Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22240
標題: 志賀氏桿菌ipaB基因產物對哺乳動物細胞毒殺作用之探討
The cytotoxic effects of Shigella flexneri ipaB on mammalian cells
作者: 陳琬蓉
Chen, Wan-Rong
關鍵字: Shigella flexneri
志賀氏桿菌
ipaB gene
mitochondria
ipaB基因
粒線體
出版社: 分子生物學研究所
引用: 鄭焯隆。2003。台灣南投地區痢疾桿菌菌株ipaB基因表現與致病性相關性之研究。國立中興大學分子生物學研究所碩士論文。 顏佩詩。2006。台灣志賀氏桿菌的 ipaB 基因及其蛋白產物功能分析。國立中興大學分子生物學研究所碩士論文。 許哲瑋。2009。志賀氏桿菌 ipaB 基因及其衍生蛋白對肺癌細胞的影響。國立中興大學分子生物學研究所碩士論文。 張瑋倫。2010。福氏志賀菌IpaB 衍生蛋白之純化及其對哺乳動物細胞的影響。國立中興大學分子生物學研究所碩士論文。 Arnoult, D. 2006. Mitochondrial fragmentation in apoptosis. Cell biology. 1: 6-12. Bernardini, M. L., J. Mounier, H. Hauteville, M. Coquis-Rondon, and P. J. Sansonetti. 1989. Identification of icsA, a plasmid locus of Shigella flexneri which governs bacterial intra- and intercellular spread through interaction with F-actin. Proc Natl Acad Sci USA.86 : 3867-3871. Buchrieser, C., P. Glaser, C. Rusniok, H. Nedjari, H. D’Hauteville, F. Kunst, P. Sansonetti, and C. Parsot. 2000. The virulence plasmid pWR100 and the repertoire of proteins secreted by the type III secretion apparatus of Shigella flexneri. Mol Microbiol. 38: 760-771. Blocker, A., P.Gounon, E. Larquet, K. Niebuhr, V. Cabiaux, C. Parsot, and P. Sansonetti. 1999. The tripartite type III secreton of Shigella flexneri inserts IpaB and IpaC into host membranes. J. Cell Biol. 147: 683-693. Chen, Y., M. R. Smith, K.Thirumalai, and A. Zychlinsky. 1996. A bacterial invasion induces macrophage apoptosis by binding directly to ICE. EMBOJ. 5: 3853-3860. Brian, K. C and B.B’Finlay . 2005. Insertion of the bacterial type III translocon:not your average needle stick. Trends Microbiol. 13: 92-95. Cossart, P ., and P. J. Sansonetti. 2004. Bacterial invasion: the paradigms of enteroinvasive pathogens.Science. 304. 242-248. Chen, C., and D.C. Chen. 2004. Mitchondrial dynamics in mammals. Curr Top Dev Biol 59:119-144. Chan, DC. 2006.Mitochondrial fusion and fission in mammals.Annu Rev Cell Dev Bio 22 : 79-99. Clark, C. S., and A.T. Maurelli. 2007. Shigella flexneri inhibits staurosporine-induced apoptosis in epithelial cell. Infect Immun. 75: 2531-2539. Dupont, H. L., M. M. Levine, R. B. Hornick, and S. B. Formal.1989. Inoculum size in shigellosis and implications for expected mode of transmission. J. Infect. Dis. 159:1126-1128. Espina, M., A. J. Olive, R. Kenjale, D. S. Moore, S. F. Ausar, R. W. Kaminski, E. V. Oaks, C. R. Middaugh, W. D. Picking, and W. L. Picking. 2006. IpaD localizes to the tip of the type III secretion system needle of Shigella flexneri. Infect Immun 74: 4391-4400. Enninga , J., J. Mounier, P. Sansonetti, and G. T.V. Nhieu. 2005. Secretion of type III effectros into host cells in real time. Nat Methods 2:959-965. Elmore, S. 2007. Apoptosis: a review of programmed cell death. Toxicol Pathol. 35: 495-516. Ernest, N. J., C.W. Habela, and H. Sontheimer. 2008. Cytoplasmic condensation is both necessary and sufficient to induce apoptotic cell death. J Cell Sci. 121: 290-297. Francois, M., V. L. Cabe, M. Dupont., P. J. Sansonetti, and I. Maridonneau-Parrini. 