In the malaria parasite Plasmodium falciparum, synthesis of isoprenoids from glycolytic intermediates is essential for survival. The antimalarial fosmidomycin (FSM) inhibits isoprenoid synthesis. In P. falciparum, we identified a loss-of-function mutation in HAD2 (P. falciparum 3D7_1226300 [PF3D7_1226300]) as necessary for FSM resistance. Enzymatic characterization revealed that HAD2, a member of the haloacid dehalogenase-like hydrolase (HAD) superfamily, is a phosphatase. Harnessing a growth defect in resistant parasites, we selected for suppression of HAD2-mediated FSM resistance and uncovered hypomorphic suppressor mutations in the locus encoding the glycolytic enzyme phosphofructokinase 9 (PFK9). Metabolic profiling demonstrated that FSM resistance is achieved via increased steady-state levels of methylerythritol phosphate (MEP) pathway and glycolytic intermediates and confirmed reduced PFK9 function in the suppressed strains. We identified HAD2 as a novel regulator of malaria parasite metabolism and drug sensitivity and uncovered PFK9 as a novel site of genetic metabolic plasticity in the parasite. Our report informs the biological functions of an evolutionarily conserved family of metabolic regulators and reveals a previously undescribed strategy by which malaria parasites adapt to cellular metabolic dysregulation.IMPORTANCE Unique and essential aspects of parasite metabolism are excellent targets for development of new antimalarials. An improved understanding of parasite metabolism and drug resistance mechanisms is urgently needed. The antibiotic fosmidomycin targets the synthesis of essential isoprenoid compounds from glucose and is a candidate for antimalarial development. Our report identifies a novel mechanism of drug resistance and further describes a family of metabolic regulators in the parasite. Using a novel forward genetic approach, we also uncovered mutations that suppress drug resistance in the glycolytic enzyme PFK9. Thus, we identify an unexpected genetic mechanism of adaptation to metabolic insult that influences parasite fitness and tolerance of antimalarials.

中文翻译：

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耐药性的抑制揭示了疟原虫代谢可塑性的遗传机制。

在疟原虫恶性疟原虫中，从糖酵解中间体合成类异戊二烯对于生存至关重要。抗疟药福斯米霉素 (FSM) 可抑制类异戊二烯的合成。在恶性疟原虫中，我们发现 HAD2 中的功能丧失突变（恶性疟原虫 3D7_1226300 [PF3D7_1226300]）是 FSM 抗性所必需的。酶学表征表明，HAD2 是卤酸脱卤酶样水解酶 (HAD) 超家族的成员，是一种磷酸酶。利用抗性寄生虫的生长缺陷，我们选择抑制 HAD2 介导的 FSM 抗性，并发现编码糖酵解酶磷酸果糖激酶 9 (PFK9) 的基因座中的低效抑制突变。代谢分析表明，FSM 抗性是通过增加磷酸甲基赤藓糖醇 (MEP) 途径和糖酵解中间体的稳态水平来实现的，并证实了受抑制菌株中 PFK9 功能的降低。我们确定 HAD2 是疟疾寄生虫代谢和药物敏感性的新型调节剂，并发现 PFK9 是疟原虫遗传代谢可塑性的新位点。我们的报告揭示了一个进化上保守的代谢调节因子家族的生物学功能，并揭示了疟原虫适应细胞代谢失调的先前未描述的策略。 重要性 寄生虫代谢的独特和重要方面是开发新型抗疟药的极好目标。迫切需要加深对寄生虫代谢和耐药机制的了解。抗生素磷米霉素的目标是从葡萄糖合成必需的类异戊二烯化合物，是抗疟药开发的候选药物。我们的报告确定了一种新的耐药机制，并进一步描述了寄生虫中的代谢调节因子家族。使用新型正向遗传方法，我们还发现了抑制糖酵解酶 PFK9 耐药性的突变。因此，我们发现了一种意想不到的适应代谢损伤的遗传机制，该机制影响寄生虫的适应性和抗疟药的耐受性。