克雷白氏肺炎桿菌可適應環境變化並具有引起多樣化的臨床感染的能力, 究竟克雷白氏肺炎桿菌如何轉換調節生理機制以允許其在特定環境中生存仍然是個謎團。近來研究證實小RNA在原核細胞適應多樣的環境變化中扮演著重要調控角色。實驗室先前的研究發現了RybB, 這個克雷白氏肺炎桿菌的sRNA, 在Sigma E大量表達的克雷白氏肺炎桿菌中, 隨之大量增加其表現量。Sigma E的缺失除造成克雷白氏肺炎桿菌的毒性喪失外亦會減損其在不同極端環境下的抗壓性。雖然Sigma E是轉錄活化因子, 全基因組DNA微陣列分析顯示在克雷白氏肺炎桿菌有45%的Sigma E-dependent的基因受到負向調控。由於RybB是可以在後轉錄的作用下來抑制基因表現, 負向調控Sigma E-regulon的基因可能是源自於RybB的後轉錄抑制作用。本研究的主要目的在於探討RybB如何在後轉錄層次上調控克雷白氏肺炎桿菌的生理與毒性。利用同源重組的技術將RybB基因剔除得到突變菌株ΔRybB。相較於野生型菌株,ΔRybB感染BALB/c小鼠的能力顯著降低。全基因組DNA微陣列分析顯示有31個克雷白氏肺炎桿菌基因在RybB的短時間脈衝表現下, mRNA的量相較於對照組顯著減少四倍以上。運用在E. coli TOP10中的Two-plasmids系統, 以偵測GFP轉譯融合蛋白的方式, 推測RybB可能可藉由干擾RNA聚合酶結合的方式負向調控GalU、OmpC、HdeB、和KP1_0760的轉譯作用。這些結果代表著RybB的負向調控可以微調克雷白氏肺炎桿菌的外膜、莢膜和抗酸蛋白的表現而影響細菌的致病力, 因此是非常重要而值得探討的因子。

Klebsiella pneumoniae adapts itself to various environments and is capable of causing a wild range of infections. How K. pneumoniae switches its physiological programs to ensure survival in a specific niche is still a mystery. Recently, it has become clear that small RNAs are crucial regulators modulating diverse cellular processes of prokaryotic cells. Previous studies in our lab found that RybB, one of K. pneumoniae sRNAs, was strongly activated by sigma E. Although sigma E is a transcriptional activator, 45% of sigma E-dependent genes were negatively regulated in K. pneumoniae. It is possible that the negative regulation of genes belonging to the sigma E-regulon is mediated through the action of RybB. Therefore, I aimed in this study to determine the role of RybB in the regulation of K. pneumoniae physiology and virulence. Using an allelic exchange technique, I generated a rybB deletion mutant and named it ΔRybB. Compared to the wild type strain,ΔRybB significantly lost its virulence to BALB/c mice. Upon pulse expression of RybB, DNA microarray analysis revealed that mRNA abundances for 31 genes were significantly decreased with more than 4-fold changes as compared to that of the vector control. Through detection of the GFP-translational fusion proteins with a Two-plasmids system in E. coli TOP10, I found that the RybB might negatively regulate the translation of GalU, OmpC, HdeB, and KP1_0760 via interfering the binding of RNA polymerase at their 5''-end untranslated region. The results suggested that the negative regulation by RybB might fine tune the expression levels of factors involved in the outer membrane, capsule, and acid-resistance. It is worth further studies to explore the detailed mechanism of RybB regulation.

中文摘要 V
Abstract VI
前言 1
一、克雷白氏肺炎桿菌之微生物學特性 1
二、克雷白氏肺炎桿菌之生化特性 1
三、克雷白氏肺炎桿菌之抗原 2
四、克雷白氏肺炎桿菌之莢膜多醣體 (capsule polysaccharide;CPS) 2
五、克雷白氏肺炎桿菌之脂多醣體 (Lipopolysaccharides; LPS) 3
六、攝鐵因子 (siderophore) 3
七、黏附因子 (Adhesins) 4
八、生物膜 (biofilm) 5
九、生物膜和游離細菌的差異性 6
十、克雷白氏肺炎桿菌與生物膜 6
十一、Sigma(σ)因子 7
十二、rybB基因 8
研究動機 9
材料和方法 10
第一節、細菌培養 10
一、實驗菌株 10
二、培養基和抗生素的配製 10
三、細菌培養 14
四、凍管製作 14
第二節、建構克雷白氏肺炎桿菌RybB基因缺失的突變菌株 15
一、Genomic DNA的抽取 15
二、利用PCR來放大K. pneumoniae CG43菌株的rybB基因上游片段 16
三、核酸電泳 17
四、TA cloning (invitrogen TOPO TA Cloning Kit) . 18
五、細胞轉型 (transformation) 實驗 . 18
六、抽細菌的質體DNA .. 19
七、利用限制酶酵素確認結果 .. 20
八、利用PCR來放大K. pneumoniae CG43菌株的RybB下游基因片段 .. 22
九、將K. pneumoniae CG43菌株的RybB的下游基因片段切限制酶酵素並利用膠體電泳萃取純化 .... 23
十、先將之前確認帶有RybB上游基因的PCRII vector切限制酶酵素並做clean up(Geneaid kit)純化 ...... 24
十一、DNA ligation (DNA接合作用) ...... 25
十二、將PCR II vector上的K. pneumoniae RybB上下游基因轉移至PKAS46 vector(圖三B) ....... 26
十三、製作勝任細胞 (competent cell) ...... 29
十四、電穿孔實驗 ........... 30
十五、接合生殖 (conjugation) ......... 31
第三節、毒性試驗 .............. 34
第四節、生長速率試驗 ............ 34
第五節、克雷白氏肺炎桿菌生物膜的檢測 ........ 35
第六節、黏性試驗 .............. 36
第七節、OmpC、GalU、HdeB、0760序列接到PXG10質體 .......... 36
第八節、Western blot ............ 37
一、製作SDS PAGE .......... 37
二、電泳 ...... 38
三、轉漬 ......... 38
四、阻斷液 (Blocking solution)、一級抗體、二級抗體 .......... 38
五、暗房壓片....... 39
第九節、抽取 total RNA .......... 39
第十節、RT PCR ......... 41
第十一節、total RNA轉成cDNA ........ 42
第十二節、Real time PCR ........ 42
研究結果 ........ 44
一、建構克雷白氏肺炎桿菌RybB基因缺失的突變菌株 ........ 44
二、檢測RybB基因缺失是否影響克雷白氏肺炎桿菌對小鼠的致病力 ...... 45
三、建構arabinose-inducible RybB 表達載體 ........ 45
四、RybB基因剔除菌株的生長曲線 ....... 45
五、RybB基因剔除菌株的生物膜形成 ............. 47
六、RybB基因剔除菌株的黏性表現 ......... 47
七、搜尋克雷白氏肺炎桿菌中受到RybB負調控的目標基因 ......... 48
八、RybB和目標基因之間的相互作用 ....... 49
九、利用RT-PCR (reverse transcription- polymerase chain reaction) 來驗證RybB和它的target mRNA之間相互影響的變化 ............ 50
十、利用Real time PCR來驗證RybB負調控其目標基因 ............ 51
討論 ............. 52
結論 ........... 57
參考文獻 ....... 58
表一、本研究使用的引子 ....... 65
表二、本研究使用的質體 ..... 66
表三、本研究使用的菌株 ........ 67
表四、RybB負調控的基因 ....... 68

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