2000. Induction of Necrosis in Human Neutrophils by Shigella flexneri Requires Type III Secretion, IpaB and IpaC Invasins, and Actin Polymerization. Infect. Immun. 68:1289-1296. Fink, S. L and B. T. Cookson. 2005. Apoptosis, Pyroptosis, and Necrosis: Mechanistic description of dead and dying eukaryotic cells. . Infect Immun.73: 1907-1916. Guichon, A., D. Hersh, M. R. Smith,and A. Zychlinsky.2001. Structure-function analysis of the shigella virulence factor IpaB. J Bacteriol. 183: 1269-1276. Hilbi , H., J.E Moss, D .Hersh, Y. Chen, and J.Arondel. 1998. Shigella-induced apoptosis is dependent on caspase-1 which binds to IpaB. J Biol Chem 273:32895–32900. Harrington, A., N. Darboe, R. Kenjale, W. L. Picking, C. R. Middaugh, S. Birket, and W. D. Picking. 2006. Characterization of the interaction of single tryptophan containing mutants of IpaC from Shigella flexneri with phospholipid membranes. Biochemistry 45: 626-636. Jennison, A. V., and N. K. Verma. 2004. Shigella flexneri infection: pathogenesis and vaccine development. FEMS Microbiol. Rev. 28:43-58. Jeong, S. Y., and D.W. Seol. 2008. The role of mitochondria in apoptosis.BMB.41(1):11-22. Jagasia, R., P. Grote, B. Westermann, and B. Conradt. 2005. DRP-1-mediated mitochondrial fragmentation during EGL-1-induced cell death in C. elegans. Natur. 433: 754-760. Kotloff , K. L., J. P. Winickoff, B. Ivanoff , J. D. Clemens, D. L. Swerdlow, P. J. Sansonetti, G. K. Adak, and M. M. Levine. 1999. Global burden of Shigella infections: implications for vaccine development and implementation of control strategies. Bull World Health Organ. 77. 651 – 666. Kuwae, A., S. Yoshida, K. Tammano, H. Mimuro, T. Suzuki, and C. Sasakawa. 2001. Shigella invasion of macrophage requires the insertion of IpaC into the host plasma membrane-functional analysis of IpaC. J Biol Chem. 276 : 32230-32239. Lafont, F., G. T. V. Nhieu, K. Hanada, P. Sansonetti, and F. G. Goot. 2002. Initial steps of Shigella infection depend on the choesterol/sphingolipid raft-mediated CD44-IpaB interaction. EMBO J. 17: 4449-4457. Le-Barillec, K., J. G. Magalhaes, E. Corcuff, A. Thuizat, P. J. Sansonetti, A. Phalipon, and J. P. Di Santo. 2005. Roles for T and NK cells in the innate immune response to Shigella flexneri. J Immunol. 175: 1735-1740. Lee,Y., SY. Jeong, M. Karbowski, C. L. Smith, and R. J. Youle. 2004. Mol Biol Cell. 15: 5001-5011. Me’nard, R., P. J. Sansonetti,C. Parsot, and T. Vasselon. 1994. Extracellular association and cytoplasmic partitioning of the IpaB and IpaC invasins of S. flecneri. Cell 79:515-525. Margineantu, D. H., W. G. Cox, L. Sundell, S. W. Sherwood, J. M. Beechem, and R. A. Capaldi. 2002. Cell cycle dependent morphology changes and associated mitochondrial DNA redistribution in mitochondrial of human cell lines. Mitochondrion.1: 425-435. Morrison, D. A. 1979. Tranformation and preservation of competent bacterial cells by freezing. Methods Enzyrnol. 68: 326-331. Paetzold, S., S. Lourido, B. Raupach, and A. Zychlinsky. 