臺灣博碩士論文加值系統

創傷弧菌是一種嗜鹽性的革蘭氏陰性菌，普遍存在於海洋、河口交界處，亦稱海洋弧菌。此菌為人體致病菌，當人體受到感染時，常常引發兩種的臨床病症分別是原發性敗血症和傷口感染，其中原發性敗血症的病患往往在到院後的48小時內死亡，致死率高達50%。然而，創傷弧菌致病機制和致病因子，至今日仍尚未清楚，但目前研究報告證實的致病因子有莢膜、磷脂酶、細胞溶解毒素、金屬蛋白酶及RTX毒素等。
巨噬細胞是身體防禦外來病原體侵略的重要成員之一，也是人體先天性免疫的第一道防線，然而，當人體遭受創傷弧菌感染而造成嚴重的病症，其主要的原因是由於創傷弧菌會毒殺巨噬細胞，導致人體免疫系統的功能降低，造成人體急劇死亡。因此，本論文目的是要選殖出創傷弧菌對巨噬細胞的毒殺基因，進而分析該毒殺基因的特性及其與其他基因的相關聯性，並提供往後探討創傷弧菌與人體免疫統系統之間關聯性的依據。
為了要選殖創傷弧菌對巨噬細胞的毒殺基因，我們以顯微鏡和細胞毒性分析，由867株創傷弧菌跳躍子 (transposon5, Tn5) 突變株中篩選出7株 (29D2、1E5、35F11、34C7、47C9、45E8及3F1) 對巨噬細胞毒殺能力下降的跳躍子突變株，其中5株突變株 (29D2、1E5、35F11、34C7及47C9)，經由基因的選殖與序列比對的分析，得知對巨噬細胞具有毒殺能力之毒殺基因是rtxA、rtxE、argD及VV3093。rtxA的核苷酸序列長度為15,621 bp，RtxA由5,206個氨基酸構成，其分子大小為~572 kDa，是一種cytotoxic toxin，屬於RTX家族的一員，而rtxE的核苷酸序列長度為2,169 bp，RtxE 由722個氨基酸組成，其分子大小為~79 kDa，RtxE的功能為ABC轉運蛋白 (ABC transporter)，與RtxB和 RtxD共同形成ABC transporter，是屬於第一型蛋白質分泌系統，幫助毒殺蛋白RtxA由菌體內運送至胞外，造成對巨噬細胞的毒殺作用，argD的核苷酸序列長度為1,212 bp，ArgD由403個氨基酸構成的蛋白質，其分子大小為~44 kDa，是一個與尿素循環及胺基酸代謝 (urea cycle and metabolison of amino acid) 有關的基因，VV3093的核苷酸序列長度為636 bp，由211個氨基酸組成的，其分子大小為~23 kDa，其功能為一種轉錄作用的調節子 (transcriptional regulator)。
綜合上述結果，我們由創傷弧菌跳躍子突變株中，共篩選出7株對巨噬細胞毒殺能力下降的突變株，經由基因選殖與序列比對分析，顯示這些毒殺巨噬細胞，分別是RTX家族的毒殺蛋白RtxA和負責轉運毒殺蛋白RtxA的RtxE、尿素循環及胺基酸代謝有關的ArgD及轉錄作用調節子的VV3093。

Vibrio vulnificus a halophilic gram-negative marine bacterium that is survied in ocean, estuarine water. V. vulnificus causes both fatal primary septicemia and wound infection in human, mortality was up to 50% in septic patients, with most of them dying within 48 h. Recently, Several putatie virulence factors of V. vulnificus are demonstrated, such as capsule, metalloprotease, phospholipase, cytolysin, iron acquisition systems and RTX toxin. To identify the genes required for killing of macrophage, we screened an insertional mutant library of V. vulnificus generated by transposon mutagenesis for reduced cytotoxicity toward macrophage. Here we reported that among 867 Tn5 mutants screened, 7 Tn5 mutants exhibited decreased cytotoxic activity toward marcrophage. Four genes inserted by transposon were cloned and sequenced. Sequence analysis revealed that the transposon was inserted into the genes : rtxA, rtxE, argD and VV3039. RtxA was composed of 5,206 amino acids with a theoretical molecular mass of ~572 kDa and is a member of RTX family. RtxE was composed of 722 amino acids with a theoretical molecular mass of ~79 kDa. The RtxE is an ABC transporter (ATP-binding cassette transporter) that belongs to a type I secretion system. ArgD was composed of 403 amino acids with a theoretical molecular mass of ~44 kDa and was involved in an urea cycle pathway and amino acid metabolism. VV3093 was composed of 636 amino acids with a theoretical molecular mass of ~23 kDa and probably was a transcription regulator. In this study, we cloned and sequenced four genes: rtxA, rtxE, argD and VV3093 and these genes probably were involved in cytotoxicity towards macrophages.

