臺灣博碩士論文加值系統

克雷伯氏肺炎桿菌 ( Klebsiella pneumoniae ) 為造成伺機性感染( opportunistic infection ) 及引起台灣化膿性肝膿瘍 ( pyogenic liver abscess ) 的主要病原菌之一。而根據相關研究表明，糖尿病 ( diabetes mellitus ) 患者為罹患 KPLA ( Klebsiella pneumoniae liver abscess )的高風險族群，但其具體致病機制還尚未清楚。許多研究指出細菌致病與生物膜 ( biofilm ) 之生成具有關聯性，細菌的附著為形成生物膜的第一步，而纖毛 ( fimbriae ) 在這扮演著一個重要的角色。第三型纖毛為克雷伯氏肺炎桿菌特有之菌毛，由 mrk 基因叢所調控，其中MrkA為構成第三型纖毛主要結構之蛋白質。在本實驗室先前研究中，發現高表達 etcABC 之克雷伯氏肺炎桿菌菌株，在高濃度葡萄糖下仍可以表達第三型纖毛。EtcABC 為三個屬於 PTS ( phosphoenolpyruvate-phosphotransferase system ) 的EⅡ complex 之蛋白質，因此，本篇論文收集 78 隻臨床菌株並透過西方墨點法偵測其 MrkA 表現量，發現約三分之一臨床菌株其 MrkA 於高濃度葡萄糖下不會被抑制。針對不會被抑制MrkA 表現量之臨床菌株，本研究使用 RT-qPCR 之方式偵測其 etcA 表達量，發現約有 75 % 之臨床菌株其 etcA 表達量明顯高於野生株 STU-1，而被高濃度葡萄糖壓抑MrkA 表現量之臨床菌株中約 90 % 其etcA 表達量呈現被壓抑的狀態。因此，本研究認為 etcABC 在葡萄糖環境下，對於調控第三型纖毛扮演著一個重要的角色。 此外，本研究同時建立線蟲模式 ( nematode model )，來加以研究細菌定殖於秀麗隱桿線蟲( Caenorhabditis elegans ) 腸道能力及生物膜之關聯性，利用電穿孔之技術使菌體帶有螢光基因並餵食給線蟲，並分別培養於高濃度葡萄糖及無糖環境下觀察其腸道定殖之情況，藉由克雷伯氏肺炎桿菌 STU-1 及臨床菌株，發現被高濃度葡萄糖抑制 MrkA 的菌株，其生物膜生成量與在腸道定殖的情況也受到葡萄糖抑制，本研究發現etcABC的高度表現造成克雷伯氏肺炎桿菌在高濃度葡萄糖下仍然大量表現第三型纖毛，而 etcABC 受壓抑的菌株則受葡萄糖的影響，壓抑第三型纖毛的表現，進而減少生物膜的形成與降低定殖於線蟲腸道的能力。

Klebsiella pneumoniae is one of the pathogens causing opportunistic infection and pyogenic liver abscess in Taiwan. According to related studies, diabetes mellitus patients are a high-risk group for Klebsiella pneumoniae liver abscess ( KPLA ), but the specific pathogenic mechanism is still unclear. Many reports suggested the pathogenesis of bacteria is related to the formation of biofilm. The attachment of bacteria is the first step in the formation of biofilm, and fimbriae play an important role in this. Type 3 fimbriae are encoded by the mrk gene cluster, of which MrkA is the protein that constitutes the main structure of type 3 fimbriae in K.pneumoniae. In the previous studies in our laboratory, overexpression of etcABC in K.pneumoniae results in expression of type 3 fimbriae in high glucose concentration. EtcABC are three proteins respectively homologous to EIIABC in phosphoenolpyruvate-phosphotransferase system ( PTS ). Therefore, in this study, we collected 78 clinical strains and performed western blotting to detect their MrkA expression and found that MrkA were not repressed by glucose in approximate one-third of the clinical strains. For the strains in which MrkA are not repressed by glucose, we tested their etcA expression by RT-qPCR and found that about 75 % of strains highly expressed etcA than wild type STU-1. In contrast, etcA expression was repressed in about 90% of strains in which MrkA are repressed by glucose. Thus, we thought etcABC maybe play an important role in type 3 fimbriae regulation by glucose.
Next in this study we also built a nematode model to examine the bacterial colonization in Caenorhabditis elegans and the relation with biofilm. First, we used electroporation to let bacteria carry fluorescence gene, and then respectively observed their colonization in intestine of nematodes in high-concentration glucose and glucose-free environments. In Klebsiella pneumoniae STU-1 and one clinical strain ( no. 042200 ), MrkA was inhibited by high concentration of glucose, and their biofilm production and colonization in the intestine were also inhibited by glucose. In this study we found that the high expression of etcABC caused Klebsiella pneumoniae to still express a large number of type III fimbriae under high concentrationof glucose. In contrast, the strains with low level of etcABC showed decreased expression of type III fimbriae in medium containing glucose, thereby reducing biofilm production and reduced ability to colonize in the nematode intestine.

