A high susceptibility to redox imbalance of the transmissible stages of Plasmodium falciparum revealed with a luciferase-based mature gametocyte assay.
A high susceptibility to redox imbalance of the transmissible stages of Plasmodium falciparum revealed with a luciferase-based mature gametocyte assay.
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DOI：
10.1111/mmi.13626
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发表时间：
2017-04
影响因子：
3.6
通讯作者：
Alano P
中科院分区：
文献类型：
--
作者：
Siciliano G;Santha Kumar TR;Bona R;Camarda G;Calabretta MM;Cevenini L;Davioud-Charvet E;Becker K;Cara A;Fidock DA;Alano P
防止恶性疟原虫从人类传播给蚊子的目标需要识别成熟（第五阶段）配子细胞（血液中循环的有性阶段）中的目标代谢过程。由于这些细胞的新陈代谢明显较低，这项任务变得复杂，这使得它们对大多数抗疟抑制剂都无效，并限制了特异性和敏感的基于细胞的测定的发展。在这里，我们鉴定并功能表征了恶性疟原虫基因 PF3D7_1234700 的调控区域，该基因编码 CPW-WPC 蛋白，并在此处命名为晚期配子细胞上调 (ULG8)，我们利用该区域在成熟雄性和雌性配子细胞中表达报告基因。使用含有 pfULG8-荧光素酶盒的转基因寄生虫，我们研究了 V 期配子细胞对特异性影响氧化还原代谢的化合物的敏感性。我们的结果揭示了成熟配子细胞对谷胱甘肽还原酶抑制剂和氧化还原循环药物亚甲蓝 (MB) 的高度敏感性。使用等效线图分析，我们发现同时抑制寄生虫酶葡萄糖-6-磷酸脱氢酶-6-磷酸葡萄糖酸内酯酶（NADPH 合成的关键成分）可有效协同 MB 活性。这些数据表明，氧化还原代谢和解毒活性在 V 期配子细胞中发挥着意想不到但至关重要的作用，使这些细胞对 NADPH 浓度的降低极其敏感。人们对恶性疟原虫成熟配子体的生理学知之甚少，我们用生物发光寄生虫进行了研究，结果表明，这些阶段几乎对所有抗疟药物都无效，如果它们对抗氧化应激的能力受损，它们就非常脆弱。
The goal to prevent Plasmodium falciparum transmission from humans to mosquitoes requires the identification of targetable metabolic processes in the mature (stage V) gametocytes, the sexual stages circulating in the bloodstream. This task is complicated by the apparently low metabolism of these cells, which renders them refractory to most antimalarial inhibitors and constrains the development of specific and sensitive cell-based assays. Here we identify and functionally characterize the regulatory regions of the P. falciparum gene PF3D7_1234700, encoding a CPW-WPC protein and named here Upregulated in Late Gametocytes (ULG8), which we have leveraged to express reporter genes in mature male and female gametocytes. Using transgenic parasites containing a pfULG8-luciferase cassette, we investigated the susceptibility of stage V gametocytes to compounds specifically affecting redox metabolism. Our results reveal a high sensitivity of mature gametocytes to the glutathione reductase inhibitor and redox cycler drug methylene blue (MB). Using isobologram analysis, we find that a concomitant inhibition of the parasite enzyme glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase, a key component of NADPH synthesis, potently synergizes MB activity. These data suggest that redox metabolism and detoxification activity play an unsuspected yet vital role in stage V gametocytes, rendering these cells exquisitely sensitive to decreases in NADPH concentration. The poorly understood physiology of the mature gametocytes of Plasmodium falciparum was investigated with bioluminescent parasites, revealing that these stages, known to be refractory to virtually all antimalarial drugs, are exquisitely vulnerable if their ability to contrast oxidative stress is impaired.