創傷弧菌中毒殺巨噬細胞基因之選殖與特性分析

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Title:	創傷弧菌中毒殺巨噬細胞基因之選殖與特性分析 Cloning and characterization of the genes required for macrophage cytotoxicity from Vibrio vulnificus
Authors:	鍾雅婷 Ya-Ting Chung
Contributors:	陳昱仲嘉南藥理科技大學：生物科技研究所
Keywords:	創傷弧菌巨噬細胞跳躍子細胞毒殺 Vibrio vulnificus macrophage transposon cytotoxicity
Date:	2008
Issue Date:	2008-12-29 15:23:57 (UTC+8)
Abstract:	創傷弧菌是一種嗜鹽性的革蘭氏陰性菌，當人類感染創傷弧菌後會造成傷口感染及原發性敗血症等嚴重的症狀。目前創傷弧菌可能的致病因子有細胞溶血毒素、蛋白酶、磷酸酯解酶和細菌表面莢膜等。巨噬細胞為人體先天免疫的第一道防線，當有外來抗原、病原體入侵人體時，巨噬細胞會吞噬並清除外來物。創傷弧菌之所以會感染人類導致嚴重病症，主要是創傷弧菌會毒殺巨噬細胞，使人體降低抵抗能力，達到感染的目的。因此，在本論文中，我們選殖出創傷弧菌對巨噬細胞的毒殺基因，並進行毒殺基因的特性分析，以進一步了解創傷弧菌與宿主的免疫系統之間的關係。為了找出創傷弧菌對巨噬細胞的毒殺因子，首先，進行跳躍子突變株庫對老鼠巨噬細胞的細胞毒性分析，在2,481株的跳躍子突變株中，共有9株突變株對巨噬細胞的毒殺能力比野生株低弱，這9株突變株經由基因的選殖及序列的分析，顯示這些巨噬細胞毒殺基因的弁鄍]括：GTPase活性、第二型蛋白質分泌能力及細胞毒殺能力。經蛋白質序列分析顯示，創傷弧菌對巨噬細胞的細胞毒殺因子ychF與大腸桿菌（Escherichia coli K-12）的GTP結合蛋白質YchF具有79%相同性，與耶爾森氏菌（Yersinia pseudotuberculosis IP32953）的老鼠致病因子VagA具有78%相同性，而與布魯氏菌（Brucella melitensis 16M）在調控鐵利用的DugA具有58%相同性。創傷弧菌YchF除了扮演著對巨噬細胞毒殺的角色外，也具有菌株對鐵利用的弁遄C綜合以上結果，創傷弧菌細胞毒殺因子ychF可能是使創傷弧菌在巨噬細胞內能夠獲得充足的鐵離子而迅速生長，造成對巨噬細胞的毒殺作用。 Vibrio vulnificus is a halophilic gram-negative bacterium that causes both wound infection and fatal primary septicemia in human. The virulence factors of V. vulnificus include cytolytic hemolysin, protease, phospholipase, and capsule. 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 2,481 Tn5 mutants screened, 9 Tn5 mutants exhibited decreased cytotoxic activity toward marcrophage. Nine genes inserted by transposon were cloned and sequenced. Sequence analysis revealed that the transposon was inserted into the genes that encode proteins with GTPase activity, protein secretion and cytotoxic activity. Here we report that one of the isolated Tn5 mutants deficient in macrophage cytotoxicity had a mutation in an open reading frame identified as ychF, a gene encoding a protein with 58% of sequence identity to that of DugA of Brucella melitensis 16M, a protein required for the acquisition of iron. At Fe3+-limiting LB broth, the ychF mutant showed attenuated growth compared to the wild-type strain. These results demonstrate that V. vulnificus may require the ychF involved in acquisition of iron to replicate within macrophage, resulting in macrophage cytotoxicity.
Relation:	校內校外均不公開
Appears in Collections:	[Dept. of Biotechnology (including master's program)] Dissertations and Theses

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