Impact of different mutations on Kelch13 protein levels, ART resistance, and fitness cost in parasites.

UNLABELLED

Reduced susceptibility to ART, the first-line treatment against malaria, is common in South East Asia (SEA). It is associated with point mutations, mostly in () but also in other genes, like . K13 and its compartment neighbors (KICs), including UBP1, are involved in endocytosis of host cell cytosol. We tested 135 mutations in KICs but none conferred ART resistance. Double mutations of C580Y with R539T or C580Y with R3138H, did also not increase resistance. In contrast, C580Y parasites subjected to consecutive RSAs did, but the sequence was not altered. Using isogenic parasites with different mutations, we found correlations between K13 protein amount, resistance, and fitness cost. Titration of K13 and KIC7 indicated that the cellular levels of these proteins determined resistance through the rate of endocytosis. While fitness cost of mutations correlated with ART resistance, R3138H caused a disproportionately higher fitness cost.

IMPORTANCE

Parasites with lowered sensitivity to artemisinin-based drugs are becoming widespread. However, even in these "resistant" parasites not all parasites survive treatment. We found that the proportion of surviving parasites correlates with the fitness cost of resistance-inducing mutations which might indicate that the growth disadvantages prevents resistance levels where all parasites survive treatment. We also found that combining two common resistance mutations did not increase resistance levels. However, selection through repeated ART-exposure did, even-though the known resistance genes, including , were not further altered, suggesting other causes of increased resistance. We also observed a disproportionally high fitness cost of a resistance mutation in resistance gene . Such high fitness costs may explain why mutations in and other genes functioning in the same pathway as are rare. This highlights that mutations are unique in their ability to cause resistance at a comparably low fitness cost.