Evaluating multiple loci modulating susceptibility of African malaria parasites to artemisinin

自21世纪初以来，全球部署了治疗疟疾的联合药物，带来了巨大的公共卫生效益。特别是非洲各地的疟疾死亡人数自20世纪90年代达到顶峰以来急剧下降，当时氯喹和磺胺嘧啶-乙胺嘧啶药物的失败导致婴儿和儿童死亡人数上升。在目前所有有效的药物组合中，一个关键成分是一种叫做青蒿素的植物衍生化学物质。青蒿素杀灭寄生虫的能力最近受到湄公河地区出现的易感性降低的威胁：这表现为青蒿素清除经治疗的疟疾患者血液中的恶性疟原虫的速度减慢。虽然在联合治疗下，这些患者在大多数情况下最终应该完全治愈，但在该地区，联合治疗药物在一些患者中也失败了。因此，需要仔细监测和警惕疟疾耐药性。在柬埔寨及其周边地区发现了一种导致青蒿素药物有效性丧失的寄生虫基因——这种基因编码kelch结构域蛋白，被称为K13，它已经积累了多种突变。这与青蒿素对这些寄生虫失去效力密切相关。然而，在非洲疟疾寄生虫中还没有观察到同样的现象——在药物治疗不起作用的少数病例中，K13突变没有被认为是原因。在过去的2年中，我们和其他人的基因编辑研究成功地证明了3个基因对降低青蒿素敏感性有直接作用：pfap2mu、pfubp1和pfcoronin。在环期青蒿素存活试验（RSA）中，这些基因的变异引起体外寄生虫存活增加。在转运接头蛋白亚基AP-2mu的情况下，我们已经证明该蛋白在恶性疟原虫中的作用与其他生物不同，因为它不与网格蛋白相互作用，但与细胞中似乎也含有K13的未知区室相关，尽管我们没有发现AP-2mu与K13蛋白直接相互作用的证据。然而，有很好的证据表明AP-2mu与另一种kelch蛋白K10直接相互作用。编码K10蛋白的基因多态性先前已被确定为湄公河地区寄生虫的遗传成分，该地区最初产生了k13依赖性的青蒿素敏感性降低。这项工作是由博士生Ryan Henrici进行的，他于2018年完成了博士学业，并继续前进。我们现在请求支持来扩展这项工作，使用基因编辑来测试新突变的影响。在田间样本和英国分离株的初步研究中，我们已经在我们目前感兴趣的五个基因——pfk13、pfap2mu、pfubp1、pfcoronin和pfk10中发现了许多这样的新突变（见支持案例）。对于每一种适应培养的新分离物（来自英国或来自我们的合作者，加纳Ho的Bismarck Dinko博士），我们将充分测试药物敏感性，并使用基因组测序对其进行遗传表征。在基因编辑实验中，我们将直接测量每种候选基因变异对已建立的实验室品系和来自英国和加纳患者的新文化的影响；因此，任何被证明产生药物敏感性表型的变异都可能有助于改变药物敏感性模式，并可开发为疟疾流行地区使用的监测标记。因此，我们的工作将有助于我们了解对目前联合药物的耐药性，并帮助我们制定在英国和非洲谨慎部署这些药物的策略，以最大限度地延长它们在治疗疟疾患者方面的使用寿命。

Combination drugs for treatment of malaria deployed globally since the start of the 21st century have provided a substantial public health benefit. Malaria deaths across Africa in particular have plummeted since a peak in the 1990s, when failure of the drugs chloroquine and sulphadoxine-pyrimethamine precipitated a rise in infant and child deaths. A key component in all the currently effective drug combinations is the plant-derived chemical called artemisinin. The parasite-killing ability of artemisinin has recently been threatened by the emergence of reduced susceptibility in the Mekong region: this is manifest as a slowing of artemisinin clearance of P. falciparum from the blood of treated malaria patients. Although under combination treatments these patients should eventually experience a full cure in most cases, in this region the combination partner drugs are also failing in some patients. Careful monitoring and vigilance of malaria drug resistance is therefore needed. A parasite gene has been identified in Cambodia and the surrounding region which contributes to the loss of effectiveness of the artemisinin drug - this gene, which encodes a kelch-domain protein and is called K13, has accumulated a variety of mutations. These are strongly associated with the loss of effectiveness of artemisinin against these parasites. However, this same phenomenon has not yet been observed in African malaria parasites - in the small number of cases where drug treatment does not work, K13 mutations are not implicated as the cause. In the past 2 years, gene-editing studies by ourselves and others have successfully demonstrated a direct effect of 3 genes in reducing susceptibility to artemisinin : pfap2mu, pfubp1 and pfcoronin. Variants of these genes elicit increased parasite survival in vitro in the ring-stage artemisinin survival assay (RSA). In the case of trafficking adaptor potein subunit AP-2mu, we have shown that the role of this protein in P. falciparum is unlike in other organisms as it does not interact with clathrin, but is associated with an as yet unknown compartment in the cell that appears to also contain K13, although we found no evidence of direct interaction between AP-2mu and the K13 protein. There is, however, good evidence of direct interaction of AP-2mu with a different kelch protein , K10. Polymorphisms in the gene encoding the K10 protein have been previously identified as a genetic component of parasites from the Mekong region in which the K13-dependent reduced artemisinin susceptibility originally arose. This work was carried out by PhD student Ryan Henrici, who completed his doctoral studies in 2018 and has moved on. We now request support to extend this work, using gene editing to test the effect of new mutations. In preliminary studies of both field samples and UK isolates, we have already identified a number of such new mutations in our current five genes of interest - pfk13, pfap2mu, pfubp1, pfcoronin and pfk10 (see Case for Support). For each new isolate (either from the UK or from our collaborator Dr Bismarck Dinko in Ho, Ghana) that is adapted to culture, we will fully test drug susceptibility, and genetically characterise them using genome sequencing. In gene-editing experiments we will then directly measure the impact of each candidate gene variant in both established laboratory lines and in our new cultures from UK and Ghanaian patients; any variant proven to generate a drug susceptibility phenotype may therefore be contributing to changing patterns of drug susceptibility, and could be developed as a surveillance marker for use in malaria endemic areas. Our work will thus assist our understanding of resistance to our current combination drugs, and help us to devise strategies for deploying these drugs carefully in both the UK and Africa, to maximise their useful life in curing malaria patients.