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

Abstract

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.

Surprising variation in the outcome of two malaria genetic crosses using humanized mice: implications for genetic mapping and malaria biology

“…Our MKK2835 x NHP1337 cross between two recent field isolates, both from Southeast Asia, produced more selfed progeny than previously reported for P. falciparum genetic crosses. Interestingly, NHP1337 dominated the selfed progeny almost entirely, consistent with bulk allele frequencies in samples taken at similar times [33]. While efforts were made to infect the mosquitos with equal number of MKK2835 x NHP1337 gametocytes, the unequal selfing rates may reflect an imbalance in the initial gametocyte ratio or in gametocyte viability between MKK2835 and NHP1337.…”

“…Previous P. falciparum genetic crosses exhibited significant segregation distortion at several loci [13, 16, 17, 25]. We explored overlap between distorted regions in all the published P. falciparum crosses and our two new crosses and included a previously published bulk analysis of selection in uncloned progeny of the MKK2835xNHP1337 cross [33] (S2 Fig). We observe overlaps on chromsomes 12, 13 and 14.…”

“…We do not yet know if the large proportion of selfed clones observed in our cross between recent field isolates will be repeated in future crosses. Using bulk segregant analysis of these same populations we suspect that these selfed clones are outcompeted over time in non-stressed in vitro culture conditions [33] this may perhaps also have been the case in the previous 3D7 x HB3 cross [39, 40].…”

“…It will be interesting to explore weather pfarps10 has a fitness cost in this genetic background. Interestingly, while we see no segregation distortion in MKK2835 x NHP1337 among the cloned progeny, we do see selection on chromosome 14 over time in a uncloned bulk culture of MKK2835 x NHP1337 cross F 1 progeny that is also centered on pfarps10 where selection is against the derived alleles in pfarps10 [33].…”

“…It is possible that pf47 and/or pfs45/48 play a key role in segregation distortion in more distantly related lines but not in a cross between allopatric recent clinical isolates. Indeed, we observed no significant segregation distortion in the MKK2835 cross and also observed no selection over time on chromosome 13 in the bulk segregant experiment using the MKK2835 x NHP1337 bulk F 1 progeny [33].…”