Apicoplast ribosomal protein S10-V127M enhances artemisinin resistance of a Kelch13 transgenic Plasmodium falciparum

Abstract

Background: The resistance of Plasmodium falciparum to artemisinin-based (ART) drugs, the front-line drug family used in artemisinin-based combination therapy (ACT) for treatment of malaria, is of great concern. Mutations in the kelch13 (k13) gene (for example, those resulting in the Cys580Tyr [C580Y] variant) were identified as genetic markers for ART-resistant parasites, which suggests they are associated with resistance mechanisms. However, not all resistant parasites contain a k13 mutation, and clearly greater understanding of resistance mechanisms is required. A genome-wide association study (GWAS) found single nucleotide polymorphisms associated with ART-resistance in fd (ferredoxin), arps10 (apicoplast ribosomal protein S10), mdr2 (multidrug resistance protein 2), and crt (chloroquine resistance transporter), in addition to k13 gene mutations, suggesting that these alleles contribute to the resistance phenotype. The importance of the FD and ARPS10 variants in ART resistance was then studied since both proteins likely function in the apicoplast, which is a location distinct from that of K13.

Methods: The reported mutations were introduced, together with a mutation to produce the k13-C580Y variant into the ART-sensitive 3D7 parasite line and the effect on ART-susceptibility using the 0-3 h ring survival assay (RSA_{0-3 h}) was investigated.

Results and conclusion: Introducing both fd-D193Y and arps10-V127M into a k13-C580Y-containing parasite, but not a wild-type k13 parasite, increased survival of the parasite in the RSA_{0-3 h}. The results suggest epistasis of arps10 and k13, with arps10-V127M a modifier of ART susceptibility in different k13 allele backgrounds.

Fig. 1

Strategy to introduce the k13-C580Y, fd-D193Y and arps10-V127M alleles. A k13-C580Y mutagenesis. Plasmid pCas9_pfk13g and linearized plasmid pMA_k13MT were co-transfected into the 3D7 parasite; pCas9_pfk13g contains expression cassettes for Cas9 and k13 specific guide RNA for generating a double-strand break in k13. The k13-C580Y mutation was introduced by homology-directed DNA repair using k13HR1 and k13HR2 sequences in pMA_k13MT. The k1/k2 primer pair was used to identify the wild-type genotype (product size = 481 bp) of the unmodified parasite and the k1/k3 pair was used to identify the modified gene (product size = 492 bp). B Genotype analysis by PCR amplification of the modified parasite after cloning. Two transgenic parasite clones (K^MTC1 and K^MTC2) were isolated and tested by PCR analysis in comparison with 3D7 (C) DNA sequence analysis of K^MTC1. The k13-C580Y mutated codon is highlighted in red (TGT(C) ◊ TaT(Y)). D fd-D193Y mutagenesis. The pCas9_pffd and pMK_fdMT plasmid pair was used for Pffd gene modification. The f1/f2 and f1/f3 primer pairs were used to identify unmodified parasites (product size = 704 bp) and modified parasites (product size = 711 bp), respectively. For the sequence analysis, the f4/f5 primer pair was used to amplify and read the sequence. E The genotype analysis of the fd-D193Y mutated parasites. E (upper) PCR result for clonal parasites (F^MTC1 and F^MTC2) with single fd-D193Y mutation. E (lower) PCR result for cloned parasite (K^MTF^MT) with double mutation k13^C580Yfd^D193Y. F DNA sequence analysis result confirming the fd-D193Y mutation: the sequence result for the F^MTC1 parasite (F, upper) and K^MTF^MT (F, lower) with the fd-D193Y mutated codon highlighted in red (GAC(D) ◊ tAt(Y)). G arps10-V127M mutagenesis. The pCas9_pfarps10 and linearized pGemT_arps10MT plasmids were used to modify the arps10 gene in 3D7 and K^MTF^MT parasites. The a1/a2 (product size = 462 bp) and a3/a2 (product size = 459 bp) primer pairs were used to identify unmodified parasite and modified parasite, respectively. The a4/a2 primer pairs were used to amplify and read the sequence for sequence analysis. H PCR genotypic analysis: (H, upper) PCR result for two clonal lines of A^MT. (H, lower) PCR result for one clonal line of K^MTF^MTA^MT. I DNA sequence analysis confirming arps10-V127M mutation. Data are shown for A^MT (I, upper) and K^MTF^MTA^MT (I, lower) clonal lines with the arps10-V127M mutated codon highlighted in red (GTG(V) ◊ aTG(M)). Pfk13-g, Pffd-g or Pfarps10-g: Pfk13, Pffd or Pfarps10 specific guide RNA gene cassette, cas9: Cas9 gene cassette, hdhfr-yfcu: drug selectable marker, HR; Homologous region, K^MT = k13^C580Y mutation, F^MT = fd^D193Y mutation, A^MT = arps10^V127M mutation

Fig. 2

Growth and development of the modified parasites. A Giemsa-stained parasites at 0, 24, 48, 72 and 96 h after initial invasion (hpi). Most parasites in each population of modified parasites were at the same stage of development as parental 3D7 parasites. B Fold increase in percentage parasitaemia over 2 cycles (96 h). The results are shown as means ± SD of 3 independent experiments. One-way ANOVA with Dunnett’s multiple comparisons test was performed. The growth of all transgenic parasites was not significantly different from 3D7 (95% CI). K^MT = k13^C580Y mutation, F^MT = fd^D193Y mutation, A^MT = arps10^V127M mutation

Fig. 3

Percentage of parasite ring survival in RSA _0−3 h. Univariate scatterplots of the % survival for 20 nM DHA treatment (left) and 700 nM DHA treatment (right) for the modified parasite lines, parental 3D7 and ART-resistance control strain (MRA1240). Mean and error bars (SD) are also shown for each sample group. One-way ANOVA with Tukey’s multiple comparisons test was performed. The dotted line at 1% survival shows the cut off used to discriminate between sensitive and resistant lines in the 700 nM DHA treatment. K^MT = k13^C580Y mutation, F^MT = fd^D193Y mutation, A^MT = arps10^V127M mutation

Fig. 4

Parasite morphology in control (mock treatment; + 0.1% DMSO) or drug treated (+ 20 nM DHA and + 700 nM DHA) cultures. A Representative images from Giemsa-stained thin smears showing morphology of parasites at 72 h of treatment. Most of the 3D7, F^MT and A^MT parasites treated with DHA were identified as pyknotic and dead (indicated by arrows). In contrast, a marked proportion of K^MT, K^MTF^MT, and K^MTF^MTA^MT parasites appeared viable and mostly at the late trophozoite and early schizont stages of development. MRA1240 parasites also appeared viable and mostly at the ring stage, with some early trophozoites, consistent with some growth delay. B The percent infected red cells containing trophozoites and schizonts (blue), rings (red) and pyknotic forms (green)