Identifying critical erythrocyte host factors for Plasmodium falciparum malaria

放大图片作者：埃根 项目概要/摘要 恶性疟原虫引起的严重疟疾是一种由蚊子传播的寄生虫病， 在发展中国家造成巨大的发病率和死亡率。大约2亿 每年约有500，000人感染疟疾，每年约有500，000人死亡，主要是 5岁以下儿童。当恶性疟原虫进入人体时， 在其生命周期的血液阶段，它侵入红细胞并在红细胞中呈指数增长。而 抗疟药物正在广泛使用，其疗效受到快速发展的威胁， 寄生虫的抗药性。由于这些药物都针对寄生虫因子， 方法可以是针对寄生虫生存所需的宿主因素。虽然长- 流行病学数据表明，某些红细胞特性在自然条件下 由于疟疾的选择，在历史上研究成熟的红细胞是具有挑战性的。 分子水平，因为这些不寻常的细胞缺乏基因组和细胞核， 难对付在这里，我们提出了一个蛋白质组范围内的正向遗传筛选，以确定主机的因素 使用来自人类造血干细胞的去核红细胞治疗疟疾至关重要 （HSC）。我们最近报道了一种利用成红细胞筛选人类血型基因的方法， 发现了寄生虫入侵的一个新的宿主因素。我们假设， 在红细胞（寄生虫在体内侵入的细胞）中进行筛选将识别新的宿主类别 决定因素我们将在HSC中使用基于RNAi和CRISPR的遗传扰动， 永生化的CD 34+细胞以产生具有基因缺失或破坏的去核红细胞， 用荧光恶性疟原虫感染细胞使用深度定量RNA测序， 我们将把shRNA/gRNA丰度与寄生虫存活和宿主细胞去核联系起来， 从而产生恶性疟原虫的候选宿主因子列表以及去核 决定因素候选人将使用补充方法进行验证，包括反向验证 遗传学、活细胞显微镜检查、体外寄生虫测定和血液学表征。我们 将利用这些候选者进一步研究使用小分子的宿主-病原体相互作用 和寄生虫抗性选择。我们预计，该项目将导致发现， 恶性疟原虫的关键宿主因子的表征，为合理的 开发针对宿主的疟疾疗法。

Egan, Elizabeth S. Project summary/Abstract Severe malaria due to Plasmodium falciparum is a parasitic disease spread by mosquitoes that causes immense morbidity and mortality in the developing world. Approximately 200 million people contract malaria each year, and there are ~500,000 deaths annually, primarily among children under the age of 5. The symptoms of severe malaria occur when P. falciparum enters the blood stage of its life cycle, where it invades and grows exponentially in erythrocytes. While antimalarial drugs are in widespread use, their efficacy is threatened by the rapid development of resistance among the parasites. As these drugs all target parasite factors, an alternative approach could be to target host factors that the parasite needs for survival. Although long- standing epidemiological data indicate that certain erythrocyte traits have been under natural selection due to malaria, it has historically been challenging to study mature erythrocytes at a molecular level as these unusual cells lack a genome and nucleus, making them genetically intractable. Here, we propose a proteome-wide forward genetic screen to identify host factors critical for malaria using enucleated erythrocytes derived from human hematopoietic stem cells (HSCs). We recently reported a screen of human blood group genes using erythroblasts, which identified a new host factor for parasite invasion. We hypothesize that comprehensive screening in erythrocytes (the cell the parasite invades in vivo) will identify new classes of host determinants. We will use RNAi- and CRISPR-based genetic perturbations in HSCs and immortalized CD34+ cells to generate enucleated erythrocytes with gene depletion or disruption, and infect the cells with fluorescent P. falciparum. Using deep, quantitative RNA-sequencing, we will correlate shRNA/gRNA abundance with parasite survival and host cell enucleation, thereby generating a list of candidate host factors for P. falciparum as well as enucleation determinants. Candidates will be validated using complementary approaches including reverse genetics, live cell microscopy, in vitro parasite assays and hematological characterization. We will use the candidates to further investigate host-pathogen interactions using small molecules and parasite resistance selection. We anticipate that this project will lead to the discovery and characterization of critical host factors for P. falciparum, laying the foundation for the rational development of host-directed therapeutics for malaria.