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

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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