Drug resistance. K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates

Abstract

The emergence of artemisinin resistance in Southeast Asia imperils efforts to reduce the global malaria burden. We genetically modified the Plasmodium falciparum K13 locus using zinc-finger nucleases and measured ring-stage survival rates after drug exposure in vitro; these rates correlate with parasite clearance half-lives in artemisinin-treated patients. With isolates from Cambodia, where resistance first emerged, survival rates decreased from 13 to 49% to 0.3 to 2.4% after the removal of K13 mutations. Conversely, survival rates in wild-type parasites increased from ≤0.6% to 2 to 29% after the insertion of K13 mutations. These mutations conferred elevated resistance to recent Cambodian isolates compared with that of reference lines, suggesting a contemporary contribution of additional genetic factors. Our data provide a conclusive rationale for worldwide K13-propeller sequencing to identify and eliminate artemisinin-resistant parasites.

Fig. 1. Genetic modification of the K13-propeller domain

Location of K13-propeller mutations and sequencing results showing the insertion of individual mutations into recombinant parasites used in the RSA_0–3h. Dd2^ctrl parasites contain only synonymous, phenotypically silent binding-site mutations and showed 0.7% survival rates, which is equivalent to those of parental Dd2 parasites (fig. S2).

Fig. 2. K13-propeller mutations confer artemisinin resistance in clinical isolates and reference lines in vitro, as defined in the RSA_0–3h

Results show the percentage of early ring-stage parasites (0 to 3 hours after invasion of human erythrocytes) that survived a 6-hour pulse of 700 nM DHA (a pharmacologically relevant concentration of the active metabolite of ARTs), as measured by microscopy 66 hours later. Data show mean ± SEM percent survival compared with control dimethyl sulfoxide–treated parasites processed in parallel. (A to D) RSA_0–3h survival for Cambodian isolates harboring native K13 mutations (shown in superscript) and ZFN-edited isogenic clones carrying wild-type K13 alleles (superscript “rev”). (E to I) RSA_0–3h survival for Cambodian isolates and reference lines harboring wild-type K13 alleles and ZFN-edited isogenic clones carrying individual K13 mutations (shown in superscripts). (J) Impact of different K13 mutations on RSA_0–3h survival in the Dd2 reference line, showing that I543Tand R539Tconfer the highest levels of resistance. (K) Introduction of C580Y into multiple Cambodian clinical isolates and reference lines, showing that this mutation confers varying degrees of in vitro resistance depending on the parasite genetic background. The geographic origins and known drug-resistance genotypes of these isolates and lines are provided in table S2. Results were obtained from 3 or 4 independent assays performed in duplicate (values provided in table S4; F32-TEM showed <0.2% RSA_0–3h survival). Two-sample t tests with unequal variances (performed with the STATA package) were used to assess for statistically significant differences between K13-edited clones and their comparator lines—the parental isolates listed on the left in (A) to (J) and the FCB^C580Y clone in (K) (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). Statistical outputs (including calculations of the SE of the difference between the means of samples being compared and the P values) are listed in the supplementary materials.