臺灣博碩士論文加值系統

困難梭狀桿菌（Clostridium difficile）為革蘭氏陽性桿菌，會產生孢子並且是人類腸道正常菌群中的專性厭氧菌。困難梭狀桿菌感染（CDI）患者常造成偽膜性結腸炎（pseudomembrane colitis）和抗生素相關性腹瀉（antibiotic-associated diarrhea）等臨床症狀。而困難梭狀桿菌的表層蛋白surface layer protein（SLP）是其中毒力因子，會附著於腸道且與免疫細胞的Toll-like receptor 4（TLR4）結合而觸發下游免疫反應。核苷酸結合寡聚化結構域蛋白2 （nucleotide-binding oligomerization domain-containing protein 2, NOD2）是在細胞質內誘導發炎重要的調節者，其能辨識外來微生物以啟動先天性免疫。本論文研究結果顯示脂質筏（lipid rafts）參與困難梭狀桿菌SLP誘導的炎症，而降低細胞膽固醇則會抑制SLP誘導的發炎反應。此外，在SLP作用下NOD2被匯集到脂質筏中。相反的，使用Lentivirus-shRNA系統敲低NOD2表現，會抑制SLP刺激的下游炎症信號傳導。本論文研究證明困難梭狀桿菌SLP誘導NOD2匯集至細胞膜脂質筏並引起腸道上皮細胞的發炎反應。

Clostridium difficile, a gram-positive bacillus, that produces spores and is an obligate anaerobe of the normal flora colonized in human intestine. C. difficile infection (CDI) patients often exhibit clinical signs of pseudomembrane colitis (PMC) or antibiotic-associated diarrhea (AAD). The surface layer protein (SLP) of C. difficile is one of the virulence factors, which attaches to the intestine and binds to Toll-like Receptor 4 (TLR4) of immune cells to trigger downstream immune responses. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is an important mediator for inducing inflammation that initiate innate immune responses by identifying microbial invaders in the cytoplasmic compartment. The cholesterol-rich microdomains on the cell membrane (also referred to lipid rafts) are thought to be crucial for bacterial adhesion and signal transduction. Our results showed that lipid rafts were involved in C. difficile SLP-induced inflammation. Depletion of cellular cholesterol inhibits SLP-induced inflammatory response. In addition, NOD2 was recruited to lipid rafts during SLP treatment. Conversely, SLP-induced downstream inflammatory signaling was inhibited by knockdown of NOD2 using the lentivirus-shRNA system. Our results demonstrate that NOD2 recruits in the lipid rafts is important for SLP-induced inflammation in colonic epithelial cells.

指導教授推薦書
口試委員會審定書
誌謝...iii
中文摘要...iv
Abstract...v
Contents ...vi
Figure contents ...viii
1.Introduction...1
1.1 Clostridium difficile infection...1
1.2 Virulence factors of C. difficile...1
1.2.1 Toxin A (TcdA) and toxin B (TcdB)...2
1.2.2 Flagella...3
1.2.3 Peptidoglycan fragments...3
1.2.4 Surface layer protein A (SlpA)...4
1.3 Importance of SLPs...4
1.4 Lipid rafts...5
1.5 Nucleotide-binding oligomerization domain-containing protein 2 (NOD2)...6
1.6 Specific aims of this study...7
2.Materials and methods...9
2.1 Cell culture...9
2.2 Bacterial strains...9
2.3 Bacterial culture...9
2.4 Purification of surface layer protein (SLP)...10
2.5 Flow cytometric analysis...10
2.6 Immunofluorescence microscopy...10
2.7 Western blot analysis...12
2.8 Lentiviral vector production and short hairpin RNA (shRNA) transfection...13
2.9 Co-immunoprecipitation (Co-IP) assay...13
2.10 Determination of IL-8 production...14
2.11 Luciferase reporter activity assay...14
2.12 Statistical analysis...15
3.Results...16
3.1Clostridium difficile SLP binds to cell membrane...16
3.2 Location of Clostridium difficile SLP in membrane rafts...16
3.3 C. difficile SLP increases NOD2 expression levels...17
3.4 C. difficile SLP induces inflammation of intestinal epithelial cells via the NOD2 signaling pathway...18
3.5 C. difficile SLP stimulates NDO2 to induce IL-8 production...19
3.6 Mobilization of NOD2 into membrane rafts in response to SLP-induced inflammation...19
3.7 Inhibition of cellular cholesterol synthesis ameliorates SLP-induced inflammation...21
4.Discussion...22
5.References...27
6.Figures...35

Figure contents
Figure 1. Purification and characterization of C. difficile SLP. ...35
Figure 2. Binding of SLP to CHO-K1 cells....36
Figure 3. Sufficient cellular cholesterol is essential for SLP binding to CHO-K1 cells....37
Figure 4. Localization of SLP in membrane lipid rafts....38
Figure 5. SLP is associated with cholesterol-rich microdomains in C. difficile 37780 infected cells....40
Figure 7. C. difficile SLP induces NOD2 expression in gastrointestine-derived epithelial cells....43
Figure 8. C. difficile SLP induces inflammatory responses in intestinal epithelial cells....44
Figure 9. C. difficile SLP-induced NOD2 expression and inflammation in intestinal epithelial cells....45
Figure 10. The association of NOD2 with membrane rafts....46
Figure 11. C. difficile infection of cells induces SLP coalescence in rafts....47
Figure 12. C. difficile SLP elicits NOD2 recruitment in rafts....48
Figure 13. Inhibition of cellular cholesterol synthesis reduces SLP-induced inflammation....49
Figure 14. Schematic diagram illustrating the functions of C. difficile SLP that enhances NOD2 to recruit in lipid rafts....50

Abrami, L., S. Liu, P. Cosson, et al. (2003). Anthrax toxin triggers endocytosis of its receptor via a lipid raft-mediated clathrin-dependent process, J Cell Biol, 160, 321-328.
