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研究生(外文):Chun-Yi Lee
論文名稱(外文):The intracellular distribution and target of IpaH4.5 and IpaH7.8
指導教授(外文):Suh-Der Tsen
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Shigella為兼性厭氧胞內寄生的革蘭氏陰性細菌,是一常見於腹瀉病人檢體中的病原菌,每年估計約有1.6億人口遭受Shigella感染,並且有100萬左右的人口死於Shigella之感染。Shigella感染過程中可由type III secretion system (T3SS)分泌數十種不同致病因子來幫助Shigella入侵至細胞內,這些致病因子的基因是位於一很大的致病質體上,在Shigella中致病因子可由不同的轉錄活化因子調控,可分為第一群分泌的致病因子以及第二群分泌的致病因子,第一群致病因子是在Shigella接觸到細胞時所分泌,這類致病因子主要是幫助Shigella入侵至細胞之中,第二類致病因子是在Shigella進入細胞後所表現,這類致病因子主要是幫助Shigella在細胞內生長。IpaH (invasive plasmid antigen gene H)家族蛋白質為第二類分泌的致病因子,IpaH家族蛋白質只存在Shigella以及EIEC (entero-invasive Escherichia coli)。IpaH的結構主要可分成具有leucine rich repeat (LRR)區域的N端以及一高度保留的區域的C端。不同的IpaH有相同的C端區域而N端則是有不同數目的LRR,目前認為N端是IpaH辨認細胞內的目標蛋白質,而C端是IpaH的功能區域。在不同的IpaH中,將IpaH4.5,IpaH7.8以及IpaH9.8突變之後,發現Shigella感染後細胞免疫反應會增強,而已知IpaH9.8會進入細胞核中並藉由和RNA剪切因子結合來影響chemokine基因表現。目前對於IpaH4.5以及IpaH7.8此二致病因子在細胞內分佈以及功能並不清楚,本研究主要針對IpaH4.5和IpaH7.8探討此二致病因子在細胞內的分佈情形,以及找尋IpaH4.5和IpaH7.8在細胞中可能的結合蛋白,結果發現IpaH4.5會聚集於細胞膜上並且有一72kD大小的蛋白質分子結合,而IpaH7.8是均勻散布細胞之中。
Shigella, a Gram-negative and facultative intracellular bacterium, is among the most frequently isolated pathogen from patients of diarrhea. It is estimated that 164.7 million cases and 1.1 million deaths were caused by Shigella infection annually. During infection, dozens of effectors were secreted by type III secretion system to help invasion of Shigella. These effectors are located on a large virulence plasmid and it expression is controlled by different transcription activators. Effectors can be divided into 2 groups. When Shigella contacts host cell, the first group of effectors are secreted and translocated into host cell. They help the invasion of Shigella. The second group of effectors are secreted after Shigella enters host cell. They help Shigella to survive in cell. IpaH family belong to the second group of effectors and were conserved in Shigella and EIEC. IpaH family have several LRR repeat regions at the N terminal and a conserved C terminal region. It has been speculated that the C terminal region is the functional domain and the variable N terminal regions allow ipaHs to interact with different targets. Deletion or mutation of IpaH4.5, IpaH7.8 and IpaH9.8 of Shigella may activate immune responses during infection. The intracellular distribution and target of IpaH4.5 and IpaH7.8 are not clear. In this study, focus was placed on intracellular distribution and cellular target of IpaH4.5 and IpaH7.8.IpaH4.5 accuMulated on cell membrane and IpaH7.8 accumulated in cytosol. In HeLa cell, IpaH4.5 binds to a 72 kD protein.
