白色念珠菌在Fluconazole和Terbinafine的壓力下所造成遺傳改變之探討

Study on Genetic Changes of Candida albicans under Stress of Fluconazole and Terbinafine

鄭瑋寧(Wei-Ning Cheng)

指導教授 ： 蘇慶華

臺北醫學大學/口腔醫學院/生醫材料暨工程研究所/碩士(2008年)

https://doi.org/10.6831/TMU.2008.00125

摘要

白色念珠菌(Candida albicans )是絕對二倍體（obligate diploid）的酵母菌， 在2004年，白色念珠菌mating type locus被發現與麵包酵母(Saccharomyces cerevisiae)具有相似的性別基因，以二倍體與二倍體交配的方式，產生了擬有性世代，但其有性世代（a/α）仍然不明。本實驗室在2007年，發現在給與Fluconazole的含有phloxine B 的PDA培養基中C. albicans會有高頻率的W/O表現型轉換。而本研究是為了更進一步了解C. albicans遺傳學變化以及發生的減數分裂的可能性，調查此遺傳的變化是否與抗藥性的機制有關，並且利用分子試驗的方式去進行佐證。初步工作先驗證在不同種之Candida在Fluconazole壓力下，是也有相同的轉換，而結果僅有C. tropicalis產生類似現象。而後在四個C. albicans菌株當中(ATCC 14053, BCRC 20512, SJ2S08 and ATCC 22816) 隨機分離出Opaque菌落（即培養基中的粉紅色菌落）。這些個別的粉紅色菌落經過第二次的培養分別經過Fluconazole 1、2、3、4週處理，利用聚合酶鏈鎖反應分析以及流式細胞儀分析。於聚合酶鏈鎖反應分析中發現31.25%的菌株至少有一個MTL基因沒有被放大出來。然而這些MTL基因有缺乏的菌落經過1至7周培養，於流式細胞儀分析發現，在培養前期菌株DNA處於單倍體有較高的趨勢，但培養後期菌株DNA則趨向二倍體。另外，在Fluconazole 至少2週處理下，我們發現高頻率的類似擔子細胞菌落群，利用顯微操作器將四個子細胞以及母細胞分離。聚合酶鏈鎖反應分析，第一次檢測發現四個子細胞菌落群有一些同型合子（homozygotes）存在，第二次進行檢測則發現所有的四個子細胞菌落群都是異型合子( heterozygotes )。這樣的結果顯示C. albicans的減數分裂不是經由類似擔孢子這樣的結構亦或是C. albicans可能是一個雌雄同株的酵母菌。

關鍵字

白色念珠菌 ； 性別基因 ； 同型合子 ； 異型合子

並列摘要

Candida albicans is known as obligate diploid yeast, which is capable of mating in a parasexual (diploid to diploid mating) way but the sexual cycle is unclear. The mating-type like loci (MTL) in C. albicans were identified in 2004 with a high similarity to mating type locus (MAT) of Saccharomyces cerevisiae. In our previous study, a high frequent morphological white-opaque switching was observed in C. albicans with the treatment fluconazole on phloxine B containing potatoes dextrose agar (PDA) plate. Therefore, the purpose of the present work was to investigate whether the morphological switching induced by the anti-fungal drug treatment was also involved in genetic changes or even the possibility of meiosis. The first work was to examine whether the species in the genus of Candida had a similar switching induced by fluconazole. The result showed that only C. tropicalis out of other 5 species had the same reaction to the drugs and that explained the switching is not general in all the species of the genus. Then four C. albicans trains (ATCC 14053, BCRC 20512, SJ2S08 and ATCC 22816) were used for random opaque colonies selection (pink in PDA phloxine B plate). The individual pink colonies were isolated from the secondary plating of cloudy zone of the fluconazole treated plate for 1, 2, 3 and 4 weeks. The isolates were selected for the examination of mating type genes (MTLa1, MTL??1 and MTL??2) by PCR as well as propidum iodide (PI) staining flow cytometry for ploidy study. An over-all ratio of 31.25% (25/80) was discovered for the opaque isolates with at least one MTL gene undetectable in PCR analysis. However, all these MTL-missed strains showed in an unstable state to increase the DNA intensity from 1 to 7 weeks and the presumed haploids became diploid strains gradually by flow cytometric analysis. In the plate of fluconazole treatment for at least 2 weeks, we also found a high ratio of basidium-like cell clusters, for which a presumed tetrad analysis was performed by micromanipulator to separate the four attached daughter cells from the empty flattened mother cell. PCR analysis, in the first step appeared to show some tetrads were homotzygotes, but further analysis revealed that all the tetrads were heterozygotes with no segregation of the MTL genes. It explains that C. albicans could not undergo meiosis through the basidium-like structure or C. albicans might be an yeast in homothallic sexual reproductive pattern.

並列關鍵字

Candida albican ； mating type locus(MTL) ； Fluconazole ； Terbinafine

參考文獻

1. Hsueh, P.R., et al., Emergence of nosocomial candidemia at a

teaching hospital in Taiwan from 1981 to 2000: increased susceptibility of Candida species to fluconazole. Microb Drug Resist, 2002. 8(4):

2. Chen, Y.C., et al., Secular trends in the epidemiology of nosocomial fungal infections at a teaching hospital in Taiwan, 1981 to 1993. Infect Control Hosp Epidemiol, 1997. 18(5): p. 369-75.

3. .Diener, A.C. and G.R. Fink, DLH1 is a functional Candida albicans

4. Hull, C.M. and A.D. Johnson, Identification of a mating type-like