Genetic and Evolutionary Basis of Fungal Drug Resistance

描述（由申请人提供）：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。真菌病原体的毒力因子，如致病性真菌耐药性，通常是数量性状。我们实验室的一个目标是绘制致病酵母菌菌株对抗真菌药物抗性的数量性状基因，并确定这种抗性的演变。这项工作将改善诊断和治疗策略，并导致更好地理解适应性复杂性状的进化。利用全基因组瓦片阵列对病原菌（YJM789）对一种抗真菌药物（氟康唑）的敏感性进行初步数据定位，成功鉴定出一个候选基因（PDR5），随后的实验表明该突变是导致YJM789对氟康唑敏感性的唯一原因（一种孟德尔性状）。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）具有耐药性，这是一种数量性状，也是由其他遗传因素决定的。目前的结果代表了一个有趣的“拮抗多效性”案例（即，某些基因在某些情况下是功能性的，但在其他情况下可能是有害的）和具有临床重要性的适应性基因丢失。在aims 1中，我们将应用QTL定位方法和几种替代酵母遗传工具来鉴定YJM789中负责AmB抗性的其他基因。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基因的单个功能，以提供AmB抗性的机制解释。一个特别的重点将是确定为什么PDR5的缺失，一个我们已经确定的遗传因素，会增加酵母对AmB的抗性。我们假设PDR5及其旁系基因参与麦角甾醇稳态，这将在酿酒葡萄球菌和一种致病酵母菌白色念珠菌中进行测试。在Aim 1中发现的其他基因的功能和进化也将被研究。在目标3中，我们将确定与耐药性相关的其他qtl。更具体地说，我们将在不同的小分子药物库下对我们的单倍体后代进行表型分析。在这些环境中负责YJM789独特生长表型的遗传位点将使用Aim 1中开发的方法进行鉴定。这些数据将用于验证以下假设，即相同类型的药物对致病性酵母生长具有相似的功能机制，以及基因组某些区域的突变可以使致病性酵母应对外源性小分子。

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.