Determinants of growth and fitness in drug resistant malaria parasites

描述（由申请人提供）：疟疾寄生虫，恶性疟原虫，继续威胁人类健康。氯喹（CQ）的过度使用导致了多药耐药（MDR）寄生虫的出现和传播，这对当前的疟疾控制工作具有重要意义。CQ抗性（CQR）基因pfcrt中的K76 T编码突变的近全局选择性扫描留下了影响寄生虫生长、适应性和快速MDR进化倾向的适应性基因组。该项目的长期目标是确定这种抗性适应基因组的分子组成部分。我们的假设是，有一个必要的遗传背景的CQR寄生虫，包括网络化的基因行动，补偿改变PfCRT功能和稳定寄生虫的生理和生长的药物压力的情况下。为了检测这些耐药适应的全基因组存在，我们专注于两种不同的寄生虫解决方案中的基本生长和正常细胞过程，以历史药物压力：1）在分离的CQR/MDR背景中保持共同pfcrt等位基因的遗传杂交后代; 2）在单一受控遗传背景中的实验室选择的pfcrt突变株系。我们的具体目标建立在对生长和生理特征的详细分析基础上，在没有药物的情况下，推动全基因组搜索控制适应性关键参数的基因网络。具体目标1。精确量化生长和生理参数，作为培养红细胞中寄生虫适应性的指标。我们将利用这些相互关联的性状的QTL定位来确定基因组区域，以便于使用强大的新兴比较数据库和工具搜索关键基因。具体目标2。鉴定pfcrt突变体转录谱中的株系特异性变化，并定位表达水平性状的表达（e）QTL控制遗传。具体目标3。识别与CQ选择相关的基因组结构变异。我们将确定已知与耐药性相关的基因的拷贝数多态性，并使用高分辨率CGH鉴定整个基因组中的基因拷贝数变异、小缺失和一些SNP。这些独立但互补的全基因组生物数据层的正式整合突出了阐明抗疟干预新途径的新方法。 项目简介：疟疾每年在非洲造成200多万儿童死亡，在全世界感染5亿多人。最近完成的/恶性疟原虫/比较基因组测序项目在布罗德研究所提供了一个广阔的视野，这个物种的遗传多样性，但是，很少有方法已经开发出使用这些信息。我们的建议使用经典的遗传连锁图谱与新的工具相结合，以研究基因表达变异和染色体结构变异周围的整个基因组。这些工具的整合将有助于绘制危险的多药耐药疟疾寄生虫的适应性和毒力的分子决定因素。这一知识将导致对这种疾病的攻击的新途径。

DESCRIPTION (provided by applicant): The malaria parasite, Plasmodium falciparum, continues to devastate human health. The emergence and spread of multi-drug resistant (MDR) parasites following decades of over-use of chloroquine (CQ) is of fundamental significance to current malaria control efforts. The near-global selective sweep of the K76T- encoding mutation in the CQ resistance (CQR) gene, pfcrt, left behind an adapted genome impacting parasite growth, fitness, and a propensity for rapid MDR evolution. The long-term goal of this project is to identify the molecular components of this resistance-adapted genome. Our hypothesis is that there is an essential genetic background of CQR parasites, comprising networked gene actions that compensate for altered PfCRT function and stabilize parasite physiology and growth in the absence of drug pressure. To detect the genome-wide presence of these drug resistance adaptations, we focus on basic growth and normal cellular processes in two different parasite solutions to historical drug pressure: 1) progeny of a genetic cross that maintain a common pfcrt allele in a segregating CQR/MDR background; 2) a spectrum of laboratory-selected pfcrt mutant lines in a single controlled genetic background. Our Specific Aims build upon a detailed analysis of growth and physiological traits in the absence of drug to drive a genome-wide search for the gene networks controlling key parameters of fitness. Specific Aim 1. To precisely quantify growth and physiological parameters as indicators of parasite fitness in cultured red blood cells. We will use QTL mapping of these inter- related traits to pinpoint genome regions to facilitate searches for key genes using powerful emerging comparative databases and tools. Specific Aim 2. To identify strain-specific shifts in pfcrt-mutant transcription profiles and to map expression (e)QTL controlling inheritance of expression level traits. Specific Aim 3. To identify genome structural variants associated with CQ-selection. We will determine copy number polymorphisms in genes known to be associated with drug resistance and use high resolution CGH identification of gene copy number variations, small deletions, and some SNPs throughout the genome. Formal integration of these layers of independent but complimentary whole-genome biological data highlights a novel approach to elucidate new avenues for antimalarial intervention. Project Narrative: Malaria kills more than 2 million kids in Africa and infects more than 500 million people worldwide each year. The recently completed /Plasmodium falciparum /comparative genome sequencing project at the Broad Institute provides a vast view of genetic diversity of this species; however, few methods have been developed to use this information. Our proposal uses a combination of classical genetic linkage mapping with new tools to study gene expression variation and chromosomal structural variation around the entire genome. An integration of these tools will facilitate the mapping of the molecular determinants of fitness and virulence in dangerous multi-drug resistant malaria parasites. This knowledge will lead to new avenues of attack against this disease.