Identifying critical erythrocyte host factors for Plasmodium falciparum malaria

确定恶性疟原虫疟疾的关键红细胞宿主因子

• 批准号: 9167283
• 负责人: Elizabeth S. Egan
• 金额: $ 235.5万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-30 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9167283
• 关键词: Age; Antimalarials; Biological Assay; Blood; Blood typing procedure; CD34 gene; Cell Nucleus; Cells; Cessation of life; Child; Clustered Regularly Interspaced Short Palindromic Repeats; Contracts; Culicidae; Data; Development; Epidemiology; Erythroblasts; Erythrocytes; Falciparum Malaria; Foundations; Genes; Genetic; Genetic Screening; Genome; Guide RNA; Hematopoietic stem cells; Human; In Vitro; Infection; Integration Host Factors; Invaded; Lead; Life Cycle Stages; Malaria; Molecular; Morbidity - disease rate; Natural Selections; Parasite resistance; Parasites; Parasitic Diseases; Pharmaceutical Preparations; Plasmodium falciparum; Pregnant Women; Properdin; Proteome; Public Health; RNA Interference; Reporting; Resistance; Resistance development; Staging; Symptoms; Therapeutic; abstracting; base; blood group; disorder control; in vivo; live cell microscopy; mortality; parasite invasion; pathogen; reverse genetics; screening; small hairpin RNA; small molecule; trait; transcriptome sequencing

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.

伊根，伊丽莎白 S. 项目概要/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。恶性疟原虫进入时会出现严重疟疾的症状 其生命周期的血液阶段，它侵入红细胞并在其中呈指数增长。尽管 抗疟药物广泛使用，其疗效受到快速发展的威胁 寄生虫之间的抵抗力。由于这些药物都针对寄生虫因子，因此需要另一种选择 方法可能是针对寄生虫生存所需的宿主因素。虽然长—— 现有的流行病学数据表明，某些红细胞特征已受到自然影响。 由于疟疾导致的选择，在历史上研究成熟红细胞一直是一项挑战 分子水平，因为这些不寻常的细胞缺乏基因组和细胞核，使它们具有遗传性 棘手的。在这里，我们提出了一种全蛋白质组正向遗传筛选来识别宿主因素 使用源自人类造血干细胞的去核红细胞对疟疾至关重要 （HSC）。我们最近报道了使用成红细胞进行的人类血型基因筛查， 确定了寄生虫入侵的新宿主因子。我们假设综合 红细胞（寄生虫在体内侵入的细胞）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 CD34+ 细胞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 RNA 测序， 我们将 shRNA/gRNA 丰度与寄生虫存活和宿主细胞去核关联起来， 从而生成恶性疟原虫候选宿主因子列表以及摘除 决定因素。将使用补充方法（包括反向方法）对候选人进行验证 遗传学、活细胞显微镜、体外寄生虫测定和血液学表征。我们 将利用候选药物进一步研究小分子宿主与病原体的相互作用 和寄生虫抗性选择。我们预计该项目将导致发现和 恶性疟原虫关键宿主因素的表征，为合理的研究奠定基础 开发针对宿主的疟疾疗法。

项目成果

Beyond Hemoglobin: Screening for Malaria Host Factors.

除血红蛋白外：筛查疟疾宿主因素。

• DOI: 10.1016/j.tig.2017.11.004
• 发表时间: 2018-03
• 期刊: Trends in genetics : TIG
• 作者: Egan ES