1. 英文摘要 1
2. 中文摘要 2
3. 介紹 3
4. 實驗材料與方法 16
5. 實驗結果 23
6. 討論 29
7. 文獻 34
8. 圖表
8-1 IpaH7.8的N端接上EGFP在HeLa細胞內的分佈 39
8-2 IpaH7.8的C端接上EGFP在HeLa細胞內的分佈 40
8-3 IpaH7.8的C端接上flag在HeLa細胞內的分佈 41
8-4 N端部分的IpaH7.8其C端接上EGFP在HeLa細胞內的分佈 42
8-5 C端部分的IpaH4.5其C端接上EGFP在HeLa細胞內的分佈 43
8-6 IpaH4.5的N端接上EGFP在HeLa細胞內的分佈 44
8-7 IpaH4.5的C端接上EGFP在HeLa細胞內的分佈 45
8-8 IpaH4.5的C端接上flag在HeLa細胞內的分佈 46
8-9 Ipah4.5的C端接上flag在HeLa細胞內以及細胞膜的分佈 47
8-10 N端部分的IpaH4.5其C端接上EGFP在HeLa細胞內的分佈 48
8-11 N端部分第1~10胺基酸剔除的IpaH4.5在HeLa細胞內的分佈 49
8-12 N端部分的IpaH4.5在HeLa細胞內以及粒線體的分佈 50
8-13 在E.coli BL21表現IpaH4.5以及IpaH7.8 51
8-14 在E.coli BL21表現IpaH4.5以及IpaH7.8並以西方點墨法分析 52
8-15 IpaH7.8進行共同沉澱以15% SDS-PHAGE分析 53
8-16 IpaH7.8進行共同沉澱以10% SDS-PHAGE分析 54
8-17 IpaH4.5進行共同沉澱以15% SDS-PHAGE分析 55
8-18 IpaH4.5進行共同沉澱以10% SDS-PHAGE分析 56
8-19 IpaH4.5進行in vivo IP以10% SDS-PHAGE分析 57
8-20 IpaH4.5進行in vivo IP以銀染以及西方點墨法分析 58
9. 附錄 59
1. Ashida, H., T. Toyotome, T. Nagai, and C. Sasakawa. 2007. Shigella chromosomal IpaH proteins are secreted via the type III secretion system and act as effectors. Molecular Microbiology 63:680-693.
2. Beatty, W., and P. Sansonetti. 1997. Role of lipopolysaccharide in signaling to subepithelial polymorphonuclear leukocytes. Infection and immunity 65:4395-4404.
3. Beutler, B., Z. Jiang, P. Georgel, K. Crozat, B. Croker, S. Rutschmann, X. Du, and K. Hoebe. 2006. Genetic analysis of host resistance: Toll-like receptor signaling and immunity at large. Annual review of immunology 24:353-89. (review)
4. Buysse, J., C. Stover, E. Oaks, M. Venkatesan, and D. Kopecko. 1987. Molecular cloning of invasion plasmid antigen (ipa) genes from Shigella flexneri: analysis of ipa gene products and genetic mapping. Journal of bacteriology 169:2561-2569.
5. Clerc, P., A. Ryter, J. Mounier, and P. Sansonetti. 1987. Plasmid-mediated early killing of eucaryotic cells by Shigella flexneri as studied by infection of J774 macrophages. Infection and immunity 55:521-527.
6. Demers, B., P. Sansonetti, and C. Parsot. 1998. Induction of type III secretion in Shigella flexneri is associated with differential control of transcription of genes encoding secreted proteins. The EMBO Journal 17:2894.
7. Falconi, M., G. Prosseda, M. Giangrossi, E. Beghetto, and B. Colonna. 2001. Involvement of FIS in the H-NS-mediated regulation of virF gene of Shigella and enteroinvasive Escherichia coli. Molecular Microbiology 42:439-452.
8. Freudenberg, M., S. Tchaptchet, S. Keck, G. Fejer, M. Huber, N. Sch tze, B. Beutler, and C. Galanos. 2008. Lipopolysaccharide sensing an important factor in the innate immune response to Gram-negative bacterial infections: benefits and hazards of LPS hypersensitivity. Immunobiology 213:193-203.
9. Gall, T., M. Mavris, M. Martino, M. Bernardini, E. Denamur, and C. Parsot. 2005. Analysis of virulence plasmid gene expression defines three classes of effectors in the type III secretion system of Shigella flexneri, Soc General Microbiol p. 951-962, vol. 151.
10. Goetz, M., A. Bubert, G. Wang, I. Chico-Calero, J. Vazquez-Boland, M. Beck, J. Slaghuis, A. Szalay, and W. Goebel. 2001. Microinjection and growth of bacteria in the cytosol of mammalian host cells. Proceedings of the National Academy of Sciences USA 98:12221.
11. Hale, T. 1991. Genetic basis of virulence in Shigella species. Microbiology and Molecular Biology Reviews 55:206-224.
12. Haraga, A., and S. Miller. 2003. A Salmonella enterica Serovar Typhimurium Translocated Leucine-Rich Repeat Effector Protein Inhibits NF-κB-Dependent Gene Expression Editor: VJ DiRita. Infection and immunity 71:4052-4058.
13. Hartman, A., M. Venkatesan, E. Oaks, and J. Buysse. 1990. Sequence and molecular characterization of a multicopy invasion plasmid antigen gene, ipaH, of Shigella flexneri. Journal of bacteriology 172:1905-1915.
14. High, N., J. Mounier, M. Prevost, and P. Sansonetti. 1992. IpaB of Shigella flexneri causes entry into epithelial cells and escape from the phagocytic vacuole. The EMBO Journal 11:1991.
