白色念株菌之EFG1和/或CPH1下游基因的分類與確認

Abstract

白色念珠菌是一種伺機型真菌類病源菌，和人類共生，在免疫系統不健全的人體中，可引起致命性的傷害。而大部份的抗真菌藥物都具有副作用，且在使用頻繁的情況下，抗藥性問題也日益嚴重。野生種白色念珠菌是屬於雙形態生物，會形成菌絲或以單細胞酵母形態存在。一般認為菌絲的形成是造成宿主細胞致病的重要步驟(43)。過去的研究發現在缺乏轉錄因子Efg1和Cph1的突變種在含有羊血清的培養液中，失去菌絲形成的能力，且對小鼠失去致病力(32)。因此推測在這兩個轉錄因子調控的下游中包括了形態變化與致病能力的基因，這些基因將是新藥物目標的研究方向。 實驗室先前運用相減雜交技術(suppression subtractive hybridization,SSH)，得到在野生種(SC5314)基因表現相對於EFG1和CPH1雙突變種(HLC54)基因表現的差異，如果在野生種中表現量較大，即表示這類基因受Efg1或Cph1正向調控，其中包含影響形態或致病之基因。我們共得到991個選殖體，但EFG1出現的比例過高，所以用限制脢圖析(restriction mapping)的方式，期望將有相同片段數量及大小的選殖體分為同一組，以減少定序的數量，接著將定序結果在Stanford's Current Assembly of Candida albicans Sequence及NCBI中做比對，把所得到的基因依照功能分類，共得到五十二個已知基因及四十八個未知基因，再經由北方墨點法(northern blot)對野生種及突變種的mRNA層次做進一步的確認，共挑選十個基因，分別為ASH1、HEM13、HWP1、KEL2、RCK2、TCA2、TPS2、UBR1、URA3、YMR128，發現HWP1、URA3、HEM13、KEL2、YMR128五個基因符合野生種的表現量較大。在其它基因晶片未問世的物種，可以使用此方法找出特定基因。

Candida albicans is commonly found as a commensal in the human population, causing infection in immunocompromised patients. Many of the antifungal drugs have undesirable side effects and their widespread usages have led to the development of drug-resistant isolates. C.albicans is dimorphic and capable of growth in filamentous form and in yeast form. Filamentous growth is believed to be an important step in the invasion and pathogenesis process(43). A mutant strain lacking the transcription factors Efg1 and Cph1 is extremely defective in filamentous growth in YPD plus goat serum and also avirulence in a mouse model(32). Hence, they are involved in both morphology and virulence pathways. Genes regulated by Efg1 and Cph1 may be new antifungal drug targets of C.albicans. Previous research in the laboratory has used the method of suppression subtractive hybridization(SSH) for identifying virulence genes by screening for difference in the expression between the wild type strain(SC5314) and the cph1/cph1 efg1/efg1 double mutant strain(HLC54) in C.albicans. If a gene has higher expression level in the wild-type strain than in the cph1/cph1 efg1/efg1 double mutant strain, this gene is downstream of the CPH1 and EFG1 and is positively regulated by them. The gene may involve in filamentous growth and may even be a virulence gene. 991 clones have come out of the SSH screening. EFG1 is the most abundant one. Restriction mapping was used to sort those clones into groups to reduce the number of samples for sequencing. After comparing those sequences with the databases of Stanford's Current Assembly of Candida albicans Sequence and NCBI, the sequences are classified by functions. Totally, there are 52 known genes and 48 unknown genes. I pick 10 samples, including ASH1、HEM13、HWP1、KEL2、RCK2、TCA2、TPS2、UBR1、URA3 and YMR128, to determine the expression level of mRNA. Five of them, HWP1、URA3、HEM13、KEL2 and YMR128, match the expected results. Their gene expression levels are higher in the wild type than in the cph1/cph1 efg1/efg1 double mutant. The SSH can be used for other organisms whose gene chips are not yet available.