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

疟疾是一种在2015年感染了2.14亿人的疾病，其中43.8万人是致命的。虽然这比近年来有了很大改善，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能够迅速对抗疟药物产生抗药性。迄今为止，该寄生虫对它曾经受到挑战的每一种药物都产生了抗药性，最近还观察到对目前的一线治疗药物，即青蒿素综合疗法的抗药性。GSK已经开发了数百万种具有不同化学活性骨架的化合物TCAM，其中许多化合物在细胞筛选中靶向恶性疟原虫。减缓耐药性发展的一个策略是不断地在寄生虫体内发现具有新靶点的新药。对抗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应性。许多使抗疟药物产生抗药性的突变会对寄生虫的适应性产生有害影响。在抗疟化合物的耐药性和突变影响的途径的最佳功能之间存在非常微妙的平衡。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测量突变体寄生虫fitness.直到最近，通过大规模基因组编辑的抗性研究是非常低效的，由于缺乏编辑恶性疟原虫基因组的工具。然而，CRISPR/Cas9的发现大大提高了编辑的速度。在该项目中，CRISPR将用于将恶性疟原虫田间分离株中发现的抗性突变引入具有相同背景的实验室菌株的相应等位基因中。已知的耐药突变产生于涉及耐药性的关键基因，包括PfCRT（氯喹），cytBC 1（阿托伐醌），kelch 13（青蒿素）和DHFR（乙胺嘧啶）-以及正在开发或临床试验中的新化合物的靶点（例如PfATP 4，PfPI 4K，PfCARL，PfeEF 2）。还将产生有限数量的双突变（例如PfATP 4/PfCDPK 5或PfPI 4 K/PfRab 11 A）。预计总共将产生约50个突变体寄生虫系的文库，利用bar-seq技术将突变体寄生虫系彼此区分开。将一系列独特的11个碱基对条形码插入寄生虫系的Rh 3基因座中。通过这种方式，通过深度测序，不同的突变寄生虫可以彼此区分，而它们在相同的培养物中，在相同的条件下相互竞争生长。通过下一代测序技术对条形码化株系进行平行定量，将精确定位产生最大适应性成本的耐药突变，识别耐受突变和不耐受突变的生物学途径。这种汇集的突变体库将允许对GSK TCAM的新型抗疟化合物进行耐药性分析。如果已经存在对该化合物的任何抗性突变，则可以通过对存活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，需要进一步研究其作用模式。还将通过在各种挑战条件下（包括氧化应激和低营养培养基）将这些突变体寄生虫一起生长来评估它们的一般适应性。

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.