| 摘要: | 克雷白氏肺炎桿菌(Kp) 為一伺機性並常引發社區性感染之病原菌。與其他病原菌 不同的是，大部份Kp 的臨床分離株皆帶有厚重的莢膜多醣體(CPS) 來躲避免疫系統的 攻擊進而成功感染宿主細胞。目前證實galU 轉譯的尿?二磷酸葡萄糖焦磷酸化? (UDP-glucose pyrophosphorylase) 參與CPS 生合成過程中尿?二磷酸葡萄糖 (UDP-glucose) 的供給。而在Kp cps 基因組中有一個與galU 相似的基因galF，我們推 測此基因產物也負責UDP-glucose 的合成。許多核?酸二次訊息傳遞分子如: cAMP、 (p)ppGpp 和c-di-GMP 在調控細菌的功能研究已相當透徹，曾有報導指出大腸桿菌體內 的UDP-glucose 濃度可影響RpoS 基因表現，並推測UDP-glucose 扮演訊息傳遞分子來 調控下游基因的表現。而在去年執行的計畫中，我們發現Fur 會抑制galU 和galF 的表 現而影響CPS 的生成。因此，本計畫目的在探討UDP-glucose 在Kp 的角色，特別是 CPS 的生合成的影響，同時證實GalF 的酵素活性，並進一步比較GalU 和GalF 在 UDP-glucose 生合成上的調控機制。下列預計於三年內完成之實驗目標: 1. 研究在Kp 中UDP-glucose 訊息的傳遞機制 (1.)研究UDP-glucose 在cps 基因表現上相關的RpoS 依賴型基因的表現上的調節機 制 (2.)利用比較基因體學和蛋白質體學找尋可受UDP-glucose 調控之下游基因 2. 研究在Kp GalF 在UDP-glucose 的生合成中所扮演的角色 (1.)在galF 的基因缺損與大量表現的情況下細菌顯型的特徵分析 (2.)在體外實驗上評估GalF 的酵素活性與基質專一性 (3.)研究GalU 和GalF 在UDP-glucose 的合成上的協調關係 3. 分析在Kp 中galU 和galF 的表現上的調控機制 (1.)分析在不同環境刺激下galU 和galF 的表現情形 (2.)分析Fur 對galU 和galF 差異表現的影響 (3.)分離鑑定影響galU 和galF 表現之未知轉錄因子
Klebsiella pneumoniae (Kp) is a common opportunistic pathogen that causes community-acquired diseases. Apart from other bacteria, most Kp clinical isolates produce large amounts of capsular polysaccharide (CPS) to protect the bacteria from attacks of the immune system for a successful infection. It has been demonstrated that galU encodes UDP-glucose pyrophosphorylase participating in the supply of UDP-glucose for CPS biosynthesis. A galU-homologue, galF, was found to locate in the cps gene cluster of Kp implying the involvement in biosyntheses of UDP-glucose. Such nucleotide-based second messengers, including cAMP, (p)ppGpp, and c-di-GMP, have been demonstrated to play important roles in versatile cellular processes. Interestingly, it has been reported that the intracellular level of UDP-glucose in Escherichia coli affected the expression of rpoS, the central regulator of general stress response in bacteria, implying UDP-glucose is a signaling molecule for controlling gene expressions. Our previous analyses in last year also have shown that the global transcriptional factor, Fur, represses the expressions of galU and galF to regulate the CPS biosynthesis in Kp. However, the detail mechanism of the UDP-glucose signaling, the regulation of galU and galF expressions, and the enzymatic activity of GalF are not yet determined. The objects of this project are to study (i) the functional roles and the regulations of GalU and GalF on the UDP-glucose biosynthesis and (ii) the involvement of UDP-glucose signaling in Kp cellular processes, especially the CPS biosynthesis. The project is to be completed in three years. The specific aims are as follows: 1. Investigation of the UDP-glucose signaling mechanism in Kp (1.)To study the mechanism of UDP-glucose-mediated expressions of RpoS-dependent genes in controlling the cps gene expression (2.)To identify target genes whose expressions are controlled by intracellular UDP-glucose level using cDNA subtractive hybridization and proteomic analysis 2. Characterization of GalF on the biosynthesis of UDP-glucose in Kp (1.)To analyze the effects of galF-knockout and galF-overexpression on biosyntheses of CPS, lipopolysaccharides (LPS), and UDP-glucose (2.)To assess the enzyme activity and substrate specificity of GalF in vitro (3.)To investigate the coordination of GalF and GalU in UDP-glucose biosynthesis 3. Analysis of the regulatory mechanism of galU and galF expressions in Kp (1.)To analyze expressions of galU and galF upon different environmental stimuli (2.)To analyze the differential regulation of Fur on the expressions of galU and galF (3.)To isolate the unknown transcriptional factors in affecting the expressions of galU and galF |