中文摘要 I
Abstract III
目錄 VI
附圖表目錄 X
第一章、前言 1
1.1克雷伯氏肺炎桿菌 (Klebsiella pneumoniae) 簡介 1
1.2 肝膿瘍簡介 1
1.3 克雷伯氏肺炎桿菌之細菌性肝膿瘍( Klebsiella pneumoniae liver abscess, KPLA ) 2
1.4 克雷伯氏肺炎桿菌之毒力因子 4
1.5 克雷伯氏肺炎桿菌之生物膜 5
1.6 克雷伯氏肺炎桿菌之纖毛 ( fimbriae ) 8
1.7 克雷伯氏肺炎桿菌之etcABCDE 操縱子 ( operon ) 9
1.8 秀麗隱桿線蟲 (Caenorhabditis elegans ) 10
1.9 秀麗隱桿線蟲之腸道 11
1.10 研究目的 12
第二章、實驗材料 15
2.1 實驗使用菌株 15
2.2 實驗使用質體 17
2.3 抗生素 17
2.4 引子 17
2.5 探針 18
2.6 實驗藥品 19
2.7 培養液殘餘葡萄糖濃度定性測試試劑 21
2.8 培養基成分 22
2.9 蛋白質膠體電泳檢體試劑 23
2.10 西方墨點法材料與試劑 23
2.11 染色體純化試劑 25
第三章、實驗方法 26
3.1 培養液殘餘葡萄糖濃度定性試驗 26
3.2 不同葡萄糖濃度菌株培養 26
3.3 蛋白質電泳分析 – 聚丙烯醯胺凝膠電泳 ( sodium dodecyl sulfate polyacrylamide gel electrophoresis , SDS – PAGE ) 27
3.4 西方墨點法 28
3.5 RNA 純化與反轉錄互補DNA 製備 29
3.5.1 RNA萃取 29
3.5.2 反轉錄作用合成互補 DNA 30
3.5.3 即時定量聚合酶鏈接反應 31
3.6 生物膜分析 31
3.7 秀麗隱桿線蟲之培養與操作 31
3.7.1 秀麗隱桿線蟲蟲卵培養 31
3.7.2 將秀麗隱桿線蟲轉移至培養基 32
3.7.3 餵食秀麗隱桿線蟲之菌株培養 33
3.7.4 餵食秀隱桿線蟲 33
3.7.5 秀隱桿線蟲之螢光顯微鏡拍攝 33
3.8 質體純化 ( 質體萃取套組法 ) 34
3.9 轉型作用 ( Transformation ) – 電穿孔法 35
3.9.1 電穿孔法之勝任細胞製備 35
3.9.2 電穿孔法之細菌轉型作用 35
3.10 ImageJ 統計軟體分析 36
第四章、實驗結果 37
4.1 定性臨床菌株在不同葡萄糖濃度培養試驗中培養液殘餘葡萄糖濃度 37
4.2 分析臨床菌株在高濃度葡萄糖培養下第三型纖毛主要結構蛋白質 MrkA 表現量之差異 37
4.3 以即時定量聚合酶鏈接反應分析臨床菌株之 etcA 表達量 38
4.3.1未被抑制之臨床菌株中約80 % 臨床菌株之 etcA 表達量較高 38
4.3.2 被壓抑之臨床菌株中約 % 之被其etcA 表達量較低 39
4.4 建立秀麗隱桿線蟲 ( Caenorhabditis elegans ) 模式觀察菌體定殖之可行性 40
4.4.1 DH5α 菌株在秀麗隱桿線蟲腸道中可成功定殖 40
4.4.2 Δcrp 菌株在秀麗隱桿線蟲 ( Caenorhabditis elegans ) 腸道中失去定殖能力 41
4.5 克雷伯氏肺炎桿菌野生株 ( wild type ) STU-1 在秀麗隱桿線蟲 ( Caenorhabditis elegans ) 腸道中之定殖能力 41
4.6 秀麗隱桿線蟲之模式可運用於不同菌株 42
4.6.1透過臨床菌株 042200 證實線蟲模式可運用於不同菌株 42
4.6.2 第三型纖毛不被葡萄糖所壓抑之臨床菌株 046273 可成功觀察到線蟲腸道定殖 43
第五章、討論 45
第六章、附圖表 49
第七章、參考資料 75

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簡銘男, 陳銘仁 ＆ 劉昌邦 糖尿病人罹患肺炎克雷白氏桿菌肝膿瘍
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