2010-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/643318摘要：吞噬作用是宿主對抗致病微生物的第一道防線，因此，對於防禦細菌入侵扮演著重要的角色。然而，細菌也發展出一些策略逃避吞噬細胞的攻擊。在宿主內，某些吞噬作用相關的基因則成為是否能抑制細菌致病的關鍵，而在細菌中同樣的也有著重要的致病基因負責抵禦吞噬作用進而成功的感染人體。克雷伯氏肺炎桿菌(Klebsiella pneumoniae)引起之化膿性肝膿瘍為台灣重要之新興感染症，其致病機制目前所知和先天免疫中之血清與吞噬抗性最相關。本實驗室原來利用黏性去篩選一突變菌株庫，由於用黏性之篩檢可能侷限於莢膜與外壁相關的基因，為了能更完整了解與白血球吞噬與殺菌抗性有關之基因，本研究使用黏菌(Dictyostelium discoideum)模式進行篩選。黏菌在其生活史中的變形蟲(amoeba)時期與哺乳類的巨噬細胞有許多相似的特徵，其一便是具有吞噬並殺死細菌的能力，目前此模式已被應用於探討某些細菌與巨噬細胞之間交互作用，例如屬於胞外細菌的綠膿桿菌(Pseudomonas aeruginosa) 以及可在細胞內複製的退伍軍人桿菌(Legionella)，但尚未用於具有厚層莢膜之細菌。本研究初步在Dictyostelium 的模式下觀察溶菌圈篩選造成肝膿瘍的克雷伯氏肺炎桿菌(NTUH-K2044)突變株庫，找尋與抵抗吞噬作用相關的致病基因，初步篩選出22 株突變株產生較野生型大的溶菌圈，這22 個篩選出來的突變株除了包含莢膜合成基因突變株以外也發現了2 個非莢膜合成基因。兩個突變株的跳躍子所嵌入之位置分別為axx (可能為一運輸蛋白)以及bxx 基因(可能為一蛋白酶)，為了確定所觀察到的結果確實因為此基因之破壞，於是再一次將基因剔除以及將此基因補回，並再次確認吞噬作用的結果。由健康人周邊血液分離的噬中性白血球與菌株作用，並以共軛焦顯微鏡證實這兩個基因的突變株的確較易被人類的噬中性白血球吞噬。因此黏菌模式確實可以篩選出莢膜之外之抗吞噬基因且在人類噬中性白血球證實為真。所以在此計畫中，我們將繼續完成篩選整個突變株庫，並藉由體外實驗(in vitro)分析候選基因之功能，用基因微陣列(microarray)檢測突變株之基因轉錄體(Transcriptome)或利用2D 之方法分析其蛋白體(proteome)，以進一步了解抗吞噬之機制，最後將在動物實驗中驗證其於致病過程中所扮演的角色。在本計畫中所發現之重要的致病基因產物可經由表現、結晶純化分析其結構，再依其重要的功能結構試找出可能的有效抑制物，提供開發新治療方法的基礎。<br> Abstract: Phagocytosis is the first line of host defense against invading microorganisms, therefore,it plays an important role in host antibacterial response. On the other hand, bacteria mustdevelop some strategies to fight or escape from the killing. Some host genes specificallyinvolved in phagocytosis contributes to arrest bacterial infection and some bacterial genesresponsible for attacking phagocytosis may be essential for their pathogenicity. Klebsiellapneumoniae causing pyogenic liver abscess is an important emerging infectious disease inTaiwan. It has been reported that serum-resistance and phagocytosis-resistance are importantfor its virulence. In our previously study, we had screened our transposon mutant library bymucoviscosity test. Because screening by mucoviscosity could be limited to discovery ofcapsular or surface-related genes, we use a Dictyostelium model to identify genes associatedwith phagocytic resistance. Since the amoeba form of Dictyostelium discoideum andmammalian macrophage share many traits such as the ability to phagocytose and kill bacteria,it has been used as a model to monitor the interaction of phagocyte-bacteria in somepathogens including extracellular bacteria (e.g. Pseudomonas aeruginosa) or bacteria withability of intracellular replication (e.g. Legionella), but has not been used inheavily-encapsulated bacteria. In our preliminary results, we use Dictyostelium model toscreen virulence factors of anti-phagocytosis genes in a mutant library which are constructedfrom a clinical strain of K. pneumoniae causing pyogenic liver abscess. Compared to the wildtype strain which was resistant to Dictyostelium, up to now, we found 22 mutants werepermissive for Dictyostelium growth (plaques were observed in these mutants). Among the 22mutants, not only capsular genes but also two non-capsular genes were identified. One is aputative transporter, the other is a putative protease (designated as Axx and Bxx, respectively).Unmark Deletion and complementation strains were constructed and used to confirm theresult. We have proved that the two mutants also became more easily to be phagocytosed byhuman neutrophil using confocal microscopy. Therefore, according to our preliminary results,Dictyostelium model can be used for screening non-capsular phagocytosis- resistance genesand these genes are also important for anti-phagocytosis to human neutrophils.In this project, we will complete our screening of the mutant library. The functions ofcandidate genes will be examined through in vitro assay. Anti-phagocytosis mechanism of thegenes will be revealed by analyzing the transcriptome by microarray or comparing theproteome by 2D. Finally, the roles of the gene in pathogenesis will be examined by in vivoanimal inoculation. Critical genes for virulence will be expressed and crystallized to analyzetheir structures. Potential inhibitors to critical functional sites will probably be identified,which will provide new therapeutic targets against this emerging disease.