請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19336
標題: | 海洋分支桿菌mmar_2318和mmar_2319基因負責生合成細胞壁脂寡聚醣與調控對黏菌的毒性 Mycobacterium marinum mmar_2318 and mmar_2319 are Responsible for Lipooligosaccharide Biosynthesis and Virulence towards Dictyostelium: screening in a transposon mutant library |
作者: | Yi-Yin Chen 陳依吟 |
指導教授: | 王錦堂(Jin-Town Wang) |
關鍵字: | 海洋分支桿菌,脂寡聚醣,致病力,巨噬細胞,黏菌, M. marinum,lipooligosaccharide,virulence,macrophage,Dictyostelium, |
出版年 : | 2016 |
學位: | 博士 |
摘要: | 抵抗吞噬細胞的吞噬作用為結核桿菌屬的細菌最主要的致病機制,故本研究利用海洋分支桿菌 (M. marinum) 作為研究材料。海洋分支桿菌是結核分支桿菌的近親,其基因體定序已被完成,在魚類造成的病症和人類結核病相類似,接著再以黏菌 (Dictyostelium discoideum) 作為宿主模式。黏菌和哺乳類巨噬細胞有許多相似的特徵,具有吞噬並殺死細菌的能力,可用來替代研究致病菌與巨噬細胞之間的交互作用,成為快速篩選的實驗平台。本研究利用跳躍子建立了1728株海洋分支桿菌突變株庫,並篩選出對黏菌具有毒力的基因。篩選後獲得30株突變株仍可使黏菌存活,此30株突變株共分別影響20個基因區段,其中有6個基因 (losA, mmar_2318, mmar_2319, wecE, mmar_2323 and mmar_2353) 位於脂寡聚醣 (lipooligosaccharide/LOS) 生合成區段中。脂寡聚醣為已知細胞壁上的醣脂抗原,在海洋分支桿菌中主要的脂寡聚醣結構為LOS-I到LOS-IV四種。
以二維色層薄層分析法 (2D-TLC) 分析醣脂抗原,結果發現mmar_2318 和 mmar_2319的基因剔除株皆為LOS-III累積量提高而LOS-IV生成缺失,其基因剔除株與基因補回株皆證實mmar_2318 和 mmar_2319會對黏菌造成毒力,但不顯著影響細菌進入黏菌的比例以及細菌在黏菌內複製的能力。進一步利用鼠類巨噬細胞株(J774a.1)及人類巨噬細胞株 (PMA-induced THP-1) 進行實驗,結果發現mmar_2318 和 mmar_2319的基因剔除株,其在細胞株內的複製能力皆不受影響,但細菌進入細胞的比例在THP-1中有顯著增加,而J774a.1則否。總結來說,雖然mmar_2319已有研究指出會參與LOS的生合成,但我們仍發現了新的基因,mmar_2318並且一樣參與LOS的生合成路徑,且mmar_2318 與mmar_2319的基因剔除株會降低對黏菌的毒力作用並細菌增加進入THP-1的細菌數。 Resistance to phagocyte killing is an important virulence factor in mycobacteria. Dictyostelium has been used to study the interaction between phagocytes and bacteria, given its similarity to the mammalian macrophage. Here, we investigated the genes responsible for virulence to Dictyostelium by screening 1728 transposon mutants of the Mycobacterium marinum NTUH-M6094 strain. A total of 30 mutants that were permissive for Dictyostelium growth were identified. These mutants revealed interruptions in 20 distinct loci. Of the 20 loci, six genes (losA, mmar_2318, mmar_2319, wecE, mmar_2323 and mmar_2353) were located in the lipooligosaccharide (LOS) synthesis cluster. LOS are antigenic glycolipids and the core LOS structure from LOS-I to LOS-IV have been reported to exist in M. marinum. Two-dimensional thin-layer chromatography (2D-TLC) glycolipid profiles revealed that deletion of mmar_2318 or mmar_2319 resulted in the accumulation of LOS-III and deficiency of LOS-IV. Deletion and complementation of mmar_2318 or mmar_2319 confirmed that both genes contributed to virulence towards Dictyostelium but not entry and replication inside Dictyostelium. Co-incubation with a murine macrophage cell line J774a.1 or PMA-induced human monocytic cell line THP-1 demonstrated that mmar_2318 or mmar_2319 deletion mutant could grow in macrophages, and their initial entry rate was not affected in J774a.1 but significantly increased in THP-1. In conclusion, although mmar_2319 has been reported to involve LOS biosynthesis in a previous study, we identified a new gene, mmar_2318 that is also involved in the biosynthesis of LOS. Deletion of mmar_2318 or mmar_2319 both exhibits reduction of virulence towards Dictyostelium and increased entry into THP-1 cells. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19336 |
DOI: | 10.6342/NTU201600926 |
全文授權: | 未授權 |
顯示於系所單位: | 微生物學科所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-105-1.pdf 目前未授權公開取用 | 2.32 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。