利用白色念珠菌短時間暴露於抗真菌藥氟康唑下造成染色體丟失做為單套染色體之研究

白色念珠菌( Candida albicans )是人體的正常菌叢，在免疫力低下的情況會造成伺機性感染，血流感染的致死率高達40%以上，是臨床上發生率與致死率最高的真菌病原菌。目前為止，白色念珠菌尚未發現其有性世代與有性胞器，普遍認為是以擬有性世代進行染色體交換作為演化的過程。本研究中，將白色念珠菌短暫的暴露於抗真菌藥物氟康唑( fluconazole ) 12-16小時，在抑菌圈內會產生具有四個核的豆莢型細胞，使用顯微操作器挑選出豆莢型細胞並持續分離其子代。結果發現分離出的子代具有形態多樣性，且性別基因與去氧核糖核酸( DNA )含量亦產生變化。我們利用新世代定序技術( next generation sequencing )以及生物資訊學的分析，發展出一個新的平台可以解析白色念珠菌八對染色體的套數，單核苷酸多型性( SNP )的變化，並建構染色體圖譜。利用這項技術進一步分析挑選出的菌株發現有單倍體( haploid )以及非整倍體( aneuploid )菌株的存在，且有異質性丟失( loss of heterozygous )的情形，這個結果說明了白色念珠菌短暫的暴露於抗真菌藥物氟康唑之下，其染色體會丟失、複製與位移，造成非整倍體以及單倍體菌株的出現，這個現象也暗示我們氟康唑在臨床上的使用需要更加小心，劑量或是處理時間不夠都將會造成白色念珠菌產生遺傳上的變異，並透過這樣的機制演化成抗藥性菌株。

關鍵字

白色念珠菌；染色體丟失；單倍體；異質性丟失；新世代定序；氟康唑

並列摘要

Candida albicans, a common fungal pathogen that can cause opportunistic 3 infections, is regarded as an apparently asexual, diploid fungus. A parasexual cycle was previously found between homozygotes with opposite mating type-like loci (MTLa/α). Here, we described an artificial approach to isolate aneuploids or haploids induced by the short-term (12-16 h) exposure of diploid reference strains SC5314 and CAI4 to the most commonly used antifungal drug, fluconazole, and followed by repeated single cell separation among small morphologically distinct colonies in the inhibition zone. The isolated strains had altered cell morphology as well as LOH events in the MTL and other marker alleles of the analyzed loci at 8 chromosomes of C. albicans with decreased DNA content. The present study employed next generation sequencing (NGS) to analyze the haploid, autodiploid and the aneuploid strains that arose from the fluconazole treatment instead of the conventional single nucleotide polymorphism/comparative genome hybridization (SNP/CGH) method. A multiple-alignment tool was also developed based on sequenced data from NGS to establish haplotype mapping for each chromosome of the selected strains. These findings revealed that C. albicans have ‘concerted chromosome loss’ to form strains with homozygous alleles and even have a haploid status after short-term exposure to fluconazole. The present study provides new insights into interactions between the pathogenic C. albicans fungus and the antifungal drug fluconazole. In clinical treatment, inappropriate dosage of fluconazole will increase the evolution and produce the fluconazole resistant strains.

並列關鍵字

candida albicans ； chromosome loss ； haploid ； loss of heterozygous ； next generation sequencing ； fluconazole

參考文獻

李楷誠 (2012). 白色念珠菌菌血症病患接受fluconazole治療前與治療後之臨床菌株特性分析, 台北醫學大學醫學科學研究所碩士論文.

Abbey, D., M. Hickman, D. Gresham and J. Berman (2011). "High-Resolution SNP/CGH Microarrays Reveal the Accumulation of Loss of Heterozygosity in Commonly Used Candida albicans Strains." G3 (Bethesda) 1(7): 523-530.

Alby, K. and R. J. Bennett (2011). "Interspecies pheromone signaling promotes biofilm formation and same-sex mating in Candida albicans." Proc Natl Acad Sci U S A 108(6): 2510-2515.

Anderson, J. M. and D. R. Soll (1987). "Unique phenotype of opaque cells in the white-opaque transition of Candida albicans." Journal of Bacteriology 169(12): 5579-5588.

Anderson, J. M. and D. R. Soll (1987). "Unique phenotype of opaque cells in the white-opaque transition of Candida albicans." J Bacteriol 169(12): 5579-5588.

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利用白色念珠菌短時間暴露於抗真菌藥氟康唑下造成染色體丟失做為單套染色體之研究

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