A Novel Tool for the Generation of Conditional Knockouts To Study Gene Function across the Plasmodium falciparum Life Cycle
A Novel Tool for the Generation of Conditional Knockouts To Study Gene Function across the Plasmodium falciparum Life Cycle
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DOI：
10.1128/mbio.01170-19
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发表时间：
2019-09-01
期刊：
影响因子：
6.4
通讯作者：
Treeck, Moritz
中科院分区：
文献类型：
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作者：
Tiburcio, Marta;Yang, Annie S. P.;Treeck, Moritz
恶性疟原虫具有复杂的生命周期，涉及与人类和蚊子宿主体内的多种组织相互作用。鉴定恶性疟原虫生命周期所有不同阶段的必需基因对于临床开发控制和根除疟疾的工具是迫切需要的。然而，恶性疟原虫的研究是有限的，无法在其整个生命周期的遗传修饰的寄生虫与目前可用的遗传工具。在这里，我们描述了一种新的无标记的恶性疟原虫寄生虫系的详细特征，该寄生虫系在整个生命周期中表达雷帕霉素诱导的Cre重组酶。使用这种寄生虫系，我们能够有条件地删除必要的入侵配体AMA1在三个不同的发展阶段的第一次。我们进一步证实了通过靶向非必需激酶FIKK 7.1的有效基因缺失。重要性研究恶性疟原虫的主要限制之一是，到目前为止，只有无性阶段适合于快速条件遗传修饰。然而，最有希望的药物靶点和候选疫苗对遗传修饰很难，因为它们在血液阶段或在蚊子媒介中传播是必不可少的。这使得我们对大多数生命周期阶段的寄生虫蛋白质的理解存在重大差距，并阻碍了药物和疫苗靶点的遗传验证。在这里，我们描述了一种方法，支持条件基因删除恶性疟原虫整个生命周期的第一次。我们通过删除不同寄生虫阶段的必需和非必需基因来展示其潜力，这为疟疾研究和药物开发开辟了全新的途径。它还可以允许使用减毒寄生虫实现新的疫苗接种策略。
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.