Genetic and Evolutionary Basis of Fungal Drug Resistance

描述(申请人提供)：真菌病原体可能是致命的，特别是在免疫功能低下的人群中，如艾滋病患者，或组织移植或化疗的接受者。真菌病原菌的毒力因子，如致病性真菌耐药性，往往是数量性状。我们实验室的一个目标是定位致病酵母菌株对抗真菌药物耐药性的基础数量性状基因，并确定这种耐药性的进化。这项工作将改进诊断和治疗策略，并导致更好地理解适应性复杂性状的进化。利用全基因组芯片定位致病菌株(YJM789)对一种抗真菌药物(氟康唑)敏感性的初步数据成功地确定了一个候选基因(PDR5)，随后的实验表明，该突变是YJM789对氟康唑敏感的唯一原因(孟德尔性状)。令人惊讶的是，同样的突变使YJM789对另一种抗真菌药物(两性霉素B，Amb)产生抗药性，这是一种也由其他遗传因素决定的数量性状。目前的结果代表了一个有趣的“拮抗多效性”(即某些基因在某些条件下具有功能，但在另一些条件下可能是有害的)和适应性基因丢失的有趣案例，具有临床意义。在目标1中，我们将应用QTL定位方法和几种替代的酵母遗传工具来鉴定YJM789中与Amb抗性有关的其他基因。贡献基因之间的相互作用也将被量化。在目标2中，我们将分析QTL基因的个体功能，以提供AMB抗性的机制解释。一个特别的焦点将是确定为什么PDR5的缺失，我们已经确定的一个起作用的遗传因素，增加酵母对Amb的耐药性。我们假设PDR5及其类似的基因参与了麦角固醇的稳态，这将在酿酒酵母和致病酵母菌白念珠菌中进行测试。目标1中确定的其他基因的功能和进化也将被研究。在目标3中，我们将确定更多与耐药性相关的QTL。更具体地说，我们将在不同小分子药物的库中对我们的单倍体后代小组进行表型。负责YJM789在这些环境中独特生长表型的遗传基因座将使用目标1中开发的方法进行识别。这些数据将用于测试以下假设：相同类型的药物对致病酵母菌的生长具有相似的作用机制，基因组某些区域的突变可以使致病酵母能够应对外源小分子。

DESCRIPTION (provided by applicant): Fungal pathogens can be lethal, especially among immunocompromised populations, such as patients with AIDS, or recipients of tissue transplantation or chemotherapy. Virulence factors for fungal pathogens, such as pathogenic fungal drug resistance, often are quantitative traits. A goal of our laboratories is to map quantitative trait genes that underlie the resistance of pathogenic yeast strains to anti-fungal agents and to determine the evolution of this resistance. This work will improve diagnosis and treatment strategies and lead to a better understanding of the evolution of adaptive complex traits. The preliminary data for mapping sensitivity of the pathogenic strain (YJM789) to one antifungal drug (Fluconazole) using whole genome tiling array successfully identified a candidate gene (PDR5), and subsequent experiments were able to show that the mutation is solely responsible for YJM789-sensitivity to Fluconazole (a Mendelian trait). Surprisingly, the same mutation confers YJM789 drug resistance to another antifungal drug (Amphotericin B, AmB), which is a quantitative trait also determined by other genetic factors. The current results represent an interesting case of "antagonistic pleiotropy" (i.e., certain genes are functional in some conditions, but could be deleterious in others) and adaptive gene loss with clinical importance. In Aim 1, we will apply QTL mapping methods and several alternative yeast genetic tools to identify other genes responsible for AmB resistance in YJM789. Interactions among contributing genes will also be quantified. In Aim 2, we will analyze the individual functions of the QTL genes to provide a mechanistic explanation for AmB resistance. A specific focus will be on determining why deletion of PDR5, a contributing genetic factor we have already identified, increases AmB resistance in yeasts. We hypothesize that PDR5 and its paralogous genes are involved in ergosterol homeostasis, which will be tested in S. cerevisiae and in a pathogenic yeast species, Candida albicans. The function and evolution of other genes identified in Aim 1 will also be investigated. In Aim 3, we will identify additional QTLs that are linked to drug resistance. More specifically, we will phenotype our panel of haploid progeny under a library of different small molecule drugs. Genetic loci which are responsible for the unique growth phenotypes of YJM789 in these environments will be identified using methodologies developed in Aim 1. These data will be used to test the hypotheses that the same types of drugs have similar functional mechanisms on pathogenic yeast growth and that mutations on certain regions of the genome can enable pathogenic yeasts to cope with exogenous small molecules.