Next-generation sequencing of barcoded Plasmodium falciparum mutants to dissect parasite fitness costs associated with drug resistance

对带条形码的恶性疟原虫突变体进行下一代测序，以剖析与耐药性相关的寄生虫适应性成本

• 批准号: 1789702
• 金额: --
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1789702
• 关键词: Next; generation; sequencing; barcoded; Plasmodium

Malaria is a disease that infected 214 million people in 2015, with 438000 of these infections being fatal. Although this is a great improvement on recent years, many challenges to the effective treatment and elimination of Malaria remain. One of the greatest challenges is the parasite's ability rapidly to develop resistance to antimalarial drugs. To date, the parasite developed resistance to every drug that it has ever been challenged with, and resistance has recently been observed to current first-line treatment drugs, artemisinin-based combination therapies. GSK has developed millions of compounds, the TCAMs, with diverse chemically active backbones, many of which target P.falciparum in cellular screens. One strategy to slow the development of resistance is to continually identify new drugs with novel targets within the parasite.Another important part of combating drug resistance is to understand how resistance develops, and how it affects the fitness of the parasite. Many mutations that confer resistance to antimalarial drugs, can have a deleterious effect on parasite fitness. There is a very delicate balance between resistance to antimalarial compounds and the optimal function of the pathway that the mutation affects. Often after a parasite stops being exposed to a drug the resistance mutation soon disappears from the population. Currently, there is no standard experiment for measuring mutant parasite fitness.Until recently the study of resistance by large-scale genome editing was very inefficient, due to the lack of tools to edit the genome in P.falciparum. The discovery of CRISPR/Cas9 however, has vastly increased the rate at which editing can occur. In this project, CRISPR will be used to introduce resistant mutations found in field isolates of P. falciparum into the respective alleles of lab strains with the same background. The known resistant mutations generated in key genes involved in drug resistance including PfCRT (chloroquine), cytBC1 (atovaquone), kelch13 (artemisinin), and DHFR (pyrimethamine) - as well as targets of new compounds under development or in clinical trials (e.g. PfATP4, PfPI4K, PfCARL, PfeEF2). A limited number of double mutations will also be created (e.g. PfATP4/PfCDPK5 or PfPI4K/PfRab11A). It is expect that a library of about 50 mutant parasite lines would be produced in all.The mutant parasite lines would be distinguished from one another by utilizing bar-seq technology. A series of unique eleven base pair barcodes would be inserted into the Rh3 locus of the parasite lines. In this way by deep sequencing, different mutant parasites can be distinguished from one another, while they grow in competition with one another in the same culture, under identical conditions. Parallel quantification of barcoded lines by next-generation sequencing, will pinpoint resistance mutations that confer the greatest fitness cost, identifying biological pathways that tolerate mutation and those that do not.This library of pooled mutants will allow profiling of resistance to novel antimalarial compounds from the GSK TCAMs. If any resistance mutation already exists to this compound this can quickly be discerned by sequencing the bar code of the surviving parasites. On the other hand, compounds that inhibit the growth of all the parasites in the pool probably target novel pathways and would require further investigation into their mode of action. The general fitness of these mutant parasites will also be assessed by growing them together under as a variety of challenge conditions including oxidative stress, and low nutrient media.

疟疾是一种疾病，2015年感染了2.14亿人，其中438000是致命的。尽管这与近几年相比有了很大的改善，但有效治疗和消除疟疾仍然面临许多挑战。最大的挑战之一是寄生虫对抗疟疾药物迅速产生抗药性的能力。到目前为止，这种寄生虫对曾经挑战过的每一种药物都产生了抗药性，最近观察到对目前的一线治疗药物--基于青蒿素的联合疗法--的抗药性。葛兰素史克已经开发了数百万种化合物，即TCAMS，它们具有不同的化学活性骨架，其中许多化合物在细胞屏幕上针对恶性疟原虫。减缓耐药性发展的一个策略是不断识别寄生虫内具有新靶点的新药。抗击耐药性的另一个重要部分是了解耐药性是如何发展的，以及它如何影响寄生虫的适合性。许多对抗疟疾药物产生抗药性的突变可能会对寄生虫的适应能力产生有害影响。在对抗疟疾化合物的抗药性和突变影响的途径的最佳功能之间存在着非常微妙的平衡。通常，在寄生虫停止接触药物后，抗药性突变很快就会从种群中消失。目前还没有标准的实验来衡量突变寄生虫的适合度，直到最近，由于缺乏编辑恶性疟原虫基因组的工具，通过大规模基因组编辑来研究抗药性的效率很低。然而，CRISPR/Cas9的发现极大地提高了编辑发生的速度。在本项目中，CRISPR将被用于将在恶性疟原虫野外分离株中发现的抗药性突变引入具有相同背景的实验室菌株的各自的等位基因中。已知的与耐药性有关的关键基因产生的耐药突变包括PfCRT(氯喹)、cytBc1(阿托瓦酮)、kelch13(青蒿素)和dhfr(乙胺嘧啶)-以及正在开发或临床试验的新化合物的靶标(例如PfATP4、PfPI4K、PfCARL、PfeEF2)。还将产生数量有限的双突变(例如PfATP4/PfCDPK5或PfPI4K/PfRab11A)。预计总共将产生约50个突变寄生虫系的文库。利用BAR-SEQ技术将突变寄生虫系彼此区分开来。一系列独特的11个碱基对的条形码将被插入寄生虫系的Rh3基因座。通过这种方式，通过深度测序，不同的突变寄生虫可以相互区分，同时它们在相同的培养条件下竞争生长。通过下一代测序对条形码线进行并行量化，将精确定位赋予最大适合度成本的抗药性突变，识别耐受突变和不耐受突变的生物途径。这个汇集的突变库将允许对来自GSK TCAM的新型抗疟疾化合物的耐药性进行分析。如果这种化合物已经存在任何抗药性突变，则可以通过对幸存寄生虫的条形码进行测序来快速识别。另一方面，抑制池中所有寄生虫生长的化合物可能针对新的途径，需要进一步研究它们的作用模式。这些突变寄生虫的一般适合性也将通过在包括氧化应激和低营养介质在内的各种挑战条件下一起生长来评估。