剔除 CaPHO112 基因對白色念珠菌型態及生長的影響

Abstract

白色念珠菌 (Candida albicans) 為雙倍體的伺機性病原菌，通常共生於人體中，但當宿主免疫力低下時，白色念珠菌將會開始增生並引起局部性或系統性感染。白色念珠菌具有酵母菌型及菌絲型兩種型態，而其致病力與能在此兩型態間自由轉換有關。由於高風險人口的增加與具有抗藥性菌株的出現，新藥開發成為一個迫切的問題。在本研究中，我試圖了解白色念珠菌特有的真菌基因，以確定其是否適合作為新的藥物標靶幫助日後新藥物的開發。發現在白色念珠菌中之 PHO112 基因，其非哺乳類編碼之蛋白質。其序列與植酸酶蛋白具有同源性。在本實驗中將利用攜帶藥物篩選標記之 SAT1 flipper cassette 基因剔除系統，將 PHO112 基因進行剔除，藉以觀察對細胞型態、生長曲線等是否有所影響。在實驗的條件下，生長曲線結果確認此基因的剔除並不影響菌株之生長，另外不論是菌絲生成、芽管生成與侵犯力測試等型態變化試驗，PHO112 的雙套剔除株與野生株並無明顯差異。此外，我也建構重組蛋白表現質體，以利後人進行更進一步的酵素活性測試。

Candida albicans is a diploid and opportunistic fungal pathogen which has often found in human. It can lead to life-threatening systemic infections in immunocompromised patients. The pathogenicity of C. albicans is associated with its ability to grow in both yeast form and filamentous/ hyphae form. As the risk population increase dramatically and the emergence of drug resistant strains, the development of new drugs become a pressing issue. In the current study, I attempt to study C. albicans genes that are unique to fungi to determine whether they are suitable as new drug targets for later development of the leads of drugs. In this study, I have found PHO112 gene, which encodes a protein homologous to phytase, not encoded by mammals. I use the SAT1 flipper cassette, carrying a drug resistance marker, to perform the PHO112 gene knockout and to observe whether there was a change in the morphogenesis and viability of C. albicans. In conclusion, there is no significant difference between the homozygous knock-out strains and the wild-type strain in their morphogenesis and growth rates under the tested conditions. Inaddition, I have constructed a recombinant plasmid for expressing of PHO112 to facilitate future studies on the enzymatic activity.