Department of Molecular Microbiology and Immunology, Johns Hopkins Universitygrid.21107.35, Baltimore, Maryland, USA.
mBio. 2021 Feb 22;13(1):e0302321. doi: 10.1128/mbio.03023-21. Epub 2022 Feb 15.
Ferredoxin (Fd) and ferredoxin-NADP+ reductase (FNR) form a redox system that is hypothesized to play a central role in the maintenance and function of the apicoplast organelle of malaria parasites. The Fd/FNR system provides reducing power to various iron-sulfur cluster (FeS)-dependent proteins in the apicoplast and is believed to help to maintain redox balance in the organelle. While the Fd/FNR system has been pursued as a target for antimalarial drug discovery, Fd, FNR, and the FeS proteins presumably reliant on their reducing power play an unknown role in parasite survival and apicoplast maintenance. To address these questions, we generated genetic deletions of these proteins in a parasite line containing an apicoplast bypass system. Through these deletions, we discovered that Fd, FNR, and certain FeS proteins are essential for parasite survival but found that none are required for apicoplast maintenance. Additionally, we addressed the question of how Fd and its downstream FeS proteins obtain FeS cofactors by deleting the FeS transfer proteins SufA and NfuApi. While individual deletions of these proteins revealed their dispensability, double deletion resulted in synthetic lethality, demonstrating a redundant role in providing FeS clusters to Fd and other essential FeS proteins. Our data support a model in which the reducing power from the Fd/FNR system to certain downstream FeS proteins is essential for the survival of blood-stage malaria parasites but not for organelle maintenance, while other FeS proteins are dispensable for this stage of parasite development. Ferredoxin (Fd) and ferredoxin-NADP+ reductase (FNR) form one of the few known redox systems in the apicoplast of malaria parasites and provide reducing power to iron-sulfur (FeS) cluster proteins within the organelle. While the Fd/FNR system has been explored as a drug target, the essentiality and roles of this system and the identity of its downstream FeS proteins have not been determined. To answer these questions, we generated deletions of these proteins in an apicoplast metabolic bypass line (PfMev) and determined the minimal set of proteins required for parasite survival. Moving upstream of this pathway, we also generated individual and dual deletions of the two FeS transfer proteins that deliver FeS clusters to Fd and downstream FeS proteins. We found that both transfer proteins are dispensable, but double deletion displayed a synthetic lethal phenotype, demonstrating their functional redundancy. These findings provide important insights into apicoplast biochemistry and drug development.
铁氧还蛋白 (Fd) 和铁氧还蛋白-NADP+还原酶 (FNR) 形成了一个氧化还原系统，该系统被假设在维持和功能疟疾寄生虫的类质体器官中发挥核心作用。Fd/FNR 系统为类质体中的各种铁硫簇 (FeS) 依赖性蛋白提供还原力，并有助于维持细胞器中的氧化还原平衡。虽然 Fd/FNR 系统已被作为抗疟药物发现的靶标，但 Fd、FNR 和依赖其还原力的 FeS 蛋白在寄生虫存活和类质体维持中可能发挥未知作用。为了解决这些问题，我们在含有类质体旁路系统的寄生虫系中生成了这些蛋白的遗传缺失。通过这些缺失，我们发现 Fd、FNR 和某些 FeS 蛋白对寄生虫存活至关重要，但发现它们都不需要类质体维持。此外，我们通过删除 FeS 转移蛋白 SufA 和 NfuApi 来解决 Fd 和其下游 FeS 蛋白如何获得 FeS 辅因子的问题。虽然这些蛋白的单独缺失表明它们是可有可无的，但双缺失导致合成致死，表明它们在向 Fd 和其他必需的 FeS 蛋白提供 FeS 簇方面具有冗余作用。我们的数据支持这样一种模型，即 Fd/FNR 系统向某些下游 FeS 蛋白提供的还原力对于血期疟原虫的存活至关重要，但对于细胞器维持则不重要，而其他 FeS 蛋白在寄生虫发育的这个阶段是可有可无的。铁氧还蛋白 (Fd) 和铁氧还蛋白-NADP+还原酶 (FNR) 构成了疟原虫类质体中少数已知的氧化还原系统之一，为细胞器内的铁硫 (FeS) 簇蛋白提供还原力。虽然 Fd/FNR 系统已被探索作为药物靶点，但该系统的必要性和作用及其下游 FeS 蛋白的身份尚未确定。为了回答这些问题，我们在类质体代谢旁路系 (PfMev) 中生成了这些蛋白的缺失，并确定了寄生虫存活所需的最小蛋白集。在该途径的上游，我们还生成了两个铁硫转移蛋白的单个和双重缺失，这些蛋白将 FeS 簇递送到 Fd 和下游 FeS 蛋白。我们发现这两个转移蛋白都是可有可无的，但双缺失显示出合成致死表型，表明它们具有功能冗余性。这些发现为类质体生物化学和药物开发提供了重要的见解。
