Ferdig, M. | Search bioRxiv

bioRxiv2020

Bulk Segregant Approaches to Nutritional Genomics in Plasmodium falciparum

What genes determine growth and nutrient utilization in asexual blood-stage malaria parasites? Competition experiments between a lab-adapted African parasite (NF54), and a recently isolated Asian parasite (NHP4026) reveal contrasting outcomes in different media: NF54 outcompetes NHP4026 in media containing human serum, while NHP4026 outcompetes NF54 in media containing AlbuMAX, a lipid-rich bovine serum formulation. We conducted parasite genetic crosses and compared genome-wide allele frequency changes in progeny populations cultured in media containing serum or AlbuMAX: this bulk segregant analysis (BSA) reveals three quantitative trait loci (QTL) underlying differential growth. The strongest QTL (chromosome 13) contains EBA-140: competition experiments between EBA-140-knockout and isogenic wildtype parasites showed fitness reversals in the two media types, validating this locus as the causative gene. These results (i) demonstrate the effectiveness of BSA for dissecting fitness traits in Plasmodium falciparum, and (ii) reveal an intimate link between red blood cell invasion and nutrient composition of growth media.

genetics↗

bioRxiv2019

Genetic mapping of fitness determinants across the malaria parasite Plasmodium falciparum life cycle

Malaria is transmitted through female Anopheline mosquitoes where gamete fusion and meiosis occurs, and humans where parasites proliferate asexually. We describe a powerful approach to identify the genetic determinants of parasite fitness across both invertebrate and vertebrate life-cycle stages in human malaria parasite Plasmodium falciparum using bulk segregant analysis (BSA). We combined experimental genetic crosses using humanized mice, with selective whole genome amplification and BSA at multiple developmental stages in both mosquito and vertebrate host to examine parasite competition and identify genomic regions under selection. We generated crosses between artemisinin resistant (ART-R, kelch13-C580Y) and ART-sensitive (ART-S, kelch13-WT) parasite clones recently isolated from Southeast Asian patients. We then quantified genome-wide changes in allele frequency in the parasite progeny population from infected midgut and salivary glands of Anopheles stephensi mosquitoes, infected livers, emerging merozoites and aliquots of in vitro cultured progeny parasites at intervals over 30 days. Three striking results emerge: we observed (i) a strong 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; (ii) highly repeatable skews in allele frequencies across the genome in blood stage parasites; (iii) 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.

evolutionary biology↗