2007. Shigella flexner phagosomal escape is independent of invasion. Infect Immun. 75: 4826-4830. Picking, W. L., J. A. Mertz, M. E. Marquart, and W. D. Picking. 1996. Protein expression and purefication. 8: 401-408. Plumb, J. A., R. Milroy, and S. B. Kaye. 1989. Effects of the pH dependence of 3-(4,5-Dimethylthoazol-2-yl)-2, 5-diphenyltetrazolium bromide-formazan absorption on chemosensitivity determined by a novel tetrazolium-based assay. Cancer research. 49 : 4435-4440. Roehrich, A. D., I. Martinez-Argudo, S. Johnson, A. J. Blocker, and A. K. J. Veenendaal. 2010. The extreme C terminus of Shigella flexneri IpaB is required for regulation of Type III secretion, needle tip composition, and binding. Immunity. 78 (4) : 1682-1691. Sansonetti. P. J. 2001. Microbes and microbial toxins: paradigms for microbial-mucosal interactions III. Shigellosis: from symptoms to molecular pathogenesis. Am. J. Physiol. Gastrointest. Liver Physiol. 280:G319-G323. Sansonetti, P. J., A. Phalipon, J. Arondel, K. Thirumalai, S. Banerjee, S. Akira, K. Takeda, and A. Zychlinsky. 2000. Caspase-1 activation of IL-1β and IL-18 are essential for Shigella flexneri-induced inflammation. Immunity 12: 581- 590. Suzuki, T., L. Franchi, C. Toma, H.Ashida, M.Ogawa, Y .Yoshikawa, H .Mimuro, N. Inohara, C .Sasakawa, and G. Nuñez. 2007. Differential Regulation of Caspase-1 Activation,Pyroptosis, and Autophagy via Ipaf and ASC in Shigella-Infected Macrophages. PLoS Pathog 3(8): e111. Schroeder, G. N., and H. Hilbi. 2008. Molecular pathogenesis of Shigella spp.: controlling host cell signaling, invsion, and death by type III secretion. Clin Microbiol. 21:134-156. Schroeder, G. N., and H. Hilbi. 2007. Cholesterol is required to trigger caspase-1 activation and macrophage apoptosis after phagosomal escape of Shigella. Cellular Microbiology. 9(1): 265-278. Schroeder, G. N., N. J. Jannt, and H. Hilbi. 2007. Intracellular type III secretion by cytoplasmic Shigella flexneri promotes caspase-1-dependent macrophage cell death. Microbiology.153: 2862-2876. Stensrud, K. F., P. R. Adam, C.D. La-Mar, A. J. Olive, G. H. Lushington, R. Sudharsan, N. L. Shelton, R. S. Givens, W. L. Picking, and W. D. Picking. 2008. Deoxycholate interacts with IpaD of Shigella flexneri in induving the recruitment of IpaB to the Type III secretion apparatus needle tip. JBC: 283(27): 18646-18654. Schuch, R., and A. T. Maurelli. 1997. Virulence plasmid instability in Shigella flexneri 2a is induced by virulence gene expression. Immunity. 65(9): 3686-3692. Nhieu. G.T,, and P. J. Sansonetti. 1999. Mechanism of Shigella entry into epithelial cells. Curr Opin Microbiol. 2: 51-55. Veenendaal , Ak., JL. Hodgkinson, L . Schwarzer, D. Stabat, SF . Zenk, and AJ .Blocker. 2007. The type III secretion system needle tip complex mediates host cell sensing and translocon insertion. Mol Microbiol. 63: 1719-1730. Zychlinsky. A., M. C. Prevost, and P. J. Sansonetti. 1992. Shigella flexneri induces apoptosis in infected macrophages. Natur 358:167-169.
