Abstract
摘要:在愛滋病人,長期住院病人,接受化療的癌症病人,或接受免疫抑制治療的病人,甚至有時候在正常的個體身上,Saccharomyces cerevisiae 酵母菌已被視為是一個重要的致病原(pathogen)。高溫的抗性及假菌絲的分化(pseudohyphal differentiation)是臨床分離菌株的兩個重要毒性特質(virulence traits)。雖然如假菌絲的分化等菌株細胞型態的轉換,可能對於其感染性、組織侵略能力和致病性具有決定性的影響,但是這些毒性特質和致病性之間的詳細機制仍然有待闡明。我們已經和國立台灣大學附設醫院檢驗醫學部合作,取得台灣地區酵母菌臨床分離株,藉由操作一些初步的實驗來觀察這些台灣臨床分離株的毒性特質;本計畫將利用基因體學,蛋白質體學,及分子遺傳學的方法,並以美國的臨床分離株比對,更進一步揭開酵母菌致病性分子機制的面紗。以下為本篇計畫的主要目標:第一年(98/81~99/7/31)-- 利用基因體學的方法,找出參與在假菌絲生長及高溫生長此兩種酵母菌毒性特質的相關未知基因,進而分析這些臨床分離菌株在遺傳物質上的演化適應。1. 以生物晶片輔助混合分組分析法(array-assisted bulk segregant analysis),針對台大醫院臨床分離酵母菌菌株的假菌絲生長性狀進行基因定位。2. 以生物晶片輔助混合分組分析法,針對台大醫院臨床分離酵母菌菌株的高溫生長性狀進行基因定位。3. 根據這些臨床分離菌種與毒性特質的相關基因的序列分析,實行親緣分析(phylogenetic analyses)的鑑定。第二年(99/8/1~100/7/31)-- 利用細胞培養檢測系統,分析臨床分離株及其衍生株的致病性,並進一步研究致病能力與其抗真菌藥物的抗藥性之間的關係。1. 建立細胞培養檢測系統以分析酵母菌臨床分離株及其衍生株之致病能力。2. 分析酵母菌臨床分離株對抗真菌藥物的抗藥性與致病能力之間的關係。第三年(100/8/1~101/7/31)-- 利用蛋白質體學的方法,定量分析臨床分離株與實驗室菌株之細胞壁結構的差異,並更進一步找出其與致病能力間的關係。1. 分析酵母菌臨床分離株與實驗室菌株細胞壁構造之差異,及其與毒性特質之關聯。2. 分析並確認酵母菌臨床分離株細胞壁構造差異的重要性,及其在致病能力上所扮演的角色。總括來說,利用上述這些策略找出可能和假菌絲形成及高溫生長相關的基因,重新建構這兩個毒性特質的訊息傳遞路徑,使我們能夠徹底地了解整個機制;而相關基因的親源分析可以告訴我們它們在演化上所代表的意義;建立細胞培養檢測系統以分析酵母菌株之致病能力,由於具致病性之黴菌也可能透過類似的方式引發宿主的免疫反應,此方法也可應用於其他真菌菌株致病能力的檢測;抗藥性機制的探討,將有助於臨床用藥之參考與新藥物的開發;研究致病能力及細胞壁結構之間的關係則能夠闡明宿主和病原之間的交互作用。總而言之,在這份計畫中的各個研究成果,除了讓我們更瞭解臨床分離酵母菌菌株的致病機轉外,更可應用至其他訊息傳遞路徑類似,且具致病性之黴菌菌株的研究上,並將對抗真菌藥物的作用標的物開發有相當的貢獻。
Abstract: Saccharomyces cerevisiae has been recognized as an important pathogen in AIDSpatients, long-term hospitalized patients, and patients undergoing cancer chemotherapy orimmunosuppressive therapy, and sometimes even in normal individuals. Temperatureresistance and pseudohyphal differentiation are considered as two major virulence traits of theclinical isolates. Although it has been shown that the ability of switch between differentcellular morphologies might be crucial for the infectivity, tissue invasion, and virulence ofpathogenic isolates, the detailed mechanism of these traits and pathogenicity still remain to beelucidated.We have collected several clinical isolates of S. cerevisiae from Taiwan under thecollaboration with the Department of Laboratory Medicine at National Taiwan UniversityHospital (NTUH). We already conducted some preliminary experiments in characterizingthese two virulence traits in NTUH isolates. Here we propose to use genomics, proteomics,and molecular genetics approach with the comparison to the isolates from the United States tofurther decipher the molecular mechanism of pathogenicity in S. cerevisiae. Our specific aimsare:Year 1 (98/8/1~99/7/31) -- using genomic approach to identify unknown genesinvolved in these two virulence traits, pseudohyphal formation and hightemperature growth (Htg) phenotype, and further analyze the genetic basis ofevolutionary adaptation in pathogenic isolates.I. Mapping the pseudohyphal formation trait by array-assisted bulk segregant analysis inNTUH clinical isolates of S. cerevisiae.II. Mapping the trait of Htg phenotype by array-assisted bulk segregant analysis in NTUHclinical isolates of S. cerevisiae.III. To carry out phylogenetic analyses of the NTUH clinical isolates for theirpathogenicity.Year 2 (99/8/1~100/7/31) -- to analyze the correlation between the pathogenicity and theeffect of antifungal drug resistance of the clinical isolates and their derived strains.I. To analyze the pathogenicity of clinical isolates of S. cerevisiae by a cell-culture basedassay system.II. To analyze the interrelation between antifungal drug resistance and virulence in theclinical isolates of S. cerevisiae.Year 3 (100/8/1~101/7/31) -- to characterize the relationship of virulence of clinicalisolates and the architecture of yeast cell wall, and using quantitative proteomicapproaches to decipher the roles of cell wall proteins in pathogenicity.I. Characterization of the relationship between the virulence of clinical isolates and thearchitecture of yeast cell wall.II. Characterization and confirmation of the importance of the target cell wall proteinsfound by SILAC.To sum up, by taking all these strategies, mapping out the possible genes involved inpseudohyphal formation and Htg phenotypes and reconstruction of the signaling pathwaysinvolved in these two virulence phenotypes will help us elucidate the mechanism thoroughly.Phylogenetic analyses of these genes will provide us more information in deciphering theirsignificance in evolution. A cell-culture based assay system will be established forpathogenicity analysis, which is highly applicable for other pathogenic fungi. Othercharacteristics of the S. cerevisiae clinical isolates, such as antifungal drug susceptibility andthe architecture of cell wall, will be further analyzed, and their correlation with pathogenicitywill be further determined. Characterization of the relationship between the virulence ofclinical isolates and the architecture of yeast cell wall sheds light on understandinghost-pathogen interaction. All the information above may provide us possible explanations forthe virulence of the clinical isolates. In addition, understanding the mechanism ofmorphologic transitions and pathogenecity in one organism may help us understand it inanother. Many key signaling pathways in model organism S. cerevisia are conserved in otherpathogenic fungi, such as C. albicans or C. neoformans. Lastly, this study may also makegreat contribution on finding specific targets for new antifungal reagents.
Keyword(s)
酵母菌
毒性特質
假菌絲
細胞壁
致病性
抗藥性
Saccharomyces cerevisiae
virulence trait
pseudohyphae
cell wall
pathogenicity
antifungal drug resistance