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  • 學位論文

克雷伯氏肺炎桿菌中受血清抑制之cad及tdc基因區功能研究

Functional analysis of serum suppressed genes cad and tdc region in Klebsiella pneumoniae

指導教授 : 王錦堂

摘要


社區型化膿性克雷伯氏肺炎桿菌造成的原發性肝膿瘍合併轉移性併發症,是全球性的新興感染症,但其致病機轉尚未完全解開。為了研究這種新興感染症的可能致病機轉,本實驗室之前以健康人血清模擬in vivo的環境並且利用基因微陣列研究可能和致病有關連的基因。結果顯示造成社區型感染之克雷伯氏肺炎桿菌代表株NTUH-K2044的cad region及tdc region在遭遇健康人血清刺激時表現量下降。本實驗針對這兩段基因區域分別研究其基因組成及功能上的特性。分析cad region及tdc region的轉錄單位,發現cad region中已知組成為轉錄活化因子cadC和 cadBA operon,其中的cadBA operon實際上尚包含yjdL及lysS。推測YjdL和LysS可能和cadBA 的蛋白質基因產物一起參與Lys代謝。而tdc region中的轉錄活化因子 tdcA和其餘tdc region的基因是分開轉錄的,和大腸桿菌中的tdc operon相異。在功能上的分析,將所建構的cad region 基因剔除株,包括cadC及cadA基因剔除株,以Moeller 培養液檢測其Lys decarboxylase 活性,發現這兩株基因剔除株的Lys decarboxylase 活性皆消失,並且於酸性壓力環境的存活率皆下降。在膽鹽壓力的抗性研究也發現cadC及tdc基因剔除株對膽鹽的耐受性較野生株差,顯示cadC可以增進克雷伯氏肺炎桿菌在遭遇酸性及膽鹽壓力環境下的存活率。以基因微陣列比較NTUH-K2044野生株及cad 基因剔除株之基因表現,發現在cad 基因剔除株中tdc region基因表現量下降,進行RT-qPCR實驗也證實tdc region的RNA表現量在cadC 基因剔除株中下降,顯示cadC對tdc region有調控的功能。在in vivo探討其對致病力的影響,採用腹腔注射及胃內感染方式在BALB/c小鼠進行體內競爭實驗。結果cad及tdc基因剔除株在肝臟、脾臟及大腸和盲腸中生存競爭結果都比野生株差。顯示cad及tdc基因區對於克雷伯氏肺炎桿菌在生物體中的生長具有重要性。

並列摘要


Community acquired primary pyogenic liver abscess with metastatic complications caused by Klebsiella pneumoniae is a global emerging infectious disease, however the pathogenesis mechanism remains unclear. To investigate the possible pathogenesis mechanism, in the previous study, we used health human serum to mimic the in vivo environment and used microarray to find novel genes associated with virulence. Results showed that the RNA expression level of cad region and tdc region in community acquired K. pneumoniae representative strain NTUH-K2044 were down-regulated under health human serum treatment. We focused on studying the genetic and functional characteristics of these two gene loci. Analyzing the transcriptional unit of cad region and tdc region, we found cad region was composed of known transcriptional activator gene cadC and cadBA operon; cadBA operon included yjdL and lysS. YjdL and LysS might involve in lysine metabolism together with protein gene products of cadBA. The transcriptional activator gene in tdc region, tdcA, was separately transcripted from other genes of tdc region, which is different from the tdc operon of E. coli. In functional study, the Lys decarboxylase activity of the constructed cad region gene mutants, including ΔcadC and ΔcadA mutants, analyzed by Moeller test was negative and the survival rate under acid environment was decreased. Also, the resistance to bile salt was decreased in ΔcadC and Δtdc mutants, suggesting cadC can increase the survival rate under acid and bile salt stress. Using microarray to compare genes expression of NTUH-K2044 wild type and Δcad mutant, we found expression level of tdc region was decreased in Δcad mutant. By RT-qPCR, we confirmed that RNA expression of tdc region was decreased in ΔcadC mutant. cadC might control tdc region expression. In in vivo virulence analysis, we used intraperitoneal inoculation and intragastric infection in BALB/c mice model to perform in vivo competition assay. The results revealed that colonization ability of Δcad and Δtdc mutants were both weaker than that of wild type in liver, spleen, colon and cecum. This suggested that cad and tdc gene region play important roles in in vivo colonization.

參考文獻


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