부산대학교 도서관

Determining the genetic basis of fitness is central to understanding evolution and transmission of microbial pathogens. In human malaria parasites (Plasmodium falciparum), most experimental work on fitness has focused on asexual blood stage parasites, because this stage can be easily cultured, although the transmission of malaria requires both female Anopheles mosquitoes and vertebrate hosts. We explore a powerful approach to identify the genetic determinants of parasite fitness across both invertebrate and vertebrate life-cycle stages of P. falciparum. This combines experimental genetic crosses using humanized mice, with selective whole genome amplification and pooled sequencing to determine genome-wide allele frequencies and identify genomic regions under selection across multiple lifecycle stages. We applied this approach to genetic crosses between artemisinin resistant (ART-R, kelch13-C580Y) and ART-sensitive (ART-S, kelch13-WT) parasites, recently isolated from Southeast Asian patients. Two striking results emerge: we observed (i) a strong genome-wide skew (>80%) towards alleles from the ART-R parent in the mosquito stage, that dropped to ~50% in the blood stage as selfed ART-R parasites were selected against; and (ii) repeatable allele specific skews in blood stage parasites with particularly strong selection (selection coefficient (s) ≤ 0.18/asexual cycle) against alleles from the ART-R parent at loci on chromosome 12 containing MRP2 and chromosome 14 containing ARPS10. This approach robustly identifies selected loci and has strong potential for identifying parasite genes that interact with the mosquito vector or compensatory loci involved in drug resistance. Malaria parasites are transmitted through female mosquitoes where gamete fusion and meiosis occurs, and humans where parasites proliferate asexually. Our work represents the first systematic analysis of malaria (Plasmodium falciparum) parasite fitness cross the complete life cycle, exploiting our ability to conduct genetic crosses in humanized mice. We use parasites recently isolated from Southeast Asia, the epicenter of the evolution and spread of P. falciparum resistance to the front line antimalarial, artemisinin. Our results provide possible insights into additional loci involved in resistance-associated malaria evolution and spread. The approach described here can be directly applied to study multiple selectable traits in the human parasite P. falciparum, such as parasite compatibility with different mosquito vectors, resistance to multiple drugs, and tolerance of temperature increase (fever). We also anticipate that this approach will accelerate genetic studies in other recombining parasites and pathogens. [ABSTRACT FROM AUTHOR]