克雷伯氏菌莢膜多醣體合成區之基因與功能研究

Abstract

疾病統計資料的分析已指出克雷伯氏菌中基因magA與rmpA與原發性肝膿瘍的成因有關，然而克雷伯氏菌刺激巨噬細胞生成cytokines與宿主免疫的部分卻仍尚待瞭解。從蔡世峰博士的研究團隊所完成克雷伯士菌NTUH-K2044的全株定序，我們經由基因序列比對，針對莢膜多醣體生合成路徑的酵素研究。根據蛋白質序列相似度的比對分析，整個基因組依照功能可區分為四類：核苷酸糖生成酵素組、糖轉移酵素、糖修飾酵素、以及莢膜生成與調控酵素。在本論文中，我們進行了前三類的生化分析，證實了第一類九個酵素的功能，但在第二類三醣單元合成及第三類修飾的部分，由於實驗的困難尚未能取得正面的結果。除此之外，我們同時進行了基因剔除菌株的基因體表現分析，發現到三個剔除菌株具有一個廣域的基因抑制現象，此一抑制現象起於kp3705至kp3767，透過觀察GDP-fucose的生成與抗生素之抗性實驗證實了此一現象與病原菌抗生素抗性有關。因此，這是發現了一個前所未見的調控機制，此一機制聯結了CPS與抗生素抗性之間的關係。

The growing number of Klebsiella pneumoniae infections, commonly acquired in hospitals, has drawn great concerns. It has been shown that the K1 and K2 capsular serotypes are the most detrimental strains, particularly to those with diabetes. The K1 cps (capsular polysaccharide) locus in the NTUH-K2044 strain of the pyogenic liver abscess (PLA) K. pneumoniae has been identified recently, but little is known about the functions of the genes therein. The active components of primary pyogenic liver abscess (PLA) Klebsiella pneumoniae in stimulating cytokine expressions in macrophages are still unclear from the host immunity and bacterial virulence, although statistical studies in pathogenesis showed that magA, rmpA, and liver abscess with diabetes, bacteremia and metastatic complications are related. In this dissertation, we demonstrated the functions of nine annotated genes in the UDP-galacturonate and GDP-fucose biosynthetic pathways. We pursued the relationship between the genes in the CPS gene cluster and virulence by studying the trisaccharide biosynthesis and decoration. In addition to the biochemical approach, we also paid attention to these genes in genetic level. Gene expression patterns of three gene deletion (kp3706, kp3709, and kp3712) strains showed a novel broad–range of gene silencing, ranging from kp3705 to kp3767. Metabolite analysis and antibiotic resistance supported the silencing effect. In summary, this work characterized the functions of genes in NDP-sugar biosynthesis and revealed an unprecedented broad spectra gene silencing effect.