Role of Plasmodium falciparum Kelch 13 Protein Mutations in P. falciparum Populations from Northeastern Myanmar in Mediating Artemisinin Resistance

Abstract

Mutations in the Plasmodium falciparum Kelch 13 (PfK13) protein are associated with artemisinin resistance. PfK13 is essential for asexual erythrocytic development, but its function is not known. We tagged the PfK13 protein with green fluorescent protein in P. falciparum to study its expression and localization in asexual and sexual stages. We used a new antibody against PfK13 to show that the PfK13 protein is expressed ubiquitously in both asexual erythrocytic stages and gametocytes and is localized in punctate structures, partially overlapping an endoplasmic reticulum marker. We introduced into the 3D7 strain four PfK13 mutations (F446I, N458Y, C469Y, and F495L) identified in parasites from the China-Myanmar border area and characterized the in vitro artemisinin response phenotypes of the mutants. We found that all the parasites with the introduced PfK13 mutations showed higher survival rates in the ring-stage survival assay (RSA) than the wild-type (WT) control, but only parasites with N458Y displayed a significantly higher RSA value (26.3%) than the WT control. After these PfK13 mutations were reverted back to the WT in field parasite isolates, all revertant parasites except those with the C469Y mutation showed significantly lower RSA values than their respective parental isolates. Although the 3D7 parasites with introduced F446I, the predominant PfK13 mutation in northern Myanmar, did not show significantly higher RSA values than the WT, they had prolonged ring-stage development and showed very little fitness cost in in vitro culture competition assays. In comparison, parasites with the N458Y mutations also had a prolonged ring stage and showed upregulated resistance pathways in response to artemisinin, but this mutation produced a significant fitness cost, potentially leading to their lower prevalence in the Greater Mekong subregion. IMPORTANCE Artemisinin resistance has emerged in Southeast Asia, endangering the substantial progress in malaria elimination worldwide. It is associated with mutations in the PfK13 protein, but how PfK13 mediates artemisinin resistance is not completely understood. Here we used a new antibody against PfK13 to show that the PfK13 protein is expressed in all stages of the asexual intraerythrocytic cycle as well as in gametocytes and is partially localized in the endoplasmic reticulum. By introducing four PfK13 mutations into the 3D7 strain and reverting these mutations in field parasite isolates, we determined the impacts of these mutations identified in the parasite populations from northern Myanmar on the ring stage using the in vitro ring survival assay. The introduction of the N458Y mutation into the 3D7 background significantly increased the survival rates of the ring-stage parasites but at the cost of the reduced fitness of the parasites. Introduction of the F446I mutation, the most prevalent PfK13 mutation in northern Myanmar, did not result in a significant increase in ring-stage survival after exposure to dihydroartemisinin (DHA), but these parasites showed extended ring-stage development. Further, parasites with the F446I mutation showed only a marginal loss of fitness, partially explaining its high frequency in northern Myanmar. Conversely, reverting all these mutations, except for the C469Y mutation, back to their respective wild types reduced the ring-stage survival of these isolates in response to in vitro DHA treatment.

Insights into the intracellular localization, protein associations and artemisinin resistance properties of Plasmodium falciparum K13

“…Our results suggested that few if any interactions were specific to either the WT or mutant K13 isoforms. Many of our candidate K13-associated proteins were also observed in a recent study that used GFP-Trap beads to affinity purify GFP-K13 followed by LC/MS-MS [53]. These proteins included S-adenosylmethionine synthetase (the most abundant protein in our dataset), elongation factor 2, and plasmepsin II.…”

“…II WT respectively (S5 Table). By comparison, a very recent study using K13-specfic polyclonal antiserum reported a PCC value of 0.58 between WT K13 and BiP [53]. Interestingly, following DHA treatment, mutant and WT parasites showed significant differences at 12h post drug pulse (Fig 3E and S5C Fig).…”

“…IEM studies also frequently detected K13 close to the perinuclear parasite ER. These results suggest that mutant K13 might act in part by enhancing ER-associated stress responses to damaged proteins [31,53]. Although BiP co-immunoprecipitated with K13 in all samples across all experiments, it was excluded from our list of putative K13-interacting partners ( Table 2 and S2 Table) due to its abundant presence in the negative control samples.…”

Plasmodium berghei K13 Mutations Mediate In Vivo Artemisinin Resistance That is Reversed by Proteasome Inhibition

“…Nevertheless, the other PB K13 mutations tested herein appear to directly reflect the impact of the equivalent mutations in PF. Both PB F458I (this study) and PF F446I K13 mutants are fitness neutral (56) and do not enhance RSA survival in vitro (56,57), yet carry ART protective phenotypes in vivo (58)(59)(60). Furthermore, PB M488I K13 mutants display a significant growth defect that has not yet been characterized in the PF equivalent (M476I) and might explain its relative scarcity in SEA (61,62).…”

Mammalian deubiquitinating enzyme inhibitors display in vitro and in vivo activity against malaria parasites and potentiate artemisinin action

“…14 Meanwhile, parallel functional and localisation studies have also revealed that Kelch13 co-localises with multiple UPR components, proteins specific to the ER and mitochondria as well as intracellular vesicular trafficking Rab GTPases. [15][16] Central to the activity of the UPR is the ubiquitin proteasome system (UPS), a conserved eukaryotic pathway that plays a role in protein homeostasis by degrading unfolded proteins. Under ART pressure, activity of the UPS is more upregulated in Kelch13 mutant parasites compared to wild type while UPS inhibitors have been shown to synergize ART action suggesting that this pathway could be selectively targeted to overcome ART resistance.…”