A forward genetic screen to identify determinants of malaria stage conversion

描述（由申请人提供）：疟疾寄生虫，包括最致命的物种恶性疟原虫，在哺乳动物宿主和蚊子载体之间转换，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为人所知的部分。传播形式，或配子体，在红细胞发育成熟后，是唯一可以通过受感染的血液传播给蚊子媒介的阶段。向配子体的阶段转化发生在红细胞晚期寄生虫的一小部分亚群中，其中一个寄生虫要么只产生无性行为的侵入性子细胞，要么只产生有性行为的侵入性子细胞。在性寄生虫侵入红细胞后，其他因素决定了发育过程中向传播能力形式的发展。在体内和体外模型中，一个复制周期产生的配子体比例（即转化率）和雌雄配子体比例（即性别比例）在不同菌株之间都是不同的。阶段转换的决定因素仍然难以捉摸，除了一些染色体缺失与体外性发育的丧失有关。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，尚未发现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。具体来说，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转染效率低，遗传学本身就很困难。我们最近建立了一个可重复和高通量兼容的方案，用于阶段转化和配子体发育，我们已经将其发展为药物分析。最近，我们还利用piggyBac转座子成功地将该检测方法应用于恶性疟原虫的第一次前向遗传筛选中作为表型读出。在这里，我们提出使用第二代piggyBac筛选系统表征参与阶段转化的途径，该筛选针对高通量突变产生和表型进行了优化。本研究结合了几种高度创新的方法，通过连接一个有效的转换系统和前向遗传筛选。结合我们正在进行的利用化学探针和转录谱研究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为止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.