摘要: Shigella flexneri為革蘭氏陰性細菌,具有毒性質體,其上帶有許多包括ipaB致病基因。本實驗室有pFLAG-CMV2-IpaB-His和pFLAG-CMV2-IpaB803-His質體,分別可在哺乳動物細胞表現重組 Flag-IpaB-His 蛋白及缺C端hydrophobic domain的Flag-IpaB803-His蛋白。本研究首先將此二質體轉殖到HepG2、CL1-5、CHO-K1三株細胞株中,培養48小時後,以免疫螢光染色觀察重組蛋白的表現。結果顯示: 將pFLAG-CMV2-IpaB-His質體送入HepG2、CL1-5、CHO-K1,表現FLAG-IpaB-His蛋白的細胞平均百分比為<1%、<2%、~10%;而將pFLAG-CMV2-IpaB803-His質體送入此三株細胞株,表現FLAG-IpaB803-His蛋白的細胞平均百分比為<1%、10%、15%。 pFLAG-CMV2-IpaB-His質體送到HepG2、CL1-5、CHO-K1、A549、HeLa,培養24小時後,除了HeLa細胞存活率增加26%,其他細胞存活率皆不變 (與送pFLAG-CMV2質體到三株細胞的對照組相比);培養48小時後,五株細胞的存活率為減少15%、不變、不變、增加20%、不變。送pFLAG-CMV2-IpaB803-His質體到五株細胞,培養24小時後,細胞存活率皆不變;培養48小時後,五株細胞存活率分別為減少15%、減少20%、不變、不變、不變。 將二質體送到CL1-0、U937、CaCO-2及COLO205四株細胞株,培養24、48小時後,細胞存活率皆不變。 送pFLAG-CMV2-IpaB-His 到CL1-5,24、48、72小時後,進行免疫螢光染色。發現送入pFLAG-CMV2-IpaB-His質體,24、48、72小時後,有FLAG-IpaB-His蛋白表現的CL1-5細胞,細胞型態改變。24小時後,有FLAG-IpaB-His蛋白表現的細胞,一半FLAG-IpaB-His蛋白在細胞質,一半FLAG-IpaB-His蛋白在細胞質/細胞核;48小時後,全部細胞FLAG-IpaB-His蛋白在細胞質;72小時後,全部細胞FLAG-IpaB-His蛋白在細胞質/細胞核。另一方面,24、48小時後,約二成的細胞,其細胞核有濃縮的現象,八成細胞,細胞核正常;72小時後,細胞核皆正常,但細胞膜有小泡的生成 (blebbing)。 送pFLAG-CMV2-IpaB803-His到CL1-5,24、48、72小時後,有FLAG-IpaB803-His蛋白表現的細胞,細胞型態皆改變,48及72小時後,還可看到凋亡小體產生。24小時後,有FLAG-IpaB-His蛋白表現的細胞,一半FLAG-IpaB-His蛋白在細胞質,一半FLAG-IpaB-His蛋白在細胞質/細胞核;48小時後,七成FLAG-IpaB803-His蛋白在細胞質,三成FLAG-IpaB803-His蛋白在細胞質/細胞核;72小時後,全部細胞FLAG-IpaB-His蛋白在細胞質。另一方面,24小時後,一半的細胞,細胞核有濃縮的現象,另一半的細胞,細胞核正常;48、72小時後,一半的細胞,細胞核有濃縮的現象,另一半的細胞,細胞核呈破碎狀。 送pFLAG-CMV2-IpaB-His和pFLAG-CMV2-IpaB803-His到CL1-5,48小時後,利用MitoTracker dye染色,觀察細胞粒線體的型狀。發現有FLAG-IpaB-His蛋白表現的細胞,粒線體型狀不變;但是有FLAG-IpaB803-His蛋白表現的細胞,粒線體明顯片斷化。 將可在細胞表現IpaB-EGFP及IpaB803-EGFP蛋白的pEGFP-N2-IpaB及pEGFP-N2-IpaB803質體轉殖到HEK-293T、CL1-0、PA-1、CHO-K1、CL1-5,48小時後,有表現IpaB-EGFP及IpaB803-EGFP蛋白的CHO-K1細胞平均百分比為<5%,有表現IpaB803-EGFP蛋白的HEK293T細胞平均百分比為<5%,表現二蛋白的其他四株細胞百分比為<1%;但送入pEGFP-N2質體到五株細胞,表現EGFP蛋白的五株細胞百分比,皆為30% ~ 40%。顯示IpaB-EGFP蛋白及IpaB803-EGFP蛋白在五株細胞株中可能易被降解,導致有蛋白表現的細胞百分比低 (<1% 或5%)。 轉殖pEGFP-N2-IpaB和pEGFP-N2-IpaB803質體到CL1-5,24、48小時後,細胞存活率皆不變;有表現IpaB-EGFP及IpaB803-EGFP蛋白的細胞,細胞型態改變,但是粒線體正常,沒有片斷化。
Shigella flexeri is a gram negative bacterium, which contains the virulence plasmid including IpaB gene. pFLAG-CMV2-IpaB-His and pFLAG-CMV2-IpaB803-His plasmids were transfected into mammalian cells, which can expression FLAG-IpaB-His recombinant protein and absence of C-terminal hydrophobic domain of FLAG-IpaB803-His protein. In this study, the HepG2, CL1-5, CHO-K1 cell lines containing the two plasmids were cultured to 48 hours and using immunofluorescence staining, recombinant protein expression was studied. The results showed that the FLAG-IpaB-His protein in the three cell lines showed <1%, 5%, 10% protein expression whereas the FLAG-IpaB803-His protein showed <1%, 10%, 20%. HepG2, CL1-5, CHO-K1, A549, HeLa cell lines contanining pFALG-CMV2-IpaB-His plasmid was cultured for 24 hrs showed 26% increased survival rate in HeLa cells but no difference in the other cell lines. For the 48 hrs, HepG2 showed 15% reduced survival rate but A549 showed 20% increased survival rate. pFLAG-CMV2-IpaB803-His plasmid was transferred to the 5 cell lines