臺灣博碩士論文加值系統

克雷白氏肺炎菌是一種臨床上極重要的細菌，經常造成化膿性感染、肺炎、尿道感染及敗血症。 為進一步了解其致病機轉，我們建立一個活體表現（IVET）系統以搜尋克雷白氏肺炎菌CG43在BALB/c小鼠體內進行感染時所特定表達的基因。 這個系統主要是針對一個galU基因缺失的克雷白氏肺炎菌株，因為喪失使用半乳糖與合成莢膜多醣體的能力，而對BALB/c小鼠具有較低致死力以及在MacConkey agar上表現非黏性白色菌落的特性來設計。 因活體表現 (IVE) 的啟動子只會在細菌感染活體的過程中被開啟，以一個具有功能的galU當作報告基因送入galU缺失的菌株，將使得該菌株在MacConkey agar上維持非黏性白色菌落表型的同時，在小鼠體內展現致死的能力。 經過數次篩選，我們得到20個活體表現（IVE）的基因序列。 在這些序列當中，五個是已知的致病相關基因，另外五個分別負責調節營養素的吸收與運送、isoprenoids的生合成、以及蛋白質的折疊等，而其餘的十個則找不到具有已知功能的相似基因序列。 二十個IVE基因當中有兩個被發現可在缺鐵的環境下開啟，有另外五個基因則會在paraquat的誘導下活化。 初步序列比對的結果發現，在體內缺鐵環境中可驅動aerobactin表現的iucA啟動子，可能被一個與Shigella flexneri SHI-2致病島群（pathogenicity island, PAI）相似的可動（mobile）區域攜帶。 基於PAIs在細菌致病上的重要性，我們以iucA啟動子為探針由CG43菌株中分離出一個大小為18,732-bp的致病相關片段，並完成其定序。 這個區域是一個具移動性的遺傳組塊攜帶兩個重要的致病決定因子，包括取得鐵離子的能力（由iucABCD iutA基因組解譯）以及莢膜黏性的增進（由rmpA2 基因負責）。 其中，rmpA2可產生成一個促莢膜多醣體產生的活化子。 因此，在本論文的第三部份探討RmpA2是否可藉由活化負責莢膜生合成基因的表現來行使其功能。 我們建構了一個rmpA2基因缺失的突變株與二個各自包含一個K2 cps基因啟動子的luxAB轉錄融合株（transcriptional fusion）。 這兩個luxAB轉錄融合株的活性在多複製（multicopy）rmpA2的存在下可大幅增加。 利用DNA電泳位移改變測定法進一步證實RmpA2蛋白質可直接黏合在這些受其調控的DNA片段上。 此外，RmpA2蛋白質的穩定性在ATP-依賴性Lon蛋白分解酵素缺失的菌株中明顯地增加。 而RmpA2被發現會抑制自己的基因表現。 因此，RmpA2在克雷白氏菌K2莢膜多醣體生合成上的促進效果可以歸因於它在K2 cps基因上的轉錄活化作用，而RmpA2本身的基因表現水平則是受到自我回饋（autoregulation）與Lon蛋白分解酵素的控制。

Klebsiella pneumoniae, an important nosocomial pathogen, causes suppurative infection, pneumonia, urinary tract infection and septicemia in humans. To gain a further understanding of its pathogenesis, a novel in vivo expression technology (IVET) was performed to identify K. pneumoniae CG43 genes that are specifically expressed during infection of BALB/c mice. The IVET employed a UDP-glucose pyrophosphorylase (galU)-deficient mutant of K. pneumoniae which is incapable of utilizing galactose and synthesizing capsular polysaccharide, as demonstrated by its low virulence to BALB/c mice and a white nonmucoid colony morphology on MacConkey-galactose agar. By using a copy of promoterless galU gene as the reporter, IVE promoters that render the galU mutant virulent while maintaining the white nonmucoid colony phenotype could be identified. A total of 20 IVE genes were obtained through the in vivo selection. Five of them have been identified previously as virulence-associated genes in other pathogens, while another five with characterized functions are involved in regulation and transportation of nutrient uptake, biosynthesis of isoprenoids, and protein folding. No known functions have been attributed to the other 10 sequences. Two of the 20 IVE genes were found to turn on under iron deprivation, whereas the expression of another five genes was activated in the presence of paraquat, a superoxide generator. Sequence comparison revealed that the iucA promoter, which drives the expression of aerobactin upon iron deprivation in vivo, might be carried on a mobile region similar to the SHI-2 pathogenicity island of Shigella flexneri. Using the iucA promoter as a probe, we have isolated and sequence determined an 18,732-bp long region, which acts as a mobile genetic element that carries two virulence determinants for Klebsiella infection, the iron-acquisition capacity (encoded by iucABCD iutA) and the mucoidy of capsule (enhanced by rmpA2). The rmpA2 locus, which encodes an activator for the production of capsular polysaccharide (CPS), was subjected to further characterization. We constructed an rmpA2 deletion mutant and two luxAB transcriptional fusions, each containing a putative promoter region of the K. pneumoniae K2 cps genes. Activity of these two Pcps::luxAB constructs was enhanced in the presence of multicopy rmpA2. The use of DNA electrophoretic mobility shift assay further demonstrated that RmpA2 protein directly interacts with the K2 cps promoters. The stability of RmpA2 protein was significantly enhanced in the strain deficient in ATP-dependent Lon protease. RmpA2 represses its own gene expression. Therefore, the enhancement of K2 CPS synthesis in K. pneumoniae CG43 by RmpA2 can be attributed to its transcriptional activation of K2 cps genes, and the expression level of rmpA2 itself is autoregulated and under the control of Lon protease.

中文摘要.........................................i
Abstract.........................................iii
Table of Contents................................v
Abbreviation.....................................vi
Chapter I.
Background and Significance......................01
Chapter II.
Identification of Genes Induced in Vivo during Klebsiella pneumoniae CG43 Infection........................05
Chapter III.
Identification of a 18-kb pathogenicity region on pLVPK in Klebsiella pneumoniae CG43.......................19
Chapter IV.
RmpA2, an RmpA2, an Activator of Capsule Biosynthesis in Klebsiella pneumoniae CG43, Regulates K2 cps Gene Expression at the Transcriptional Level........................31
Chapter V.
Conclusion and Perspective.......................71
Reference........................................74
Publication......................................89

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