Identifying critical erythrocyte host factors for Plasmodium falciparum malaria

题名/责任者：Elan，Elizabeth S. 项目摘要/摘要 由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。约2亿 每年都有人感染疟疾，每年约有50万人死亡，主要是 5岁以下的儿童。恶性疟原虫进入时出现严重疟疾的症状 血吸虫生命周期的血液阶段，在这一阶段它会侵入红细胞并呈指数增长。而当 抗疟疾药物被广泛使用，其疗效受到快速发展的威胁 寄生虫之间的抗药性。由于这些药物都针对寄生虫因素，因此另一种选择 方法可能是针对寄生虫生存所需的宿主因素。尽管很长时间- 长期的流行病学数据表明，某些红细胞特征一直处于自然状态 由于疟疾，研究成熟的红细胞历来是具有挑战性的。 分子水平，因为这些不寻常的细胞缺乏基因组和核，使它们在基因上 很难对付。在这里，我们提出了一种蛋白质组范围的正向遗传筛选来识别宿主因子。 使用来自人造血干细胞的去核红细胞对疟疾至关重要 (HSCs)。我们最近报道了一种使用红细胞的人类血型基因筛查，这是一种 确定了寄生虫入侵的一个新的宿主因素。我们假设，全面的 对红细胞(寄生虫体内侵入的细胞)进行筛查将识别新的宿主类别 决定因素。我们将在造血干细胞中使用基于RNAi和CRISPR的遗传扰动 使CD34+细胞永生化，以产生基因缺失或破坏的去核红细胞， 并用荧光恶性疟原虫感染细胞。使用深度、定量的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， 从而生成恶性疟原虫的候选宿主因素列表以及去核 决定因素。候选人将使用包括反向在内的补充方法进行验证 遗传学、活细胞显微镜、体外寄生虫分析和血液学特征。我们 将使用这些候选分子进一步研究宿主与病原体之间的相互作用 以及对寄生虫的抗性选择。我们预计这个项目将导致发现和 恶性疟原虫关键寄主因子的表征，为合理利用 疟疾宿主导向疗法的发展。

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.