Experimentally Engineered Mutations in a Ubiquitin Hydrolase, UBP-1, Modulate In Vivo Susceptibility to Artemisinin and Chloroquine in Plasmodium berghei

Abstract

As resistance to artemisinins (current frontline drugs in malaria treatment) emerges in Southeast Asia, there is an urgent need to identify the genetic determinants and understand the molecular mechanisms underpinning such resistance. Such insights could lead to prospective interventions to contain resistance and prevent the eventual spread to other regions where malaria is endemic. Reduced susceptibility to artemisinin in Southeast Asia has been primarily linked to mutations in the Plasmodium falciparum Kelch-13 gene, which is currently widely recognized as a molecular marker of artemisinin resistance. However, two mutations in a ubiquitin hydrolase, UBP-1, have been previously associated with reduced artemisinin susceptibility in a rodent model of malaria, and some cases of UBP-1 mutation variants associated with artemisinin treatment failure have been reported in Africa and SEA. In this study, we employed CRISPR-Cas9 genome editing and preemptive drug pressures to test these artemisinin susceptibility-associated mutations in UBP-1 in Plasmodium berghei sensitive lines in vivo. Using these approaches, we show that the V2721F UBP-1 mutation results in reduced artemisinin susceptibility, while the V2752F mutation results in resistance to chloroquine (CQ) and moderately impacts tolerance to artemisinins. Genetic reversal of the V2752F mutation restored chloroquine sensitivity in these mutant lines, whereas simultaneous introduction of both mutations could not be achieved and appears to be lethal. Interestingly, these mutations carry a detrimental growth defect, which would possibly explain their lack of expansion in natural infection settings. Our work provides independent experimental evidence on the role of UBP-1 in modulating parasite responses to artemisinin and chloroquine under in vivo conditions.

Plasmodium bergheiK13 Mutations MediateIn VivoArtemisinin Resistance That is Reversed by Proteasome Inhibition

“…In earlier efforts to introduce UBP-1 mutations in PB, we found that pre-emptive drug pressure to which the engineered mutation is anticipated to confer protective advantage can selectively enrich for the mutant in a mixed, transfected parasite population even when the mutant population is <1% in the mixture (40). Using this approach, we subjected a larger inoculum (2 x 10 7 ) of the G2022 C592Y.1* , G2023 C592Y.2* , G2024 I555T* and G2025 R551T* lines to AS at 20 or 64 mg/kg to see if any enrichment in the recrudescent parasite populations could be achieved (Fig.…”

“…S3D) and sequencing. The V2721F UBP-1 mutant line, which we previously found to mediate reduced susceptibility to ARTs in PB (40), was also generated in the 1804cl1 background and cloned (Table S1). PB K13 mutants display reduced susceptibility to DHA in 24-hour assays and increased survival in PB-adapted RSAs.…”

“…Unlike in PF, PB can only be maintained in one blood stage cycle in vitro, which restricts drug susceptibility assays to one 24-hour developmental cycle. Drug susceptibility readouts are, therefore, based on single-generation flow cytometry quantification of schizont maturation (40,45,46). Using this approach, we aimed to characterize the DHA dose-response profiles of the PB K13 mutants as compared to wild-type parasites or to a previously reported UBP-1 mutant with reduced ART susceptibility (40).…”

“…Drug susceptibility readouts are, therefore, based on single-generation flow cytometry quantification of schizont maturation (40,45,46). Using this approach, we aimed to characterize the DHA dose-response profiles of the PB K13 mutants as compared to wild-type parasites or to a previously reported UBP-1 mutant with reduced ART susceptibility (40). Interestingly, in contrast with the equivalent PF K13 mutants, PB M488I, R551T and Y505H K13-mutant parasites displayed reduced susceptibility to DHA in standard growth inhibition assays with 3.3, 1.4 and 1.2-fold IC50 increases, respectively, as compared to isogenic K13 wild-type parasites ( Fig.…”

“…K13 mutations are thought to lead to a partial loss of protein function, which subsequently impairs hemoglobin endocytic uptake, thereby lessening ART activation and conferring ART resistance (37). This has pointed towards a K13mediated hemoglobin-centric mechanism of ART resistance, which could be possibly shared with other drugs such as CQ that act by binding to heme moieties in the digestive vacuole, following cytostome-mediated hemoglobin endocytosis (38,(40)(41)(42). Of note, mutant K13-mediated ART resistance phenotypes are associated with upregulated cellular stress responses, which can be targeted by selective inhibition of the parasite 26S proteasome (43,44).…”