Evaluating multiple loci modulating susceptibility of African malaria parasites to artemisinin

自21世纪世纪以来，全球使用的治疗疟疾的复方药物已经提供了大量的公共卫生益处。特别是非洲各地的疟疾死亡人数自20世纪90年代达到高峰以来大幅下降，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。目前所有有效的药物组合中的一个关键成分是一种名为青蒿素的植物衍生化学物质。青蒿素杀死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：这表现为青蒿素从接受治疗的疟疾患者血液中清除恶性疟原虫的速度减慢。尽管在联合治疗下，这些患者在大多数情况下最终应该完全治愈，但在该地区，联合伴侣药物在一些患者中也失败了。因此，需要认真监测和警惕疟疾抗药性。在柬埔寨及其周边地区发现了一种寄生虫基因，这种基因导致青蒿素药物失去效力-这种基因编码一种kelch结构域蛋白，称为K13，已经积累了各种突变。这些与青蒿素对这些寄生虫的有效性丧失密切相关。然而，在非洲疟疾寄生虫中尚未观察到同样的现象-在少数药物治疗不起作用的病例中，K13突变并不是原因。在过去的两年里，我们和其他人的基因编辑研究成功地证明了3个基因在降低青蒿素敏感性方面的直接作用：pfap 2 mu，pfubp 1和pfcoronin。这些基因的变体在环阶段青蒿素存活测定（RSA）中引起体外寄生虫存活增加。在运输衔接子蛋白亚基AP-2 mu的情况下，我们已经表明，这种蛋白质在恶性疟原虫中的作用与其他生物体不同，因为它不与网格蛋白相互作用，但与细胞中似乎也含有K13的未知区室有关，尽管我们没有发现AP-2 mu和K13蛋白之间直接相互作用的证据。然而，有很好的证据表明AP-2 mu与不同的kelch蛋白K10直接相互作用。编码K10蛋白的基因中的多态性先前已被鉴定为来自湄公河地区的寄生虫的遗传组分，其中K13依赖性降低的青蒿素敏感性最初出现。这项工作是由博士生Ryan Henrici完成的，他于2018年完成了博士研究并继续前进。我们现在请求支持扩展这项工作，使用基因编辑来测试新突变的影响。在对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中发现了许多这样的新突变-pfk 13，pfap 2 mu，pfubp 1，pfcoronin和pfk 10（见支持病例）。对于每一个适应培养的新分离株（无论是来自英国还是来自我们的合作者俾斯麦丁科博士在加纳的Ho），我们将全面测试药物敏感性，并使用基因组测序对其进行遗传测序。在基因编辑实验中，我们将直接测量每个候选基因变体在已建立的实验室品系和我们来自英国和加纳患者的新培养物中的影响;因此，任何被证明产生药物敏感性表型的变体都可能有助于改变药物敏感性模式，并可被开发为疟疾流行地区的监测标志物。因此，我们的工作将有助于我们了解对我们目前组合药物的耐药性，并帮助我们制定在英国和非洲谨慎部署这些药物的策略，以最大限度地延长其治疗疟疾患者的使用寿命。

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.