15. Jurica, M., and M. Moore. 2003. Pre-mRNA splicing awash in a sea of proteins. Molecular cell 12:5-14.
16. Kane, C., R. Schuch, W. Day, and A. Maurelli. 2002. MxiE regulates intracellular expression of factors secreted by the Shigella flexneri 2a type III secretion system. Journal of bacteriology 184:4409-4419.
17. Kobe, B., and J. Deisenhofer. 1994. The leucine-rich repeat: a versatile binding motif. Trends in biochemical sciences(Regular ed.) 19:415-421.
18. Kobe, B., and A. Kajava. 2001. The leucine-rich repeat as a protein recognition motif. Current opinion in structural biology 11:725-732.
19. Kotloff, K., J. Winickoff, B. Ivanoff, J. Clemens, D. Swerdlow, P. Sansonetti, G. Adak, and M. Levine. 1999. Global burden of Shigella infections: implications for vaccine development and implementation of control strategies. Bulletin of the World Health Organization 77:651-666.
20. Mavris, M., A. Page, R. Tournebize, B. Demers, P. Sansonetti, and C. Parsot. 2002. Regulation of transcription by the activity of the Shigella flexneri type III secretion apparatus. Molecular Microbiology 43:1543-1553.
21. McDonald, C., P. Vacratsis, J. Bliska, and J. Dixon. 2003. The Yersinia virulence factor YopM forms a novel protein complex with two cellular kinases. Journal of Biological Chemistry 278:18514-18523.
22. Miao, E., C. Scherer, R. Tsolis, R. Kingsley, L. Adams, A. Baumler, and S. Miller. 1999. Salmonella typhimurium leucine-rich repeat proteins are targeted to the SPI1 and SPI2 type III secretion systems. Mol Microbiol 34:850-864.
23. Niyogi, S., M. Vargas, and J. Vila. 2004. Prevalence of the sat, set and sen genes among diverse serotypes of Shigella flexneri strains isolated from patients with acute diarrhoea. Clinical Microbiology & Infection 10:574-576.
24. O'Brien, A., C. Fernandez-Prada, D. Hoover, B. Tall, A. Hartman, J. Kopelowitz, and M. Venkatesan. 2000. Shigella flexneri IpaH7. 8 facilitates escape of virulent bacteria from the endocytic vacuoles of mouse and human macrophages. Infection and immunity 68:3608-3619.
25. Ogawa, M., Y. Handa, H. Ashida, M. Suzuki, and C. Sasakawa. 2008. The versatility of Shigella effectors. Nature Reviews Microbiology 6:11-16.
26. Okuda, J., T. Toyotome, N. Kataoka, M. Ohno, H. Abe, Y. Shimura, A. Seyedarabi, R. Pickersgill, and C. Sasakawa. 2005. Shigella effector IpaH9. 8 binds to a splicing factor U2AF35 to modulate host immune responses. Biochemical and Biophysical Research Communications 333:531-539.
27. Pacheco, T., A. Gomes, N. Barbosa-Morais, V. Benes, W. Ansorge, M. Wollerton, C. Smith, J. Valcarcel, and M. Carmo-Fonseca. 2004. Diversity of Vertebrate Splicing Factor U 2 AF 35: IDENTIFICATION OF ALTERNATIVELY SPLICED U 2 AF 1 mRNAs. Journal of Biological Chemistry 279:27039-27049.
28. Pacheco, T., L. Moita, A. Gomes, N. Hacohen, and M. Carmo-Fonseca. 2006. RNA interference knockdown of hU2AF35 impairs cell cycle progression and modulates alternative splicing of Cdc25 transcripts. Molecular biology of the cell 17:4187.
29. Paetzold, S., S. Lourido, B. Raupach, and A. Zychlinsky. 2007. Shigella flexneri Phagosomal Escape Is Independent of Invasion. Infection and immunity 75:4826.
30. Page, A., H. Ohayon, P. Sansonetti, and C. Parsot. 1999. The secreted IpaB and IpaC invasins and their cytoplasmic chaperone IpgC are required for intercellular dissemination of Shigella flexneri. Cellular Microbiology 1:183-193.
31. Payne, S., and R. Finkelstein. 1977. Detection and differentiation of iron-responsive avirulent mutants on Congo red agar. Infection and immunity 18:94-98.
32. Pedron, T., C. Thibault, and P. Sansonetti. 2003. The Invasive Phenotype of Shigella flexneri Directs a Distinct Gene Expression Pattern in the Human Intestinal Epithelial Cell Line Caco-2*. Journal of Biological Chemistry 278:33878-33886.
