Authors

Discussion

Point mutations in the endoplasmic reticulum (ER) and Golgi-associated transporters CARL, UGT, and ACT have been identified as key mediators of resistance to imidazolopiperazines, such as KAF156 (ganaplacide) and GNF179. In Plasmodium falciparum, the cyclic amine resistance locus (pfcarl) encodes a highly conserved protein characterized by seven transmembrane domains and precise localization to the cis-Golgi apparatus. Although its exact physiological role remains elusive, CARL is implicated in broader multidrug resistance mechanisms across different antimalarial classes. To investigate these dynamics in the Plasmodium berghei model, CRISPR-Cas9 was employed to introduce equivalent single nucleotide polymorphisms (SNPs) observed in pfcarl into the orthologous gene pbcarl (PBANKA_1216600).  While attempts to introduce the P708L and V948L mutations indicated they were lethal to the parasite - a conclusion supported by the successful recovery of only silent control mutations (P708P and V948V) - the S921I substitution was successfully established. Phenotypic analysis of the S921I variant revealed no observable defects in gametocyte development or ookinete formation either in vitro or in vivo. However, the mutation appeared to impose a significant fitness cost; mutant parasites were rapidly outcompeted by wild-type strains within seven days and failed to demonstrate successful transmission. Most notably, while the S921I mutation conferred an insignificant 1.87-fold shift in sensitivity to dihydroartemisinin (DHA), it resulted in a profound 416-fold increase in resistance to GNF179. This confirms that CARL is a determinant of high-level resistance to this novel class of compounds, but critically this resistance appears to result in un-transmittable parasites.