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.

Egan，Elizabeth S. 项目摘要/摘要 由于恶性疟原虫引起的严重疟疾是蚊子传播的一种寄生疾病， 引起发展中国家的巨大发病率和死亡率。约2亿 人们每年收缩疟疾，每年约有500,000人死亡，主要是 5岁以下的儿童。 其生命周期的血液阶段，它在红细胞中侵入和成倍增长。尽管 抗疟药在广泛使用中，其疗效受到快速发展的威胁 寄生虫之间的抗性。由于这些药物都是靶向寄生虫因素，这是一种替代 方法可能是寄生虫需要生存所需的宿主因素。虽然很长 站立的流行病学数据表明某些红细胞特征一直在自然之下 由于疟疾而选择的选择，从历史上看，研究成熟的红细胞在 分子水平，因为这些异常细胞缺乏基因组和核，使它们在遗传上使它们成为 棘手。在这里，我们提出了一个全蛋白质组的前向遗传筛选，以识别宿主因素 使用源自人类造血干细胞的浓核红细胞的疟疾至关重要 （HSC）。我们最近报道了使用红细胞的人类血型基因的屏幕，该基因 确定了寄生虫入侵的新宿主因素。我们假设这一全面 在红细胞中筛查（寄生虫入侵体内的细胞）将识别新的宿主类 决定因素。我们将在HSC中使用基于RNAi的基于RNAI-and CRISPR的遗传扰动 永生的CD34+细胞生成具有基因耗竭或破坏的浓缩红细胞， 并用荧光恶性疟原虫感染细胞。使用深，定量的RNA测序， 我们将将shrna/grna丰度与寄生虫存活和宿主细胞摘除相关联， 从而产生候选宿主因素的列表，用于恶性疟原虫 决定因素。候选人将使用互补方法进行验证 遗传学，活细胞显微镜，体外寄生虫测定和血液学特征。我们 将使用候选者进一步研究使用小分子的宿主 - 病原体相互作用 和寄生虫的选择。我们预计该项目将导致发现，并且 对恶性假单胞菌的关键宿主因素的表征，为理性奠定了基础 开发疟疾宿主定向的治疗剂。

项目成果

Beyond Hemoglobin: Screening for Malaria Host Factors.

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