中文摘要 .............................................. I
英文摘要 .............................................. III
本文目錄 .............................................. V
圖目錄 .............................................. IX
表目錄 .............................................. X
附錄目錄 .............................................. XI
縮寫表 .............................................. XII
第一章 緒論 .......................................... 1
1.1 創傷弧菌 ......................................... 1
1.2 創傷弧菌毒力因子 ................................. 4
1.3 創傷弧菌跳躍子突變株的建構 ....................... 8
1.4 研究動機 ......................................... 9
第二章　材料與方法 .................................... 11
2.1 材料與藥品 ....................................... 11
2.1.1 菌株與質體 ..................................... 11
2.1.2 細胞 ........................................... 11
2.1.3 培養基 ......................................... 11
2.1.3.1 細菌培養基 ................................... 11
2.1.3.2 細胞培養基 ................................... 12
2.1.4 抗生素 ......................................... 13
2.1.5 溶液 .......................................... 13
2.1.6 DNA標準品 ...................................... 15
2.1.7 酵素 ........................................... 16
2.1.8 套組 ........................................... 16
2.2 實驗方法 ......................................... 17
2.2.1 菌株培養技術 ................................... 17
2.2.1.1 菌株的培養條件 ............................... 17
2.2.1.2 菌株的保存 ................................... 17
2.2.1.3 跳躍子突變株於96孔盤的保存 ................... 17
2.2.1.4 細菌生長曲線之測定 ........................... 18
2.2.2 細胞培養技術 ................................... 18
2.2.2.1 冷凍細胞之解凍活化 ........................... 18
2.2.2.2 細胞的保存方法 ............................... 18
2.2.2.3 細胞的繼代培養 ............................... 19
2.2.2.4 細胞計數的方法 ............................... 19
2.2.2.5 細胞培養於96孔培養盤的方法 ................... 19
2.2.2.6 細胞毒性分析 ................................. 20
2.2.3 基本的分生技術 ................................. 21
2.2.3.1　套組純化質體DNA .............................. 21
2.2.3.2　鹼裂解法純化質體DNA .......................... 22
2.2.3.3　套組純化細菌染色體DNA ........................ 22
2.2.3.4　DNA電泳 ...................................... 23
2.2.3.5　聚合酶連鎖反應 ............................... 23
2.2.3.6　限制酵素切割DNA .............................. 24
2.2.3.7　DNA片段膠體分離回收及純化 .................... 24
2.2.3.8　接合反應 ..................................... 25
2.2.3.9　勝任細胞製備 ................................. 25
2.2.3.10 轉形作用 ..................................... 25
2.2.4　創傷弧菌中巨噬細胞毒殺基因之選殖 ............... 26
2.2.4.1 對巨噬細胞毒性降低的跳躍子突變株之篩選 ....... 26
2.2.4.2　跳躍子突變株對巨噬細胞毒性測試 ............... 26
2.2.4.3 選殖巨噬細胞毒殺基因 ......................... 26
第三章　結果 .......................................... 28
3.1 創傷弧菌對巨噬細胞毒殺基因之選殖 .................. 28
3.1.1 篩選創傷弧菌對巨噬細胞毒殺能力下降之突變株 ...... 28
3.1.2 創傷弧菌跳躍子突變株於LB broth培養基之生長情形 .. 30
3.1.3 選殖創傷弧菌對巨噬細胞之毒殺基因 ................ 31
3.1.4 創傷弧菌對巨噬細胞之毒殺基因序列分析 ............ 32
第四章　討論 .......................................... 34
第五章　結論 .......................................... 37
參考文獻 .............................................. 38
圖 .................................................... 46
表 .................................................... 52
附錄 .................................................. 56

1.Strom MS, Paranjpye RN. Epidemiology and pathogenesis of Vibrio vulnificus. Microbes Infect. 2000;2(2):177-188.
2.Kaspar CW, Tamplin ML. Effects of temperature and salinity on the survival of Vibrio vulnificus in seawater and shellfish. Appl Environ Microbiol. 1993;59(8):2425-2429.
3.Oliver JD, Hite F, McDougald D, Andon NL, Simpson LM. Entry into, and resuscitation from, the viable but nonculturable state by Vibrio vulnificus in an estuarine environment. Appl Environ Microbiol. 1995;61(7):2624-2630.
4.Blake PA, Merson MH, Weaver RE, Hollis DG, Heublein PC. Disease caused by a marine Vibrio. Clinicalcharacteristics and epidemiology. N Engl J Med. 1979;300(1):1-5.
