Clin Microbiol, 1990. 28(12): p. 2627-31.
61. Marolda, C.L., et al., Micromethods for the characterization of lipid A-core and O-antigen lipopolysaccharide. Methods Mol Biol, 2006. 347: p. 237-52.
62. Tsai, C.M. and C.E. Frasch, A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem, 1982. 119(1): p. 115-9.
63. Wang, Y. and D.E. Taylor, Chloramphenicol resistance in Campylobacter coli: nucleotide sequence, expression, and cloning vector construction. Gene, 1990. 94(1): p. 23-8.
64. Moese, S., et al., Helicobacter pylori Induces AGS Cell Motility and Elongation via Independent Signaling Pathways. Infect Immun, 2004. 72(6): p. 3646-9.
65. Abu-Lail, N.I. and T.A. Camesano, Role of lipopolysaccharides in the adhesion, retention, and transport of Escherichia coli JM109. Environ Sci Technol, 2003. 37(10): p. 2173-83.
66. Kitchens, R.L. and R.S. Munford, CD14-dependent internalization of bacterial lipopolysaccharide (LPS) is strongly influenced by LPS aggregation but not by cellular responses to LPS. J Immunol, 1998. 160(4): p. 1920-8.
67. Wright, S.D., et al., CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science, 1990. 249(4975): p. 1431-3.
68. Wang, Z., et al., Influence of Core Oligosaccharide of Lipopolysaccharide to Outer Membrane Behavior of Escherichia coli. Mar Drugs, 2015. 13(6): p. 3325-39.
69. Sturla, L., et al., Expression, purification and characterization of GDP-d-mannose 4,6-dehydratase from Escherichia coli. FEBS Letters, 1997. 412(1): p. 126-130.
70. Somoza, J.R., et al., Structural and kinetic analysis of Escherichia coli GDP-mannose 4,6 dehydratase provides insights into the enzyme's catalytic mechanism and regulation by GDP-fucose. Structure, 2000. 8(2): p. 123-35.
71. Koplin, R., et al., Genetics of xanthan production in Xanthomonas campestris: the xanA and xanB genes are involved in UDP-glucose and GDP-mannose biosynthesis. J Bacteriol, 1992. 174(1): p. 191-9.
72. Appelmelk, B.J., et al., Potential role of molecular mimicry between Helicobacter pylori lipopolysaccharide and host Lewis blood group antigens in autoimmunity. Infect Immun, 1996. 64(6): p. 2031-40.
73. Chang, P.C., et al., Effects of a HP0859 (rfaD) knockout mutation on lipopolysaccharide structure of Helicobacter pylori 26695 and the bacterial adhesion on AGS cells. Biochem Biophys Res Commun, 2011. 405(3): p. 497-502.