Akashi, S., S. Saitoh, Y. Wakabayashi, et al. (2003). Lipopolysaccharide interaction with cell surface Toll-like receptor 4-MD-2: higher affinity than that with MD-2 or CD14, J Exp Med, 198, 1035-1042.
Aktories, K., C. Schwan, and T. Jank. (2017). Clostridium difficile Toxin Biology, Annu Rev Microbiol, 71, 281-307.
Alonso, M. A., and J. Millan. (2001). The role of lipid rafts in signalling and membrane trafficking in T lymphocytes, J Cell Sci, 114, 3957-3965.
Awad, M. M., P. A. Johanesen, G. P. Carter, et al. (2014). Clostridium difficile virulence factors: Insights into an anaerobic spore-forming pathogen, Gut Microbes, 5, 579-593.
Barketi-Klai, A., M. Monot, S. Hoys, et al. (2014). The flagellin FliC of Clostridium difficile is responsible for pleiotropic gene regulation during in vivo infection, PLoS One, 9, 96876.
Barnich, N., J. E. Aguirre, H. C. Reinecker, et al. (2005). Membrane recruitment of NOD2 in intestinal epithelial cells is essential for nuclear factor kappa B activation in muramyl dipeptide recognition, J Cell Biol, 170, 21-26.
Batah, J., C. Deneve-Larrazet, P. A. Jolivot, et al. (2016). Clostridium difficile flagella predominantly activate TLR5-linked NF-kappa B pathway in epithelial cells, Anaerobe, 38, 116-124.
Ben-Zaken, O., S. Gingis-Velitski, I. Vlodavsky, and N. Ilan. (2007). Heparanase induces Akt phosphorylation via a lipid raft receptor, Biochem Biophys Res Commun, 361, 829-834.
Bianco, M., G. Fedele, A. Quattrini, et al. (2011). Immunomodulatory activities of surface-layer proteins obtained from epidemic and hypervirulent Clostridium difficile strains, J Med Microbiol, 60, 1162-1167.
Binion, D. (2016). Clostridium difficile Infection and Inflammatory Bowel Disease, Gastroenterol Hepatol (N Y), 12, 334-337.
Butera, A., M. Di Paola, L. Pavarini, et al. (2018). Nod2 Deficiency in mice is Associated with Microbiota Variation Favouring the Expansion of mucosal CD4+ LAP+ Regulatory Cells, Sci Rep, 8, 14241.
Calabi, E., F. Calabi, A. D. Phillips, and N. F. Fairweather. (2002). Binding of Clostridium difficile surface layer proteins to gastrointestinal tissues, Infect Immun, 70, 5770-5778.
Caruso, R., N. Warner, N. Inohara, and G. Nunez. (2014). NOD1 and NOD2: signaling, host defense, and inflammatory disease, Immunity, 41, 898-908.
Collins, L. E., M. Lynch, I. Marszalowska, et al. (2014). Surface layer proteins isolated from Clostridium difficile induce clearance responses in macrophages, Microbes Infect, 16, 391-400.
Dingle, K. E., X. Didelot, M. A. Ansari, et al. (2013). Recombinational switching of the Clostridium difficile S-layer and a novel glycosylation gene cluster revealed by large-scale whole-genome sequencing, J Infect Dis, 207, 675-686.
Eaton, K. A., S. Suerbaum, C. Josenhans, and S. Krakowka. (1996). Colonization of gnotobiotic piglets by Helicobacter pylori deficient in two flagellin genes, Infect Immun, 64, 2445-2448.
