探討剔除白色念珠菌CaORF19.4906、CaORF19.4975和CaORF19.4761對白色念珠菌之影響

Abstract

白色念珠菌(Candida albicans)為伺機型的病原真菌，平時共生於人體的黏膜、消化道、生殖泌尿道中。當宿主免疫能力下降時，便會產生局部性的感染，但如果宿主免疫力缺陷時，便很有可能造成全身系統性的感染，嚴重者甚至會致命。目前已知在啤酒酵母菌(Saccharomyces cerevisiae)中，FLO為具有串聯重複現象之基因，影響了酵母菌黏附、入侵、假菌絲形成、生物膜形成能力。而在白色念珠菌中，ALS亦為具有串聯重複現象基因，影響了白色念珠菌(Candida albicans)的黏附能力進而影響菌體之致病能力。因此，本實驗挑選了CaORF19.4906、CaORF19.4975、CaORF19.4761三個具有串聯重複序列基因，利用含有藥物篩選標記的SAT1 flipper cassette，分別建構出此三基因的剔除株，並進行生長曲線、生物膜定性、芽管生成能力、菌落型態變化、侵犯力測試、DNA修復測試。結果中顯示，三個基因的個別剔除株與野生株相似。在侵犯力測試中，CaORF19.4906及CaORF19.4761此二基因的單套及雙套剔除株之菌落較野生株容易被流水沖落但不如對照組的HLC54，而CaORF19.4975則與野生株相似。在生物膜定性、芽管生成能力、菌落型態變化、DNA修復測試中，此三基因剔除株皆與野生株相似。

Candida albicans is an opportunistic fungal pathogen. It is commensal in human mucosal、gastrointestinal tract and genitourinary system. When the host immunity is weak, it could cause local infection. But if the host is immunocompromised, it could cause systemic infection even life threatening. In Saccharomyces cerevisiae, FLO gene contains tandem repeats. It is involved in adhesion, invasion, pseudohyphae and biofilm formation. In Candida albicans, ALS contains tandem repeat and it is involved in cell adhesion. Hence, tandem repeats may be associated with pathogenesis. In this study, I used SAT1 flipper cassette, which contains drug resistance marker (SAT1), to knock out three tandem repeat genes in Candida albicans, CaORF19.4906, CaORF19.4975 and CaORF19.4761.Then I compared the knock- out strains with SC5314b(wild type) on growth rates, biofilm formation, invasion, morphogenesis and DNA repair ability. I found that the all three homozygous knock-out strains have their log-phase doubling time similar to the wild type. In the invasion test, the colonies of CaORF19.4906, CaORF19.4761 heterozygous and homozygous strains were easier removed by running water than the wild type was. The CaORF19.4975 knock-out strain was the same with the wild type. And there was no significant difference between the wild type and the knock-out in other tests.