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Abstract：
Plasmodium falciparum has a complex life cycle that involves interaction with multiple tissues inside the human and mosquito hosts. Identification of essential genes at all different stages of the P. falciparum life cycle is urgently required for clinical development of tools for malaria control and eradication. However, the study of P. falciparum is limited by the inability to genetically modify the parasite throughout its life cycle with the currently available genetic tools. Here, we describe the detailed characterization of a new marker-free P. falciparum parasite line that expresses rapamycin-inducible Cre recombinase across the full life cycle. Using this parasite line, we were able to conditionally delete the essential invasion ligand AMA1 in three different developmental stages for the first time. We further confirm efficient gene deletion by targeting the nonessential kinase FIKK7.1.IMPORTANCE One of the major limitations in studying P. falciparum is that so far only asexual stages are amenable to rapid conditional genetic modification. The most promising drug targets and vaccine candidates, however, have been refractory to genetic modification because they are essential during the blood stage or for transmission in the mosquito vector. This leaves a major gap in our understanding of parasite proteins in most life cycle stages and hinders genetic validation of drug and vaccine targets. Here, we describe a method that supports conditional gene deletion across the P. falciparum life cycle for the first time. We demonstrate its potential by deleting essential and nonessential genes at different parasite stages, which opens up completely new avenues for the study of malaria and drug development. It may also allow the realization of novel vaccination strategies using attenuated parasites.
摘要：
恶性疟原虫具有复杂的生命周期，涉及与人类和蚊子宿主内部的多种组织相互作用。临床开发疟疾控制和根除工具迫切需要在恶性疟原虫生命周期的所有不同阶段鉴定必需基因。然而,恶性疟原虫的研究受到目前可用的遗传工具无法在其整个生命周期中对寄生虫进行遗传修饰的限制。这里,我们描述了在整个生命周期中表达雷帕霉素诱导的Cre重组酶的新的无标记恶性疟原虫寄生虫系的详细表征。利用这个寄生虫线，我们首次能够在三个不同的发育阶段有条件地删除必需的入侵配体AMA1。我们通过靶向非必需激酶FIKK7.1进一步证实了有效的基因缺失。重要性研究恶性疟原虫的主要限制之一是到目前为止，只有无性阶段才能进行快速条件遗传修饰。最有希望的药物靶点和候选疫苗，然而,由于它们在血液阶段或在蚊子媒介中传播是必不可少的，因此对遗传修饰是难以理解的。这在我们对大多数生命周期阶段的寄生虫蛋白的理解上留下了很大的差距，并阻碍了药物和疫苗靶标的遗传验证。这里,我们首次描述了一种支持恶性疟原虫生命周期中条件性基因缺失的方法。我们通过删除不同寄生虫阶段的必需和非必需基因来证明其潜力，这为疟疾和药物开发的研究开辟了全新的途径。它还可以允许使用减毒的寄生虫实现新的疫苗接种策略。