and cultured for 24 hrs, showed no difference in the cell survival while 48hrs showed 15% and 20% reduced survival rate in HepG2 and CL1-5 respectively. The two plasmids were transferred to CL1-0, U937, CaCO-2 and COLO205 cell lines and cultured for 24 hrs and 48 hrs. Cell survival was the no difference. The pFLAG-CMV2-IpaB-His plasmid was tranfected in CL1-5 cell line and protein expression was studied using immunofluorescence staining. The CL1-5 was cultured for 24 hr, 48 hr, and 72 hr. After 24 hrs, half of the FLAG-IpaB-His protein was found in the cytoplasm and other half in the cytoplasm/nucleus; 48 hrs, FLAG-IpaB-His protein in all the cells was found in the cytoplasm; 72 hrs, FLAG-IpaB-His protein was found in the cytoplasm/nucleus in all cells. It was found out that FLAG-IpaB-His protein expression inhibited the cell differentiation of the CL1-5. On the other hand, after 24 hr and 48 hr, 20% of the cells show nuclei condensation. After 72 hrs, the nucleus is normal but showed blebbing in the cell membrane. pFLAG-CMV2-IpaB803-His plasmid was transferred to CL1-5 cell line and cultured to 24 hr, 48 hr, and 72 hrs. After 24 hrs, half of the FLAG-IpaB-His protein is found in the cytoplasm and the rest in the cytoplasm/nucleus. In 48 hrs, 70% of the protein is in cytoplasm and 30% in the cytoplasm/nucleus. In 72 hrs, all the protein is in the cytoplasm. Apoptotic bodies can be seen in the 48 hrs and 72 hrs. Cell differentiation is inhibited by the expression of FLAG-IpaB803-His protein. On the other hand, after 24 hrs, nucleus is condensed in 50% of cells but after 48 hr and 72 hrs, half of the cell's nuclei are condensed and the rest have broken nucleus. The CL1-5 line was transfected with pFlAG-CMV2-IpaB-His and pFLAG-CMV2-IpaB803-His to check the effect of the protein expression to the mitochondria. It was found that the FLAG-IpaB-His protein did not change the shape of the mitochondria but the FLAG-IpaB803-His protein caused mitochondrial fragmentation. The pEGFP-N2-IpaB and pEGFP-N2-IpaB803 plasmids were transfected into HEK-293T, CL1-0, PA-1, CHO-K1, CL1-5 cell lines and cultured for 48 hrs. The expression of IpaB-EGFP and IpaB803-EGFP recombinant protein expression was studied. The percentage of the 2 proteins in CHO-K1 cells is <5% but in the other cell lines is <1% but the pEGFP-N2 plasmid to the 5 cell lines contains 30% to 40% of EGFP protein. This shows that expression of IpaB-EGFP protein and IpaB803-EGFP protein in the five cell lines is susceptible to degradation resulting in the low levels of protein expression. CL1-5 cell line containing the pEGFP-N2-IpaB and pEGFP-N2-IpaB803 plasmid was cultured for 24 hr and 48 hr. The cell survival was the no different but the recombinant protein expression inhibited the cell differentiation but showed no fragmentation of the mitochondria.
URI: http://hdl.handle.net/11455/22240
其他識別: U0005-2208201114304300
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2208201114304300
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