Fagan, R. P., and N. F. Fairweather. (2014). Biogenesis and functions of bacterial S-layers, Nat Rev Microbiol, 12, 211-222.
Ghose, C. (2013). Clostridium difficile infection in the twenty-first century, Emerg Microbes Infect, 2, 62.
Grogono-Thomas, R., M. J. Blaser, M. Ahmadi, and D. G. Newell. (2003). Role of S-layer protein antigenic diversity in the immune responses of sheep experimentally challenged with Campylobacter fetus subsp. fetus, Infect Immun, 71, 147-154.
Haiko, J., and B. Westerlund-Wikstrom. (2013). The role of the bacterial flagellum in adhesion and virulence, Biology (Basel), 2, 1242-1267.
Hartlova, A., L. Cerveny, M. Hubalek, et al. (2010). Membrane rafts: a potential gateway for bacterial entry into host cells, Microbiol Immunol, 54, 237-245.
Hasegawa, M., T. Yamazaki, N. Kamada, et al. (2011). Nucleotide-binding oligomerization domain 1 mediates recognition of Clostridium difficile and induces neutrophil recruitment and protection against the pathogen, J Immunol, 186, 4872-4880.
Heine, H., C. J. Kirschning, E. Lien, et al. (1999). Cutting edge: cells that carry A null allele for toll-like receptor 2 are capable of responding to endotoxin, J Immunol, 162, 6971-6975.
Hutton, M. L., M. Kaparakis-Liaskos, L. Turner, et al. (2010). Helicobacter pylori exploits cholesterol-rich microdomains for induction of NF-kappaB-dependent responses and peptidoglycan delivery in epithelial cells, Infect Immun, 78, 4523-4531.
Juul, F. E., K. Garborg, M. Bretthauer, et al. (2018). Fecal Microbiota Transplantation for Primary Clostridium difficile Infection, N Engl J Med, 378, 2535-2536.
Kabi, A., and C. McDonald. (2012). FRMBP2 directs NOD2 to the membrane, Proc Natl Acad Sci U S A, 109, 21188-21189.
Kim, S., A. Covington, and E. G. Pamer. (2017). The intestinal microbiota: Antibiotics, colonization resistance, and enteric pathogens, Immunol Rev, 279, 90-105.
Kirk, J. A., O. Banerji, and R. P. Fagan. (2017). Characteristics of the Clostridium difficile cell envelope and its importance in therapeutics, Microb Biotechnol, 10, 76-90.
Kowalsky, G. B., D. Beam, M. J. Oh, et al. (2011). Cholesterol depletion facilitates recovery from hypotonic cell swelling in CHO cells, Cell Physiol Biochem, 28, 1247-1254.
Kufer, T. A., E. Kremmer, D. J. Banks, and D. J. Philpott. (2006). Role for erbin in bacterial activation of Nod2, Infect Immun, 74, 3115-3124.
Lafont, F., G. Tran Van Nhieu, K. Hanada, et al. (2002). Initial steps of Shigella infection depend on the cholesterol/sphingolipid raft-mediated CD44-IpaB interaction, EMBO J, 21, 4449-4457.
Lai, C. H., H. J. Wang, Y. C. Chang, et al. (2011). Helicobacter pylori CagA-mediated IL-8 induction in gastric epithelial cells is cholesterol-dependent and requires the C-terminal tyrosine phosphorylation-containing domain, FEMS Microbiol Lett, 323, 155-163.
Lecine, P., S. Esmiol, J. Y. Metais, and C. Nicoletti. (2007). The NOD2-RICK complex signals from the plasma membrane, J Biol Chem, 282, 15197-15207.
Lecine, P., S. Esmiol, J. Y. Metais, et al. (2007). The NOD2-RICK complex signals from the plasma membrane, J Biol Chem, 282, 15197-15207.
Leffler, D. A., and J. T. Lamont. (2015). Clostridium difficile infection, N Engl J Med, 372, 1539-1548.
Liu, Y. H., Y. C. Chang, L. K. Chen, et al. (2018). The ATP-P2X7 Signaling Axis Is an Essential Sentinel for Intracellular Clostridium difficile Pathogen-Induced Inflammasome Activation, Front Cell Infect Microbiol, 8, 84.
Long, M., S. H. Huang, C. H. Wu, et al. (2012). Lipid raft/caveolae signaling is required for Cryptococcus neoformans invasion into human brain microvascular endothelial cells, J Biomed Sci, 19, 19.
Merrigan, M. M., A. Venugopal, J. L. Roxas, et al. (2013). Surface-layer protein A (SlpA) is a major contributor to host-cell adherence of Clostridium difficile, PLoS One, 8, 78404.
