臺灣博碩士論文加值系統

奇異變形桿菌(Proteus mirabilis)為格蘭氏陰性腸內菌，在健康人體腸道屬於正常菌叢，但在長期使用導尿管病患身上，會造成伺機性感染，嚴重甚至可能導致腎臟病、肺炎等併發症，目前研究發現P. mirabilis在導尿管形成生物膜後具有顯著的感染能力，造成導尿管病患嚴重感染，然而生物膜合成的機制至今還是不清楚。本篇利用transposon mutagenesis的方式建構zapD突變株，實驗結果發現zapD突變株生物膜合成能力大幅下降，探究其可能原因，我們發現zapD突變株的cpxAR基因表現量有顯著的降低，先前實驗室的研究發現，cpxAR的突變株，會影響細菌MR/P fimbriae的表現，進而導致生物膜合成能力下降，所以，我們觀察zapD突變株 MR/P fimbriae的蛋白表現量，我們也發現zapD突變株其MR/P fimbriae蛋白表現量有明顯的下降；此外，我們也觀察zapD上游的zapA基因，我們發現細菌的potease活性下降且zapA基因的表現明顯的降低，我們利用reporter assay及EMSA發現，zapA基因會直接受到Cpx pathway的調控；另外，我們探討其他毒力因子發現zapD突變株的細胞，FlaA蛋白表現量也有明顯的上升。總結而論，我們發現zapD突變會影響P. mirabilis生物膜合成能力，其機制是因為zapD突變導致cpxAR基因表現下降，影響MR/P fimbriae蛋白的表現使生物膜合成能力降低，同時，Cpx pathway會直接調控zapD的上游基因zapA的表現。

Proteus mirabilis, a facultative Gram-negative bacterium, is a common cause of catheter-associated urinary tract infections. Recently studies have showed that P. mirabilis can form biofilm on catheter leading to blockage of catheter and cause severe infections. There has been a report that mannose-resistant proteus like (MR/P) fimbriae contributes P. mirabilisbiofilm formation. However, the genes and its mechanisms in P. mirabilisbiofilm formation remain unclear. In previous study, we have showed that mrp operon was regulated by Cpx pathway. In this study, we constructedzapD- mutant by transposon mutagenesis. Data from biofilm formation test demonstrated that, in the absence of zapD, P. mirabilisbiofilm formation dramatically decreased. Further, we also found thatthe expression of MR/P fimbriae and cpxAR gene expression significantly decreased in zapD mutant. Besides, we revealed that zapA, an upstream gene of zapD, was directly regulated by CpxR by use of reporter assay and EMSA. In sum, this study shows that zapD plays an important role in P. mirabilisbiofilm formation involving in Cpx pathway which can impair mrp operon expression. Moreover, we also show that zapD has an influence on its upstream gene, zapA, which contains CpxR-binding site on its promoter and directly regulated by Cpx pathway.&;#8195;

誌謝 i
摘要 ii
Abstract iii
目錄 iv
圖目錄 vi
表目錄 viii
第一章 緒論 1
第二章 實驗材料與方法 11
第一節 實驗材料 11
第二節 跳躍子卡匣突變方法 (transposon mutagenesis) 13
第三節 分析表現型及毒力因子 14
第三章 實驗結果 48
第一節 利用跳躍子突變方法篩選基因並鑑定 48
第二節 zapD 基因毒力因子表現之分析 49
第三節 分析zapD可能影響的路徑 50
第四節 相關phenotype及zapBCD的調控探討 53
第五節 zapD突變株的補回(complementation)試驗 (圖三) 55
第四章 結論與討論 56
第一節 結論 56
第二節 zapD生物膜合成及機制 56
第三節Cpx pathway與生物膜的合成 58
第四節Cpx pathway和zapA的調控 59
第五章 未來展望 60
第六章 圖 61
第七章 表 77
第八章 附錄 80
一P. mirabilis 80
二 virulence factors 81
三 P. mirabilis在固體培養基上的表面移行 (swarming) 週期 82
四 zapEABCD基因相似性比較圖 83
五E. coli中Cpx的調控 84
六 pUT-Tn5 85
七 pGEM-T Easy 86
八 ATP transporter 87
九 Virulence factors in biofilm formation 88
Reference 89
&;#8195;
圖目錄
圖一zapD突變株Kanamycin cassette插入突變處及PCR確認圖 61
圖二野生株與zapD突變株生長曲線之比較: 62
圖三分析野生株與、zapD突變株生物膜生成能力： 63
圖四分析野生株和突變株MR/P fimbriae的蛋白表現量 63
圖五野生株與zapD突變株泳動能力(swimming) 64
圖六zapD突變株與野生株在各個時間點的細胞長度比較: 67
圖七分析野生株與zapD突變株的FlaA蛋白表現量: 68
圖八分析野生株與zapD突變株protease activity: 68
圖九分析野生株與zapD突變株的cpxAR reporter assay: 69
圖十分析野生株與zapD突變株的zapA reporter assay: 69
圖十一分析野生株與cpxAR突變株的zapA reporter assay: 70
圖十二、CpxR protein與zapA promoter 的EMSA結果圖 71
圖十三zapA啟動子定序 72
圖十四分析野生株與zapD突變株SDS感受性 73
圖十五野生株與zapD突變株NTUB1細胞入侵能力 74
圖十六Urea對野生株與zapD突變株生物膜合成能力 74
圖十七PolymixinB對野生株與zapD突變株生物膜合成能力 75
圖十八分析野生株與cpxAR突變株zapBCD reporter assay 75
圖十九分析野生株與hfq突變株zapBCD reporter assay 76
圖二十分析野生株與qseEGF突變株zapBCD reporter assay 76

&;#8195;
表目錄
表一實驗中所使用的菌株及質體 77
表二實驗中所使用的引子 78
表三野生株和zapD突變株對各種抗生素的感受性(最小抑菌濃度；MIC)： 79

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