Moderate ART resistance mutations with low fitness cost in malaria parasites from Africa

Reduced susceptibility to ART, the first-line treatment against malaria, is common in South East Asia (SEA). It is caused by point mutations, mostly in kelch13 (k13) but also in other genes, like upb1. 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 k13C580Y with k13R539T or k13C580Y with ubp1R3138H, did also not increase resistance. In contrast, k13C580Y parasites subjected to consecutive RSAs did, but the k13 sequence was not altered. Using isogenic parasites with different k13 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 k13 mutations correlated with ART resistance, ubp1R3138H caused a disproportionately higher fitness cost. SignificanceART resistance is only a partial resistance with a proportion of ring stages surviving physiological ART levels. The correlation of resistance with fitness cost in isogenic lines indicates that fitness cost likely prevents resistance levels permitting survival of all ring stages under physiological ART concentrations. We also found no indication that double mutations in k13, including the two most common resistance mutations in SEA, or with non-k13 genes, are a threat to lead to higher resistance. However, repeated ART exposure increased resistance by mechanisms not altering k13 gene sequence, indicating changes in the background of these parasites. The disproportionally high fitness cost of ubp1R3138H may explain why kic mutations affecting resistance are rare and highlights the unique property of K13 to influence endocytosis only in ring stages.