Motzkus-Feagans, C. A., A. Pakyz, R. Polk, et al. (2012). Statin use and the risk of Clostridium difficile in academic medical centres, Gut, 61, 1538-1542.
Munro, S. (2003). Lipid rafts: elusive or illusive?, Cell, 115, 377-388.
Ogura, Y., D. K. Bonen, N. Inohara, et al. (2001). A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease, Nature, 411, 603-606.
Papatheodorou, P., D. Hornuss, T. Nolke, et al. (2013). Clostridium difficile binary toxin CDT induces clustering of the lipolysis-stimulated lipoprotein receptor into lipid rafts, MBio, 4, e00244-00213.
Paredes-Sabja, D., A. Shen, and J. A. Sorg. (2014). Clostridium difficile spore biology: sporulation, germination, and spore structural proteins, Trends Microbiol, 22, 406-416.
Pellegrini, E., A. Desfosses, A. Wallmann, et al. (2018). RIP2 filament formation is required for NOD2 dependent NF-kappaB signalling, Nat Commun, 4043.
Plociennikowska, A., A. Hromada-Judycka, K. Borzecka, and K. Kwiatkowska. (2015). Co-operation of TLR4 and raft proteins in LPS-induced pro-inflammatory signaling, Cell Mol Life Sci, 72, 557-581.
Predrag, S., K. Branislava, S. Miodrag, et al. (2012). Clinical importance and representation of toxigenic and non-toxigenic Clostridium difficile cultivated from stool samples of hospitalized patients, Braz J Microbiol, 43, 215-223.
Rogers, T. J., C. M. Thorpe, A. W. Paton, and J. C. Paton. (2012). Role of lipid rafts and flagellin in invasion of colonic epithelial cells by Shiga-toxigenic Escherichia coli O113:H21, Infect Immun, 80, 2858-2867.
Ryan, A., M. Lynch, S. M. Smith, et al. (2011). A role for TLR4 in Clostridium difficile infection and the recognition of surface layer proteins, PLoS Pathog, 7, 1002076.
Schaffler, H., and A. Breitruck. (2018). Clostridium difficile - From Colonization to Infection, Front Microbiol, 9, 646.
Schneitz, C., L. Nuotio, and K. Lounatma. (1993). Adhesion of Lactobacillus acidophilus to avian intestinal epithelial cells mediated by the crystalline bacterial cell surface layer (S-layer), J Appl Bacteriol, 74, 290-294.
Schwan, C., T. Nolke, A. S. Kruppke, et al. (2011). Cholesterol- and sphingolipid-rich microdomains are essential for microtubule-based membrane protrusions induced by Clostridium difficile transferase (CDT), J Biol Chem, 286, 29356-29365.
Shen, A. (2012). Clostridium difficile toxins: mediators of inflammation, J Innate Immun, 4, 149-158.
Sidiq, T., S. Yoshihama, I. Downs, and K. S. Kobayashi. (2016). Nod2: A Critical Regulator of Ileal Microbiota and Crohn's Disease, Front Immunol, 7, 367.
Simons, K., and R. Ehehalt. (2002). Cholesterol, lipid rafts, and disease, J Clin Invest, 110, 597-603.
Simons, K., and D. Toomre. (2000). Lipid rafts and signal transduction, Nat Rev Mol Cell Biol, 1, 31-39.
Smits, W. K., D. Lyras, D. B. Lacy, et al. (2016). Clostridium difficile infection, Nat Rev Dis Primers, 2, 16020.
Sun, X., and S. A. Hirota. (2015). The roles of host and pathogen factors and the innate immune response in the pathogenesis of Clostridium difficile infection, Mol Immunol, 63, 193-202.
Tang, Y. M., and C. D. Stone. (2017). Clostridium difficile infection in inflammatory bowel disease: challenges in diagnosis and treatment, Clin J Gastroenterol, 10, 112-123.
Travassos, L. H., L. A. Carneiro, M. Ramjeet, et al. (2010). Nod1 and Nod2 direct autophagy by recruiting ATG16L1 to the plasma membrane at the site of bacterial entry, Nat Immunol, 55-62.
Voth, D. E., and J. D. Ballard. (2005). Clostridium difficile toxins: mechanism of action and role in disease, Clin Microbiol Rev, 18, 247-263.
Wright, A., R. Wait, S. Begum, et al. (2005). Proteomic analysis of cell surface proteins from Clostridium difficile, Proteomics, 5, 2443-2452.
Xu, X., H. Nagarajan, N. E. Lewis, et al. (2011). The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line, Nat Biotechnol, 29, 735-741.
Yamamoto, S., and X. Ma. (2009). Role of Nod2 in the development of Crohn's disease, Microbes Infect, 11, 912-918.