Duplication of superoxide dismutase and a mutation in aquaglyceroporin mediate the sensitivity of Plasmodium falciparum to cryptosporin, a natural product derived from Acaromyces ingoldii
Cryptosporin, a fungal metabolite, exhibited potent antimalarial activity in both asexual blood stage Plasmodium falciparum and liver-stage Plasmodium berghei with minimal human HepG2 toxicity. Unlike atovaquone, cryptosporin's mechanism is independent of mitochondrial electron transport. Minimum inoculum of resistance experiments showed a low risk of resistance development. RNA-Seq analysis revealed the upregulation of genes associated with sexual development including many canonical markers such as Pfs25 and PfCCp3, likely indicating a stress response that is also seen when parasites are treated with artemisinin. In vitro evolution and whole genome sequencing and analysis identified a mutation (F138Y) in PfAQP (PF3D7_1132800) and duplications in both parasite superoxide dismutase genes, PfSOD-1 (PF3D7_0814900) and PfSOD-2 (PF3D7_0623500). CRISPR-Cas9 editing confirmed that the F138Y mutation in PfAQP was sufficient to confer cryptosporin resistance. Alignment of the P. falciparum structure to that of hsAQP3 suggests that the mutation may impact transport of hydrogen peroxide and the transition between open and closed conformations. Indeed, studies with BY4742 fps1 knockout yeast expressing PfAQP showed that the permeability of PfAQP was not affected by cryptosporin and that it is likely not a direct target. Taken together, this study highlights the role of PfAQP in resistance to cryptosporin. In addition, cryptosporin likely induces high level of oxidative stress which results in the duplication of oxidative dismutase genes as part of the parasite's defense response. These findings highlight the role of PfAQP in mediating drug resistance, the mechanism of which warrants further research.