Insights into the intracellular localization, protein associations and artemisinin resistance properties of Plasmodium falciparum K13
Insights into the intracellular localization, protein associations and artemisinin resistance properties of Plasmodium falciparum K13
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DOI：
10.1371/journal.ppat.1008482
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发表时间：
2020-04-01
期刊：
影响因子：
6.7
通讯作者：
Fidock, David A.
中科院分区：
文献类型：
--
作者：
Gnadig, Nina F.;Stokes, Barbara H.;Fidock, David A.
恶性疟原虫耐药性的发展对全球抗击疟疾的斗争构成了重大障碍。目前一线药物的核心成分——青蒿素部分耐药已席卷东南亚。在恶性疟原虫感染患者中，抗逆转录病毒药物耐药性寄生虫在接受抗逆转录病毒衍生物或基于抗逆转录病毒药物的联合治疗后清除率缓慢。东南亚也出现了对伴侣药物的耐药性，导致治疗经常失败。恶性疟原虫K13蛋白的单氨基酸突变是抗逆转录病毒药物耐药性的主要遗传原因，并预示着治疗失败的风险增加。通过产生单克隆抗体，我们研究了K13在双氢青蒿素处理或未处理的寄生虫中的亚细胞定位。分析显微镜数据表明，K13位于或靠近内质网和介导细胞内运输的囊泡，包括向寄生虫输入宿主血红蛋白的质膜相关细胞口。K13特异性单克隆抗体的共免疫沉淀实验鉴定了与内质网、囊泡运输、细胞质或线粒体相关的多种蛋白质，K13突变型和野生型寄生虫之间没有明显差异。我们还观察到K13突变体寄生虫中突变型或野生型K13的过度表达可以恢复易感性，支持K13突变导致功能丧失的假设。恶性疟原虫（Plasmodium falciparum）红细胞内寄生虫出现青蒿素耐药性，导致东南亚以青蒿素为基础的一线联合治疗失败率上升。寄生虫易感性降低是由K13突变引起的，这在临床上与患者体内寄生虫清除延迟有关，在体外则与环期寄生虫短暂暴露于活性抗逆转录病毒药物代谢物双氢青蒿素的生存能力增强有关。在这里，我们描述了一组K13特异性单克隆抗体和基因编辑的寄生虫系，它们在反式中共同表达K13的表位标记版本。通过应用分析定量成像管道，我们将K13定位于寄生虫内质网、rabb阳性囊泡和邻近细胞口的部位。后一种结构在寄生虫的质膜上形成，并将血红蛋白运送到消化液泡，在那里释放青蒿素激活血红素部分。我们还提供证据表明，在用双氢青蒿素治疗后，K13部分定位在寄生虫线粒体附近。K13特异性单克隆抗体产生的免疫沉淀数据在K13突变型和野生型等基因系中鉴定出多种推测的K13相关蛋白，包括内质网居住分子、线粒体蛋白和Rab gtpase。我们还发现突变型K13介导的抗性在野生型或突变型K13的共同表达下是相反的。这些数据有助于确定K13的生物学特性及其在介导恶性疟原虫对抗逆转录病毒治疗的耐药性中的作用。
Author summaryThe development of drug resistance in Plasmodium falciparum parasites presents a significant impediment to the global fight against malaria. Partial resistance to artemisinin (ART), the core component of current first-line drugs, has swept across Southeast Asia. In P. falciparum-infected patients, ART-resistant parasites show slow rates of clearance following treatment with an ART derivative or ART-based combination therapy. Resistance to partner drugs has also emerged in Southeast Asia, leading to frequent treatment failures. Single amino acid mutations in the P. falciparum K13 protein constitute the primary genetic cause of ART resistance and predict an increased risk of treatment failure. By generating monoclonal antibodies, we have investigated the subcellular localization of K13 in dihydroartemisinin-treated or untreated parasites. Analytical microscopy data localize K13 to or near the endoplasmic reticulum and vesicles that mediate intracellular trafficking, including plasma membrane-associated cytostomes that import host hemoglobin into the parasite. Co-immunoprecipitation experiments with K13-specific monoclonal antibodies identified multiple proteins associated with the endoplasmic reticulum, vesicular trafficking, the cytosol, or the mitochondria, with no apparent differences between K13 mutant and wild-type parasites. We also observed that overexpression of mutant or wild-type K13 in K13 mutant parasites could restore susceptibility, supporting the hypothesis that K13 mutations cause loss of function.The emergence of artemisinin (ART) resistance in Plasmodium falciparum intra-erythrocytic parasites has led to increasing treatment failure rates with first-line ART-based combination therapies in Southeast Asia. Decreased parasite susceptibility is caused by K13 mutations, which are associated clinically with delayed parasite clearance in patients and in vitro with an enhanced ability of ring-stage parasites to survive brief exposure to the active ART metabolite dihydroartemisinin. Herein, we describe a panel of K13-specific monoclonal antibodies and gene-edited parasite lines co-expressing epitope-tagged versions of K13 in trans. By applying an analytical quantitative imaging pipeline, we localize K13 to the parasite endoplasmic reticulum, Rab-positive vesicles, and sites adjacent to cytostomes. These latter structures form at the parasite plasma membrane and traffic hemoglobin to the digestive vacuole wherein artemisinin-activating heme moieties are released. We also provide evidence of K13 partially localizing near the parasite mitochondria upon treatment with dihydroartemisinin. Immunoprecipitation data generated with K13-specific monoclonal antibodies identify multiple putative K13-associated proteins, including endoplasmic reticulum-resident molecules, mitochondrial proteins, and Rab GTPases, in both K13 mutant and wild-type isogenic lines. We also find that mutant K13-mediated resistance is reversed upon co-expression of wild-type or mutant K13. These data help define the biological properties of K13 and its role in mediating P. falciparum resistance to ART treatment.