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，继续危害人类健康。数十年来过度使用氯喹（chloroquine， CQ）导致耐多药寄生虫（MDR）的出现和传播，对当前疟疾控制工作具有重要意义。CQ抗性（CQR）基因pfcrt中编码K76T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，适应性和快速MDR进化的倾向。该项目的长期目标是确定这种适应抗性的基因组的分子成分。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性在全基因组中的存在，我们关注了两种不同的寄生虫解决方案在历史药物压力下的基本生长和正常细胞过程：1)在分离的CQR/MDR背景下保持共同pfcrt等位基因的遗传杂交后代；2)在单一可控遗传背景下实验室选择的PFCRT突变系谱。我们的具体目标建立在对生长和生理性状的详细分析上，在没有药物的情况下，推动对控制关键适应度参数的基因网络的全基因组搜索。具体目标精确量化生长和生理参数作为寄生虫在培养红细胞中的适应度指标。我们将使用这些相互关联的性状的QTL定位来精确定位基因组区域，以便使用强大的新兴比较数据库和工具来促进关键基因的搜索。具体目标2。确定pfcrt突变体转录谱的菌株特异性变化，并绘制表达图谱(e)控制表达水平性状遗传的QTL。具体目标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基因的拷贝数多态性，并使用高分辨率CGH鉴定基因拷贝数变异、小缺失和基因组中的一些snp。这些独立但互补的全基因组生物学数据层的正式整合凸显了阐明抗疟疾干预新途径的新方法。

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.