5.Hollis DG, Weaver RE, Baker CN, Thornsberry C. Halophilic Vibrio species isolated from blood cultures. J Clin Microbiol. 1976;3(4):425-431.
6.Yuan CY, Yuan CC, Wei DC, Lee AM. Septicemia and gangrenous change of the legs caused by marine vibrio, Vibrio vulnificus—report of a case. Taiwan Yi Xue Hui Za Zhi. 1987;86(4):448–451.
7.Chuang YC, Yuan CY, Liu CY, Lan CK. Huang AH. Vibrio vulnificus infection in Taiwan: report of 28 cases and review of clinical manifestations and treatment. Clin Infect Dis. 1992;15(2):271–276.
8.Hsueh PR, Lin CY, Tang HJ, Lee HC, Liu JW, Liu YC, Chuang YC. Vibrio vulnificus in Taiwan. Emerg Infect Dis. 2004;10(8):1363-1368.
9.Amaro C, Biosca EG, Fouz B, and Garay E. Electrophoretic analysis of heterogeneous lipopolysaccharides from various strains of Vibrio vulnificus biotype 1 and 2 by silver staining and immunoblotting. Curr Microbi. 1992;25(2):99-104.
10.Biosca EG, Oliver JD, Amaro C. Phenotypic characterization of Vibrio vulnificus biotype 2, a lipopolysaccharide-based homogeneous O serogroup within Vibrio vulnificus. Appl Environ Microbiol. 1996;62(3):918-927.
11.Tison DL, Nishibuchi M, Greenwood JD, Seidler RJ. Vibrio vulnificus biogroup 2: new biogroup pathogenic for eels. Appl Environ Microbiol. 1982;44(3):640–646.
12.Bisharat N, Agmon V, Finkelstein R, et al. Clinical, epidemiological, and microbiological features of Vibrio vulnificus biogroup 3 causing outbreaks of wound infection and bacteraemia in Israel. Lancet. 1999;354(9188):1421-1424.
13.Hlady WG, Klontz KC. The epidemiology of Vibrio infections in Florida, 1981-1993. J Infect Dis. 1996;173(5):1176-1183.
14.Klontz KC, Lieb S, Schreiber M, Janowski HT, Baldy LM, Gunn RA. Syndromes of Vibrio vulnificus infections. Clinical and epidemiologic features in Florida cases, 1981-1987. Ann Intern Med. 1988;109(4):318-323.
15.Tacket CO, Brenner F, Blake PA. Clinical features and an epidemiological study of Vibrio vulnificus infections. J Infect Dis. 1984;149(4):558-561.
16.Tamplin ML, Specter S, Rodrick GE, Friedman H. Vibrio vulnificus resists phagocytosis in the absence of serum opsonins. Infect Immun. 1985;49(3): 715-718.
17.Yoshida S, Ogawa M, Mizuguchi Y. Relation of capsular materials and colony opacity to virulence of Vibrio vulnificus. Infect Immun. 1985;47(2): 446-451.
18.Wright AC, Simpson LM, Oliver JD, Morris JJ. Phenotypic evaluation of acapsular transposon mutants of Vibrio vulnificus. Infect Immun. 1990; 58(6):1769-1773.
19.Powell JL, Wright AC, Wasserman SS, Hone DM, Morris JJ. Release of tumor necrosis factor alpha in response to Vibrio vulnificus capsular polysaccharide in in vivo and in vitro models. Infect Immun. 1997;65(9): 3713-3718.
20.McPherson VL, Watts JA, Simpson LM, Oliver JD. Physiological effects of the lipopolysaccharide of Vibrio vulnificus on mice and rats. Microbios. 1991;67(272-273):141-149.
21.Espat NJ, Auffenberg T, Abouhamze A, Baumhofer J, Moldawer LL, Howard RJ. A role for tumor necrosis factor-alpha in the increased mortality associated with Vibrio vulnificus infection in the presence of hepatic dysfunction. Ann Surg. 1996;223(4):428-433.
22.Paranjpye RN, Lara JC, Pepe JC, Pepe CM, Strom MS. The type IV leader peptidase/N-methyltransferase of Vibrio vulnificus controls factors required for adherence to HEp-2 cells and virulence in iron-overloaded mice. Infect Immun. 1998;66(12):5659-5668.
23.Kim HR, Rho HW, Jeong MH, et al. Hemolytic mechanism of cytolysin produced from V. vulnificus. Life Sci. 1993;53(7):571-577.
24.Wright AC, Morris JG Jr. The extracellular cytolysin of Vibrio vulnificus: inactivation and relationship to virulence in mice. Infect Immun. 1991;59(1):192-197.
25.Park JW, Ma SN, Song ES, et al. Pulmonary damage by Vibrio vulnificus cytolysin. Infect Immun. 1996;64(7):2873-2876.
