Single-cell functional and population genomic analysis of Plasmodium knowlesi malaria parasites

摘要对寄生虫的基因组分析可以帮助我们了解它们对不同宿主的适应机制。感染人类的诺氏疟原虫有不同的遗传类型，现在发现与不同的猕猴宿主物种有关。序列分析揭示了这些类型之间的大量全基因组差异，尽管这在某些染色体区域特别高。诺氏疟原虫的分析将通过流式细胞术从猕猴和人类新鲜分离物的血液中进行分选，序列分析的结果将有助于设计遗传操作实验，以测试参与宿主适应和可能增强生殖隔离的位点。寄生虫的基因组和功能研究有助于发现适应不同寄主物种的机制。人畜共患寄生虫诺氏疟原虫越来越普遍地被视为东南亚人类疟疾的一个病因，其不同的遗传类型（称为聚类1和聚类2）现在被发现主要与不同的猴宿主物种（分别为长尾猕猴和长尾猕猴）有关。序列分析揭示了这些类型之间的全基因组差异，其水平相当于亚种，尽管在这些类型之间存在更高水平的固定差异的染色体区域。许多感染包含多种基因型，猕猴感染经常包含额外的寄生虫种类。该项目将通过流式细胞术对猕猴和人类新鲜分离的血液进行诺氏疟原虫的分析，并对单个细胞和单个分类细胞池进行测序。对寄生虫的分析将来自天然猕猴和人类感染，并且可能还包括对来自受感染蚊子的寄生虫的分析，以检验媒介对不同的诺氏疟原虫类型的特异性易感性的假设。将选择人类感染样本，包括：(a)第1类寄生虫，(b)第2类寄生虫，和(c)推定混合型感染。来自猕猴的样本将被分别选择，这些样本要么有第1类寄生虫，要么有第2类寄生虫——这将是首次尝试从野生猕猴寄生虫中获得全基因组序列。长期存在于恒河猴体内的“H株”寄生虫已经适应了在人类红细胞中生长——它不同于第1和第2簇寄生虫，将用于等位基因替代研究。技术：寄生虫的流式细胞分选和克隆；基因组测序;转录组分析;寄生虫的遗传操作；基因结构和功能的生物信息学分析

Strategic Research Priority: Bioscience for HealthAbstract Genomic analyses of parasites can give insights into mechanisms of adaptation to different hosts. There are divergent genetic types of Plasmodium knowlesi infecting humans, now discovered to be associated with different macaque monkey reservoir host species. Sequence analysis reveals substantial genome-wide divergence between these types, although this is particularly high in some chromosomal regions. Analysis of P. knowlesi will be undertaken by flow cytometric sorting from blood of macaque and human fresh isolates, and results of sequence analyses will enable design of genetic manipulation experiments to test for loci that are involved in host adaptation and potentially enhancing reproductive isolation. Project Genomic and functional studies of parasites enable discovery of mechanisms of adaptation to different host species. The zoonotic parasite Plasmodium knowlesi is increasingly commonly seen as a cause of malaria in humans in Southeast Asia, with divergent genetic types (termed Cluster 1 and Cluster 2) now discovered to be predominantly associated with different monkey reservoir host species (long-tailed and pig-tailed macaques respectively). Sequence analysis reveals genome-wide divergence between these types of a level equating to sub-species, although there are chromosomal regions with even higher levels of fixed differences between the types. Many infections contain multiple genotypes, and macaque infections frequently contain additional parasite species. This project will undertake analysis of P. knowlesi by flow cytometric sorting from blood of macaque and human fresh isolates, and sequencing from single cells and pools of individual sorted cells. The analyses of parasites will be from natural macaque as well as human infections, and may potentially also involve analysis of parasites from infected mosquitoes to test a hypothesis of vector-specific susceptibility to the divergent P. knowlesi types. Samples of human infections will be selected to include : (a) Cluster 1 type parasites, (b) Cluster 2 type parasites, and (c) putatively mixed type infections. Samples from macaques will be separately chosen that have either Cluster 1 or Cluster 2 parasites - these will be the first full genome sequences attempted from wild macaque parasites. The 'H strain' parasite long maintained in rhesus macaques has been adapted for growth in human erythrocytes - this is divergent from both cluster 1 and cluster 2 parasites and will be used for allelic replacement studies. Techniques: Flow cytometric sorting and cloning of parasites; Genome sequencing; Transcriptome analysis; Genetic manipulation of parasites; Bioinformatic analyses of gene structure and function