野生種白色念珠菌與cph1/cph1 efg1/efg1雙基因突變種於可誘發野生種形成菌絲環境下之基因表現比較

Abstract

白色念珠菌 (Candida albicans) 是一種真菌類伺機性病源菌，在免疫系統不健全的人體中引起病害，嚴重時足以致命。在型態上，野生種白色念珠菌會形成菌絲，並且對寄主有致病力，一般也認為形成菌絲與否跟有無致病能力有相當程度的關係；而在過去的研究中發現一株CPH1 EFG1 雙基因突變種沒有形成菌絲的能力，亦無法致病。

本研究藉由抑制相減雜交技術 (Suppression Subtractive hybridization, SSH) 得到在能誘發野生種產生菌絲的條件下，野生種和突變種中表現不同之基因。其中包含影響形態或/及致病之基因，我們期望從中篩選出確實賦予白色念珠菌致病能力之基因，提供藥物設計者更精準的藥物標的 (drug target)。針對白色念珠菌特有之基因設計藥物，可以減少對寄主本身的危害，並且增加治療的效率，避免抗藥性的產生。

在本實驗之SHH結果中, 逢機挑選32個基因片段進行分析, 分離出種12種不同基因。 其中包括與形成菌絲有關的EFG1基因，證實了此技術的可行性。在已分析的32個基因片段中，EFG1共出現18次，可能表示該基因在野生種細胞內所被轉譯之套數。其他十二種基因，有些已有相關文獻或是可能牽涉形態改變或致病，例如：serine/threonine kinase已知與真菌形成菌絲有關；白色念珠菌之squalene epoxidase (CAERG1) 因為與細胞膜生合成有關，所以可能涉及型態上的轉換；以及白色念珠菌之hxt6 gene for galactose/glucose transporter與能量的代謝可能影響致病機制….等等。在未來的研究中，本實驗室將設計更多試驗以尋出與白色念珠菌致病直接相關之基因。

Candida albicans (C. albicans) is an opportunistic pathogenic fungus and one of the most commonly isolated human pathogens. Especially in immunocompromised patients, it can lead to lethal candidiasis. It is believed that filamentous growth is involved in virulence. Researchers have developed the avirulent cph1/cph1 efg1/efg1 C. albicans, which is unable to form filaments. In recent years, the abuse of antibiotics and the emerging of resistance to existing antifungal drugs have made the treatment of fungal infection more and more difficult. To search for a new drug with higher efficiency and less toxicity, I have screened and isolated the virulence genes of C. albicans with the technique of suppression subtractive hybridization in the hope of unveiling suitable new targets for drug development. Two sets of suppression substrate hybridization were performed. One subtracted mutant RNA from wild type RNA (WT-Mut). The other subtracted wild-type RNA from mutant RNA (Mut-WT). Genes in the WT-Mut group are expressed only in the wild type C. albicans or expressed at a higher level in wild-type than in mutant strain. Virulence genes could be in this group. 32 islated cDNA fragments were subjected for sequence analysis. Among them, 18 were EFG1 DNA fragments. Since EFG1 gene was included in the WT-Mut group as expected, this performance of this technique is reliable. It demonstrated that this suppression subtractive hybridization can indicate not only whether certain genes are expressed or not but also the difference in the expression level. In the remaining 14 clones, grouped into 12 different genes, some have been reported to be associated in morphology or virulence: Serine/threonine kinase is involved in filamentous growth; squalene epoxidase (CAERG1) is in the pathway of synthesis of cell membrane; hxt6 gene for galactose/glucose transporter is responsible for the metabolism of energy.