Title

測定RybB在後轉錄調控克雷白氏肺炎桿菌的生理與毒性上扮演的角色

Translated Titles

Determination of the role of RybB in the post-transcriptional control of Klebsiella pneumoniae physiology and virulence

Authors

楊竣凱

Key Words

克雷白氏肺炎桿菌 ; small RNAs ; sigma E ; RybB ; DNA微陣列 ; Two-plasmids系統 ; GalU ; OmpC ; HdeB ; KP1_0760 ; Klebsiella pneumoniae ; small RNAs ; sigma E ; RybB ; DNA microarray ; Two-plasmids system ; GalU ; OmpC ; HdeB ; KP1_0760

PublicationName

中山醫學大學生化微生物免疫研究所學位論文

Volume or Term/Year and Month of Publication

2013年

Academic Degree Category

碩士

Advisor

賴怡琪

Content Language

繁體中文

Chinese Abstract

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。

English Abstract

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.

Topic Category 醫藥衛生 > 基礎醫學
醫學院 > 生化微生物免疫研究所
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