廖淑貞臺灣大學:醫學檢驗暨生物技術學研究所林紫禕Lin, Tzu-YiTzu-YiLin2010-05-112018-07-062010-05-112018-07-062009U0001-1208200918054100http://ntur.lib.ntu.edu.tw//handle/246246/182966Proteus mirabilis (奇異變形桿菌) 為腸桿菌科，革蘭氏陰性的兼性厭氧菌，在健康人類腸道屬於正常菌叢，但在長期使用導尿管之免疫低下患者，會造成伺機性感染，常見如泌尿道感染，甚至可能導致腎臟病、肺炎或敗血症的發生。olymyxin B (多黏菌素B) 屬抗菌胜肽的一種，以其帶正電荷的結構與細菌細胞外膜的負電荷結合，破壞細胞膜的穩定或形成孔洞，使細胞內的物質外漏進而達到殺菌的效果。研究發現，革蘭氏陰性菌藉由在lipopolysaccharide (LPS) 的lipid A上以4-amino-4-deoxy-L-arabinose (L-Ara4N) 修飾，使細胞表面負電性降低，造成polymyxin B與細胞膜結合減少而產生抗性。. mirabilis天生就對polymyxin B具有很高的抗性，但其抗藥機制仍不甚清楚。本研究利用mini-Tn5 transposon mutagenesis來篩選出對polymyxin B具感受性之突變株，結果發現突變處為預測可轉譯出UDP-glucose dehydrogenase 的ugd gene；在Escherichia coli與Salmonella Typhimurium研究中，ugd與polysaccharide及L-Ara4N的合成有關。分析其他表現型，發現ugd突變株有以下特徵：1.表面移行能力喪失 2.鞭毛蛋白合成減少 3.分化成游走細胞的細胞數減少 4.脂多醣缺失 5.溶血酶活性降低 6.入侵細胞能力下降 7.細胞表面構造改變。利用pACYC-184質體將ugd基因補回，發現原先突變株受影響之毒力因子均回復與野生株相當，表示ugd基因影響這些毒力因子的表現。究ugd基因與表面移行能力的關係，以real time RT-PCR結果推測，P. mirabilis ugd基因突變造成正向調控flhDC表現之umoA、B、D 膜蛋白以及與鞭毛合成相關的flhDC、fliA及flaA mRNA表現減少；此外，reporter assay結果發現，ugd突變株sigma factor rpoE大量表現，推測ugd基因突變改變細胞表面使RpoE活化。本實驗室先前研究，已知在P. mirabilis中雙組成調控系統RppA/RppB與調控polymyxin B抗性有關，以real time RT-PCR、reporter assay發現在有polymyxin B環境下RppA會正向調控ugd基因的表現。論文期望藉由研究與polymyxin B抗性有關的ugd基因，了解P. mirabilis的抗藥機轉，並探討ugd基因突變造成表面移行能力喪失的可能原因。Proteus mirabilis is a facultative Gram-negative bacterium and a member of the family Enterobacteriaceae. It’s a normal flora in intestines of healthy human. However, it’s an opportunistic pathogen in immunodificent patients who use urethral catheters in long-term therapy. It frequently causes urinary tract infection (UTI), even kidney disease, pneumonia and septicemia.olymyxin B is a kind of cationic antimicrobial peptides. While the positive-charge structure of polymyxin B combine with the negative-charge bacterium membrane, the bacterium membrane is disrupted by fatty-acid chains of polymyxin B and leak the cytoplasm contents out. It is known that the modification of the lipid A of the lipopolysaccharides (LPSs) in Gram-negative bacteria by 4-amino-4-deoxy-L-arabinose (L-Ara4N) decreases negative charge of the membrane. In this way, polymyxin B couldn’t bind the membrane, then leading to the resistance of polymyxin B. . mirabilis is naturally resistant to polymyxin B, but the underlying mechanism of drug resistance is not known clearly. By mini-Tn5 transposon mutagenesis, we selected a mutant that was susceptible to polymyxin B. The Tn5-inserted site was ugd gene that was predicted to encode the UDP-glucose dehydrogenase in P. mirabilis. ugd gene is associated with polysaccharides and L-Ara4N synthesis in Escherichia coli and Salmonella Typhimurium. The ugd mutant exhibited reduced swarming ability, decreased flagellin synthesis, decreased number of swarmer cells, decreased haemolysin activity, decreased cell invasion ability, and alterations of LPS and membrane integrity. Complementation of ugd gene by pACYC-184 vector restored all virulence factor expression in the ugd mutant.e used real time RT-PCR to investigate the mechanism of nonswarming of the ugd mutant. We found the reduced mRNA expression of umoA、B、D membrane proteins which could up-regulate the flhDC operon, and flhDC, fliA and flaA which are class I, II and III genes of flagellar synthesis in the ugd mutant. This implies that ugd gene mutation will affect the expression of umoA、B、D proteins and then decrease the expression of downstream flhDC, fliA and flaA. In addition, reporter assay indicated ugd gene mutation led to increased expression of rpoE. It implies ugd mutation could create a stress condition and can be sensed by RpoE. It has been shown that RppA/RppB, a two-component system, is associated with polymyxin B resistance in P. mirabilis. By real-time RT-PCR and reporter assay, we investigate if ugd gene is under the control of RppA/RppB. The data indicated that RppA regulated ugd gene expression in the presence of polymyxin B.n this study, we investigated the roles of ugd gene in polymyxin B resistance, swarming ability and virulence factor expression in P. mirabilis.致謝 i錄 ii要 viibstract ix一章 緒論 1 第一節 奇異變形桿菌 (Proteus mirabilis) 介紹 1 第二節 多黏菌素B (Polymyxin B) 的介紹 9 第三節 脂多醣 (Lipopolysaccharides；LPSs) 的介紹 13 第四節 ugd基因的相關研究 14 第五節 研究動機與目的 16 第六節 實驗設計 17二章 實驗材料與方法 18 第一節 實驗材料 18 第二節 跳躍子卡匣突變方法 (transposon mutagenesis) 21 第三節 突變基因之鑑定 (identification) 27 第四節 分析突變株毒力因子 (virulence factor) 表現 36 第五節 補償 (complementation) 試驗 58 第六節 探究ugd基因 61三章 實驗結果 67 第一節 利用跳躍子突變方法篩選基因並鑑定 67 第二節 ugd基因與毒力因子表現之分析 69 第三節 ugd與表面移行關係之探討以及分析可能的雙組成調控系統 75四章 結論與討論 78 第一節 結論 78 第二節 串聯ugd基因與表面移行能力之間的關係 80 第三節 其他菌種ugd基因之研究 82 第四節 Ugd影響的表現型在不同物種間的異同 83 第五節 ugd的調控 84五章 表 85六章 圖 90七章 附錄 111考資料 120application/pdf2503259 bytesapplication/pdfen-US奇異變形桿菌多黏菌素BProteus mirabilispolymyxin BUDP-glucose dehydrogenase[SDGs]SDG3http://ntur.lib.ntu.edu.tw/bitstream/246246/182966/1/ntu-98-R96424021-1.pdf