26.Smith GC, Merkel JR. Collagenolytic activity of Vibrio vulnificus: potential contribution to its invasiveness. Infect Immun. 1982;35(3):1155-1156.
27.Kothary MH, Kreger AS. Purification and characterization of an elastolytic protease of Vibrio vulnificus. J Gen Microbiol. 1987;133(7):1783-1791.
28.Miyoshi S, Nakazawa H, Kawata K, Tomochika K, Tobe K, Shinoda S. Characterization of the hemorrhagic reaction caused by Vibrio vulnificus metalloprotease, a member of the thermolysin family. Infect Immun. 1998; 66(10):4851-4855.
29.Lin W, Fullner KJ, Clayton R, et al. Identification of a vibrio cholerae RTX toxin gene cluster that is tightly linked to the cholera toxin prophage. Proc Natl Acad Sci U S A. 1999;96(3):1071-1076.
30.Fullner KJ, Mekalanos JJ. In vivo covalent cross-linking of cellular actin by the Vibrio cholerae RTX toxin. EMBO J. 2000;19(20):5315-5323.
31.Cordero CL, Kudryashov DS, Reisler E, Satchell KJ. The Actin cross- linking domain of the Vibrio cholerae RTX toxin directly catalyzes the covalent cross-linking of actin. J Biol Chem. 2006;281(43):32366-32374.
32.Pukatzki S, Ma AT, Sturtevant D, et al. Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc Natl Acad Sci U S A. 2006;103(5):1528-1533.
33.Lee JH, Kim MW, Kim BS, et al. Identification and characterization of the Vibrio vulnificus rtxA essential for cytotoxicity in vitro and virulence in mice. J Microbiol. 2007;45(2):146-152.
34.Liu M, Alice AF, Naka H, Crosa JH. The HlyU protein is a positive regulator of rtxA1, a gene responsible for cytotoxicity and virulence in the human pathogen Vibrio vulnificus. Infect Immun. 2007;75(7):3282-3289.
35.Weinberg ED. Microbial pathogens with impaired ability to acquire host iron. Biometals. 2000;13(1):85-89.
36.Hlady WG, Klontz KC. The epidemiology of Vibrio infections in Florida, 1981-1993. J Infect Dis. 1996;173(5):1176-1183.
37.Wright AC, Simpson LM, Oliver JD. Role of iron in the pathogenesis of Vibrio vulnificus infections. Infect Immun. 1981;34(2):503-507.
38.Simpson LM, Oliver JD. Siderophore production by Vibrio vulnificus. Infect Immun. 1983;41(2):644-649.
39.Hensel M, Shea JE, Gleeson C, Jones MD, Dalton E, Holden DW. Simultaneous identification of bacterial virulence genes by negative selection. Science. 1995;269(5222):400-403.
40.Tsuchiya T, Mitsuo E, Hayashi N, et al. Vibrio vulnificus damages macrophages during the early phase of infection. Infect Immun. 2007;75(9):4592-4596.
41.Kashimoto T, Ueno S, Hanajima M, et al. Vibrio vulnificusinduces macrophage apoptosis in vitro and in vivo. Infect Immun. 2003;71(1):533-555.
42.Chuang YC, Chang TM, Chang MC. Cloning and characterization of the gene (empV) encoding extracellular metalloprotease from Vibrio vulnificus. Gene. 1997;189(2):163-168.
43.Lally ET, Hill RB, Kieba IR, Korostoff J. The interaction between RTX toxins and target cells. Trends Microbiol. 1999;7(9):356-361.
44.Boardman BK, Satchell KJ. Vibrio cholerae strains with mutations in
an atypical type I secretion system accumulate RTX toxin intracellularly. J Bacteriol. 2004;186(23):8137-8143.
45.Kim YR, Lee SE, Kook H, et al. 2008. Vibrio vulnificus RTX toxin kills host cells only after contact of the bacteria with host cells. Cell Microbiol. 10(4):848–862.
46.Lee BC, Choi S, HKim TS. Vibrio vulnificus RTX toxin plays an important role in the apoptotic death of human intestinal epithelial cells exposed to Vibrio vulnificus. Microbes Infect. 2008;10(14-15):1504-1513.
47.Lee BC, Le JHe, Kim MW, et al. Vibrio vulnificus rtxE is important for virulence and its expression is induced by exposure to host cells. Infect Immun. 2008;76(4):1509–1517.
48.Okada U, Kondo K, Hayashi T, et al. Structural and functional analysis of the TetR-family transcriptional regulator SCO0332 from Streptomyces coelicolor. Acta Crystallogr D Biol Crystallogr. 2008;64(2):198-205.