Mutations in the Plasmodium falciparum Kelch 13 (PfK13) protein are associated with artemisinin resistance. PfK13 is essential for asexual erythrocytic development, but its function is not known. We tagged the PfK13 protein with green fluorescent protein in P. falciparum to study its expression and localization in asexual and sexual stages. We used a new antibody against PfK13 to show that the PfK13 protein is expressed ubiquitously in both asexual erythrocytic stages and gametocytes and is localized in punctate structures, partially overlapping an endoplasmic reticulum marker. We introduced into the 3D7 strain four PfK13 mutations (F446I, N458Y, C469Y, and F495L) identified in parasites from the China-Myanmar border area and characterized the in vitro artemisinin response phenotypes of the mutants. We found that all the parasites with the introduced PfK13 mutations showed higher survival rates in the ring-stage survival assay (RSA) than the wild-type (WT) control, but only parasites with N458Y displayed a significantly higher RSA value (26.3%) than the WT control. After these PfK13 mutations were reverted back to the WT in field parasite isolates, all revertant parasites except those with the C469Y mutation showed significantly lower RSA values than their respective parental isolates. Although the 3D7 parasites with introduced F446I, the predominant PfK13 mutation in northern Myanmar, did not show significantly higher RSA values than the WT, they had prolonged ring-stage development and showed very little fitness cost in in vitro culture competition assays. In comparison, parasites with the N458Y mutations also had a prolonged ring stage and showed upregulated resistance pathways in response to artemisinin, but this mutation produced a significant fitness cost, potentially leading to their lower prevalence in the Greater Mekong subregion.IMPORTANCE Artemisinin resistance has emerged in Southeast Asia, endangering the substantial progress in malaria elimination worldwide. It is associated with mutations in the PfK13 protein, but how PfK13 mediates artemisinin resistance is not completely understood. Here we used a new antibody against PfK13 to show that the PfK13 protein is expressed in all stages of the asexual intraerythrocytic cycle as well as in gametocytes and is partially localized in the endoplasmic reticulum. By introducing four PfK13 mutations into the 3D7 strain and reverting these mutations in field parasite isolates, we determined the impacts of these mutations identified in the parasite populations from northern Myanmar on the ring stage using the in vitro ring survival assay. The introduction of the N458Y mutation into the 3D7 background significantly increased the survival rates of the ring-stage parasites but at the cost of the reduced fitness of the parasites. Introduction of the F446I mutation, the most prevalent PfK13 mutation in northern Myanmar, did not result in a significant increase in ring-stage survival after exposure to dihydroartemisinin (DHA), but these parasites showed extended ring-stage development. Further, parasites with the F446I mutation showed only a marginal loss of fitness, partially explaining its high frequency in northern Myanmar. Conversely, reverting all these mutations, except for the C469Y mutation, back to their respective wild types reduced the ring-stage survival of these isolates in response to in vitro DHA treatment.

中文翻译：

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缅甸东北部恶性疟原虫 Kelch 13 蛋白突变在介导青蒿素抗性中的作用。

恶性疟原虫 Kelch 13 (PfK13) 蛋白的突变与青蒿素抗性有关。PfK13 对于无性红细胞发育至关重要，但其功能尚不清楚。我们在恶性疟原虫中用绿色荧光蛋白标记 PfK13 蛋白，以研究其在无性和有性阶段的表达和定位。我们使用了一种针对 PfK13 的新抗体来显示 PfK13 蛋白在无性红细胞阶段和配子体中普遍表达，并且位于点状结构中，部分重叠内质网标记。我们将四个 PfK13 突变（F446I、N458Y、C469Y 和 F495L）引入到 3D7 菌株中，这些突变在中缅边境地区的寄生虫中鉴定出，并表征了这些突变体的体外青蒿素反应表型。我们发现所有引入 PfK13 突变的寄生虫在环期存活试验 (RSA) 中都显示出比野生型 (WT) 对照更高的存活率，但只有带有 N458Y 的寄生虫显示出显着更高的 RSA 值 (26.3%)比 WT 控制。在这些 PfK13 突变在田间寄生虫分离株中恢复为 WT 后，除具有 C469Y 突变的那些以外的所有回复寄生虫显示出显着低于其各自亲本分离株的 RSA 值。尽管在缅甸北部引入了 F446I 的 3D7 寄生虫（缅甸北部的主要 PfK13 突变）并没有显示出明显高于 WT 的 RSA 值，但它们具有延长的环期发育，并且在体外培养竞争测定中显示出非常小的适应性成本。相比下，具有 N458Y 突变的寄生虫也有一个延长的环形阶段，并显示出对青蒿素的反应上调的抗性途径，但这种突变产生了显着的健康成本，可能导致它们在大湄公河次区域的流行率较低。 东南亚出现了青蒿素抗性，危及全球消除疟疾的实质性进展。它与 PfK13 蛋白的突变有关，但 PfK13 如何介导青蒿素抗性尚不完全清楚。在这里，我们使用了一种针对 PfK13 的新抗体来显示 PfK13 蛋白在无性红细胞内循环的所有阶段以及配子细胞中表达，并且部分位于内质网中。通过将四个 PfK13 突变引入 3D7 菌株并在野外寄生虫分离物中恢复这些突变，我们使用体外环存活试验确定了这些突变在来自缅甸北部的寄生虫种群中对环阶段的影响。将 N458Y 突变引入 3D7 背景显着提高了环形阶段寄生虫的存活率，但代价是寄生虫的适应性降低。F446I 突变的引入是缅甸北部最普遍的 PfK13 突变，在暴露于双氢青蒿素 (DHA) 后并未显着增加环期存活率，但这些寄生虫表现出延长的环期发育。此外，带有 F446I 突变的寄生虫仅表现出轻微的适应性损失，部分解释了其在缅甸北部的高频率。相反，将所有这些突变（C469Y 突变除外）恢复为它们各自的野生型会降低这些分离株响应体外 DHA 治疗的环期存活率。