A forward genetic screen to identify determinants of malaria stage conversion

描述(申请人提供)：疟疾寄生虫，包括毒性最强的物种恶性疟原虫，在哺乳动物宿主和蚊子媒介之间切换，在此过程中经历多个发育阶段和宿主组织。如果要实现消除疟疾，阻断传播将是必不可少的，但这是疟疾周期中最不被了解的部分。在红细胞中发育成熟后，传播形式或配子体是唯一可以通过受感染的血餐传播给蚊子媒介的阶段。在红细胞期晚期寄生虫的一小部分亚群中，阶段转化为配子体，其中一种寄生虫只产生无性或有性侵犯的侵袭子细胞。在性寄生虫入侵红细胞后，其他因素决定了发育过程中向有传播能力的形式的发展过程。在体内和体外模型中，来自一个复制周期的配子体的比例(即转化率)和雌雄配子体的比例(即性别比)在不同品系之间是不同的。阶段转换的决定因素仍然难以捉摸，除了几个染色体缺失与体外性发育丧失有关。因此，我们假设疟原虫阶段转化具有很强的遗传成分。到目前为止，还没有鉴定出参与疟原虫阶段转化的寄生虫(和宿主)因素，主要是由于缺乏系统研究这一过程的方法学。具体地说，目前还没有一个有效的、可重复的系统来研究恶性疟原虫的转化，而且由于恶性疟原虫的低转染率，遗传上的困难是固有的。我们最近建立了一个可重复性和高通量兼容的阶段转换和配子体发育的方案，我们已经开发出一种药物分析。最近，我们还成功地将这种方法应用于恶性疟原虫第一次正向遗传筛查中的表型读数，使用的是iggyBac转座子。在这里，我们建议使用针对高通量突变产生和表型分析而优化的第二代iggyBac屏幕，系统地表征参与阶段转换的途径。这项研究结合了几种高度创新的方法，通过将有效的转换系统和正向遗传筛选联系起来。结合我们正在进行的利用化学探针和转录图谱调查阶段转化途径的努力，这项研究为全面分析疟疾生命周期中一个重要但迄今难以捉摸的过程提供了独特的机会。

DESCRIPTION (provided by applicant): Malaria parasites including the most virulent species, Plasmodium falciparum, switch between a mammalian host and a mosquito vector, passing through multiple developmental stages and host tissues in the process. If malaria elimination is to be achieved, interrupting transmission will be essential yet it is the least understood part of the malaria cycle. Transmission forms, or gametocytes, upon reaching maturity after development in red blood cells, are the only stages that can be transmitted to the mosquito vector via an infected blood meal. Stage conversion into gametocytes occurs in a small subpopulation of late red blood cell stage parasites, whereby one parasite either produces only asexually or sexually committed invasive daughter cells. After invasion of sexual parasites into red blood cells, additional factors determine developmental progression during development toward transmission competent forms. The proportion of gametocytes derived from one replication cycle (i.e., conversion rate) and the ratio of female and male gametocytes (i.e., sex ratio) vary between strains, both in vivo and in in vitro models. Determinants of stage conversion remain elusive except that several chromosomal deletions have been linked to the loss of sexual development in vitro. We therefore hypothesize that Plasmodium stage conversion has a strong genetic component. No parasite (and host) factors involved in Plasmodium stage conversion have been identified so far, mostly due to lack of methodology to study this process systematically. Specifically, there has not been an efficient and reproducible system for studying conversion and genetics are inherently difficult because of the low transfection efficiency in P. falciparum. We have recently established a reproducible and high throughput compatible protocol for stage conversion and gametocyte development, which we have developed into a drug assay. More recently, we have also successfully applied this assay as a phenotypic readout in the first forward genetic screen in P. falciparum, using the piggyBac transposon. Here we propose systematic characterization of the pathways involved in stage conversion using a second-generation piggyBac screen that is optimized for high throughput mutant generation and phenotyping. This study combines several highly innovative approaches by linking an efficient conversion system and a forward genetic screen. In combination with our ongoing efforts to investigate pathways involved in stage conversion using chemical probes and transcriptional profiling, this study provides the unique opportunity for a comprehensive analysis of an essential yet so far elusive process in the malaria life cycle.