The genetic dissection of differential growth in Plasmodium falciparum and its relationship to chloroquine drug selection

Plasmodium falciparum malaria is responsible for severe morbidity and mortality throughout the world. Anti-malarials used to treat malaria, such as chloroquine (CQ), exhibit increased failure and have an effect on the parasite genome potentially resulting in differences in parasite growth. Differences in erythrocytic growth rates between parasite clones affect parasite virulence (Chotivanich et al., 2000; Dondorp et al., 2005). Parasite growth is complex, composed of discrete biological steps contributing to differences in overall 'growth.' To identify genes and mechanisms regulating differential growth, a series of phenotypes ranging from bulk growth (e.g. blood smears and Hx-incorporation) to its nested steps (merozoite production, invasion efficiency, and cycle time) were measured in progeny of a cross between a CQ resistant and CQ sensitive parasite, Dd2 and HB3 respectively, and used in quantitative trait loci (QTL) mapping. Progeny exhibit quantitative distributions of growth in all five growth assays. Significant QTLs from the two bulk growth assays and cycle time produce a preliminary list of 167 candidate genes. An unbiased method using whole genome datasets and bioinformatics was developed to prioritize the list. Putative functions were assigned using protein network datasets, gene expression correlations, and gene ontology enrichment resulting in a final list of 22 candidates.

A relationship between parasite growth and drug sensitivity is observed in CQR parasites but not CQS parasites; suggessting CQ selection has altered the parasite genome and parasite fitness as measured by growth. Progeny inheriting 'D' alleles at three random markers are absent less often compared to progeny inheriting mixed alleles. This suggests the Dd2 genome is fitter. When progeny inherit a resistant pfcrt allele, there is an increase in the percent of absent progeny with 'D' alleles at three markers. Therefore CQ resistant progeny suffer from a fitness cost. Specific alleles implicated in the 'CQR background' were used to ask about parasite fitness and lethality support the idea that CQ selection alters the parasite genome by causing compensatory mutations in response to a cost to resistance. These compensatory mutations affect other parasite phenotypes such as growth. This hypothesis is further supported by observed growth differences in CQ-selected mutant lines.