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研究生(外文):Szu-Yu Lai
論文名稱:探討small RNA Spot42在奇異變形桿菌中所扮演的角色
論文名稱(外文):Investigation of the small RNA Spot42 in uropathogenic Proteus mirabilis
外文關鍵詞:Proteus mirabilissmall RNASpot42
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  奇異變形桿菌 (Proteus mirabilis)是革蘭氏陰性的兼性厭氧菌,於健康人體腸道內屬正常菌叢,然而在腸道以外的地方則會造成伺機性感染 (opportunistic infection),主要會造成長期插導尿管或泌尿道功能缺失的病人之泌尿道感染(urinary tract infection, UTI)。細菌發展出許多調控機制,使其能夠適應週遭多樣且波動的環境。目前在Escherichia coli已被發現有許多sRNA會參與調控細菌的壓力反應,其中包含sRNA Spot42 (或稱spf)。在E. coli中發現當環境中葡萄糖濃度增加時Spot42會大量表現,抑制galK (encodes a galactokinase)轉譯,此外也參與在調控至少22個和次級碳源的攝取與代謝相關基因表現。在壓力調控方面則是發現Spot42會正向調控general stress response regulator, RpoS的mRNA表現,使細菌增加抵抗酸性壓力的能力,但是否會影響細菌適應其他不利的環境及調控機制仍不甚了解。本研究旨在探討Spot42在P. mirabilis中所扮演的角色。我們建構spf突變株,在表現型方面發現spf突變並不影響細菌生長,此外也發現spf突變會降低細菌的抗酸及抗氧化壓力的能力、在巨噬細胞 (THP-1)的存活率及對膀胱細胞 (NTUB1)及腎臟細胞 (A498)的入侵及貼附能力。而利用pGEM-T easy載體在野生株中過度表現spf則發現不會影響細菌移動性、抗藥性、和生物膜的形成。在基因調控方面,先前的研究指出CRP-cAMP complex會結合到spf的啟動子區域而抑制其轉錄並且負向調控rpoS mRNA表現,我們分別利用EMSA及realtime PCR證實CRP-cAMP complex會結合至spf的啟動子區域並負向調控其表現;利用reporter assay發現CRP會促進rpoS轉錄但realtime PCR的結果則顯示CRP會負向調控rpoS mRNA表現;分析Spot42對rpoS的調控發現Spot42會正向調控rpoS mRNA的表現,我們進一步利用生物資訊網站預測也在 rpoS發現Spot42 binding site。綜合上述結果我們認為在P. mirabilis中Spot42受到CRP的直接調控,進而影響rpoS表現造成壓力抵抗相關的表現型。

Proteus mirabilis, a Gram-negative, facultative anaerobic bacteria, is one of the most common cause of urinary tract infections, especially in patients with indwelling catheters or structural abnormalities of the urinary tract. The bacteria have developed diverse response systems to survive in the harsh and changing conditions. Many Hfq-binding small RNAs have been found to play roles in stress responses in E. coli and S. typhimurium. The Spot42 RNA, a 109 nucleotides long, Hfq-dependent small non-coding RNA, has been reported to block the translation of galK gene in E. coli under growth in the presence of glucose and regulate over twenty-two genes associated with uptake and catabolism of non-favored carbon sources. On the other hand, Spot42 was also found to participate in bacterial acid resistance and upregulate the general stress response regulator, RpoS. But the connection between Spot42 and the bacterial stress responses is still poorly understood. In this study, we constructed spf deletion mutant to investigate the effect of Proteus mirabilis Spot42 on adaption to stress responses and the regulatory mechanisms. We found that spf mutation decreaced acidic (pH=3) and oxidative stress resistance (30 mM H2O2), the survival in macrophage, and the ability to invade and adhere to NTUB1 and A498 cells compared with wild-type strain. In addition, we also noticed that spf has nothing to do with cell growth, swarming motility, biofilm formation and the sensitivity to antibiotics. In the aspect of gene regulation, we confirmed that CRP-cAMP binds to spf promoter region and negatively controls the expression of Spot42 in P. mirabilis by EMSA and realtime PCR respectively. Further, the realtime PCR data indicates that Spot42 would positively regulate the expression of rpoS mRNA level. We used bioinformatic tools to predict RNA-RNA interactions, and also found out the binding site of Spot42 on rpoS mRNA. The realtime PCR data also demonstrates that CRP negatively regulates the expression of rpoS mRNA level. In summury, our data suggest that Spot42 is important in P. mirabilis stress resistance by either directly or indirectly modulating the expression of RpoS.

誌謝 i
摘要 ii
Abstract iii
目錄 iv
表目錄 v
圖目錄 vi
第一章 緒論 1
第一節 奇異變形桿菌(Proteus mirabilis)介紹 1
第二節 CRP、Spot42 sRNA及RpoS的基本介紹 7
第三節 研究動機與目的 11
第四節 實驗設計 12
第二章 實驗材料與方法 13
第一節 實驗材料 13
第二節 Spot42突變株建構方法 16
第三節 分析表現型 (phenotype)及毒力因子 (virulence factor) 32
第四節 Spot42參與之基因調控 47
第三章 結果 56
第一節 P. mirabilis spf突變株之建立與確認 56
第二節 spf突變菌株之表現型分析 57
第三節 spf突變株毒力因子之分析 59
第四節 分析spf可能調控的路徑 60
第四章 結論與討論 63
第一節 結論 63
第二節 討論 64
第三節 未來展望 67
第五章 表 68
第六章 圖 72
附錄 87
參考文獻 96

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