33. Rohde, J., A. Breitkreutz, A. Chenal, P. Sansonetti, and C. Parsot. 2007. Type III secretion effectors of the IpaH family are E3 ubiquitin ligases. Cell Host & Microbe 1:77-83.
34. Sakai, T., C. Sasakawa, and M. Yoshikawa. 1988. Expression of four virulence antigens of Shigella flexneri is positively regulated at the transcriptional level by the 30 kilo Dalton virF protein. Molecular Microbiology 2:589-597.
35. Sansonetti, P., A. Ryter, P. Clerc, A. Maurelli, and J. Mounier. 1986. Multiplication of Shigella flexneri within HeLa cells: lysis of the phagocytic vacuole and plasmid-mediated contact hemolysis. Infection and immunity 51:461-469.
36. Schroeder, G., and H. Hilbi. 2008. Molecular pathogenesis of Shigella spp.: controlling host cell signaling, invasion, and death by type III secretion. Clinical Microbiology Reviews 21:134.
37. Singer, A., J. Rohde, R. Lam, T. Skarina, O. Kagan, R. DiLeo, N. Chirgadze, M. Cuff, A. Joachimiak, and M. Tyers. 2008. Structure of the Shigella T3SS effector IpaH defines a new class of E3 ubiquitin ligases. Nature Structural & Molecular Biology 15:1293-1301.
38. Suzuki, T., H. Mimuro, S. Suetsugu, H. Miki, T. Takenawa, and C. Sasakawa. 2002. Neural Wiskott-Aldrich syndrome protein (N-WASP) is the specific ligand for Shigella VirG among the WASP family and determines the host cell type allowing actin-based spreading. Cellular Microbiology 4:223-233.
39. Toyotome, T., T. Suzuki, A. Kuwae, T. Nonaka, H. Fukuda, S. Imajoh-Ohmi, T. Toyofuku, M. Hori, and C. Sasakawa. 2001. Shigella protein IpaH9. 8 is secreted from bacteria within mammalian cells and transported to the nucleus. Journal of Biological Chemistry 276:32071-32079.
40. Veenendaal, A., J. Hodgkinson, L. Schwarzer, D. Stabat, S. Zenk, and A. Blocker. 2007. The type III secretion system needle tip complex mediates host cell sensing and translocon insertion. Molecular Microbiology 63:1719-1730.
41. Venkatesan, M., J. Buysse, and A. Hartman. 1991. Sequence variation in two ipaH genes of Shigella flexneri 5 and homology to the LRG-like family of proteins. Molecular Microbiology 5:2435-2445.
42. Venkatesan, M., J. Buysse, and D. Kopecko. 1989. Use of Shigella flexneri ipaC and ipaH gene sequences for the general identification of Shigella spp. and enteroinvasive Escherichia coli. Journal of Clinical Microbiology 27:2687-2691.
43. Venkatesan, M., M. Goldberg, D. Rose, E. Grotbeck, V. Burland, and F. Blattner. 2001. Complete DNA sequence and analysis of the large virulence plasmid of Shigella flexneri. Infection and immunity 69:3271.
44. Wassef, J., D. Keren, and J. Mailloux. 1989. Role of M cells in initial antigen uptake and in ulcer formation in the rabbit intestinal loop model of shigellosis. Infection and immunity 57:858-863.
45. West, N., P. Sansonetti, J. Mounier, R. Exley, C. Parsot, S. Guadagnini, M. Prevost, A. Prochnicka-Chalufour, M. Delepierre, and M. Tanguy. 2005. Optimization of virulence functions through glucosylation of Shigella LPS, p. 1313-1317, vol. 307. American Association for the Advancement of Science.
46. Yang, F., J. Yang, X. Zhang, L. Chen, Y. Jiang, Y. Yan, X. Tang, J. Wang, Z. Xiong, and J. Dong. 2005. Genome dynamics and diversity of Shigella species, the etiologic agents of bacillary dysentery. Nucleic Acids Research 33:6445.
47. Yoshida, S., E. Katayama, A. Kuwae, H. Mimuro, T. Suzuki, and C. Sasakawa. 2002. Shigella deliver an effector protein to trigger host microtubule destabilization, which promotes Rac1 activity and efficient bacterial internalization. The EMBO Journal 21:2923-2935.
48. Zurawski, D., K. Mumy, C. Faherty, B. McCormick, and A. Maurelli. 2009. Shigella flexneri type III secretion system effectors OspB and OspF target the nucleus to downregulate the host inflammatory response via interactions with retinoblastoma protein. Molecular Microbiology 71:350-368.
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