Culture adaptation of malaria parasites selects for convergent loss-of-function mutants

Abstract

Cultured human pathogens may differ significantly from source populations. To investigate the genetic basis of laboratory adaptation in malaria parasites, clinical Plasmodium falciparum isolates were sampled from patients and cultured in vitro for up to three months. Genome sequence analysis was performed on multiple culture time point samples from six monoclonal isolates, and single nucleotide polymorphism (SNP) variants emerging over time were detected. Out of a total of five positively selected SNPs, four represented nonsense mutations resulting in stop codons, three of these in a single ApiAP2 transcription factor gene, and one in SRPK1. To survey further for nonsense mutants associated with culture, genome sequences of eleven long-term laboratory-adapted parasite strains were examined, revealing four independently acquired nonsense mutations in two other ApiAP2 genes, and five in Epac. No mutants of these genes exist in a large database of parasite sequences from uncultured clinical samples. This implicates putative master regulator genes in which multiple independent stop codon mutations have convergently led to culture adaptation, affecting most laboratory lines of P. falciparum. Understanding the adaptive processes should guide development of experimental models, which could include targeted gene disruption to adapt fastidious malaria parasite species to culture.

Multiplication rate variation in the human malaria parasite Plasmodium falciparum

“…Many parasites that have been in culture for a long time are loss-of-function mutants, with particular genes having acquired premature stop codons 15 , or having been lost in subtelomeric deletions 16 . Determining the true range of variation in asexual multiplication rate of P. falciparum would be an important step in being able to identify determinants of growth and virulence factors.…”

Single-cell RNA sequencing reveals a signature of sexual commitment in malaria parasites

“…Six of these shared hits have been linked to interaction with the host cell either via protein export or during the reinvasion process, while seven putatively function in gene regulation based on their predicted interaction with either RNA, DNA, or chromatin. These included two additional ApiAP2-family members, PF3D7_1139300 and PF3D7_1222400, a primate specific ApiAP2 recently found to acquire loss of function mutations in laboratory-adapted strains 17 . Four additional hits are implicated in the regulation of chromatin structure: two SNF2 family helicases (ISWI, SNF2L) that function in chromatin remodeling 18 and two putative histone modifying enzymes: the histone deacetylase HDA1 7 , and a previously un-annotated putative histone lysine-specific demethylase PfLSD2 (PF3D7_0801900, see Extended Data Fig.…”

Plasmodium falciparum schizont stage transcriptome variation among clinical isolates and laboratory-adapted clones

“…In transcriptomic studies of malaria parasites, achieving large numbers of replicate sample preparations is difficult, particularly from clinical isolates [5], and most current understanding is derived from a small number of parasite lines that have been culture-adapted for many years. Examination of genome sequences has identified premature stop codon mutants affecting some transcriptional factor genes and cell signalling protein genes [20,21], as well as particular gene deletions and amplifications only observed in parasites adapted to in vitro culture [22,23]. Although culture-adapted parasites are phenotypically and transcriptionally diverse [6,12,24], it is not clear to what extent they reflect the diversity of parasites causing clinical malaria.…”

“…Much of our understanding of malaria parasite biology is derived from parasite lines that have been adapted to in vitro culture for decades [27-30]. Genome sequencing studies have identified polymorphisms in transcriptional factors and cell signalling proteins [31, 32], as well as genetic deletions and amplifications [33, 34] that are associated with adaptation of parasites to in vitro culture. While laboratory-adapted isolates are antigenically and transcriptionally diverse [3, 17, 26, 35, 36], it is not clear to what extent they reflect the diversity among ex vivo parasites.…”