Determinants of growth and fitness in drug resistant malaria parasites

描述(申请人提供)：疟疾寄生虫恶性疟原虫继续破坏人类健康。在过度使用氯喹(CQ)几十年后出现和传播多药耐药(MDR)寄生虫，对当前的疟疾控制工作具有根本意义。对CQ抗性(CQR)基因pfcrt中K76T编码突变的近全球选择性扫描，留下了一个适应的基因组，影响寄生虫的生长、适合性和快速MDR进化的倾向。该项目的长期目标是确定这种抗性适应基因组的分子成分。我们的假设是，CQR寄生虫有一个基本的遗传背景，包括网络基因作用，补偿改变的PfCRT功能，并在没有药物压力的情况下稳定寄生虫的生理和生长。为了检测这些耐药适应在全基因组范围内的存在，我们关注在两种不同的寄生虫解决方案中对历史药物压力的基本生长和正常的细胞过程：1)在分离的CQR/MDR背景下保持共同pfcrt等位基因的遗传杂交的后代；2)在单一受控遗传背景下实验室选择的pfcrt突变系的范围。我们的具体目标是在缺乏药物的情况下对生长和生理特征进行详细分析，以推动对控制健康关键参数的基因网络的全基因组搜索。具体目的1.精确量化培养红细胞中寄生虫的生长和生理参数，作为寄生虫适合性的指标。我们将使用这些相互关联的性状的QTL作图来精确定位基因组区域，以便于使用强大的新兴比较数据库和工具搜索关键基因。具体目的2.鉴定pfcrt-突变型转录图谱中的品系特异性变化，并定位控制表达水平性状遗传的表达(E)QTL。具体目的3.鉴定与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，并使用高分辨率CGH识别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学数据的正式整合突出了一种新的方法来阐明抗疟疾干预的新途径。 项目简介：疟疾每年夺去非洲200多万儿童的生命，感染全球5亿多人。布罗德研究所最近完成的恶性疟原虫/比较基因组测序项目提供了对该物种遗传多样性的广泛看法；然而，几乎没有开发出利用这些信息的方法。我们的建议使用经典的遗传连锁图谱和新的工具相结合的方法来研究整个基因组周围的基因表达变异和染色体结构变异。这些工具的整合将有助于绘制危险的多重抗药性疟疾寄生虫的适合性和毒性的分子决定因素图。这一知识将导致针对这种疾病的新的攻击途径。

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.