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.
Highlights
As resistance to artemisinins emerges in Southeast Asia, there is an urgent need to identify the genetic determinants and understand the molecular mechanisms underpinning such resistance
Due to the complexity of the selection procedure with multiple drugs, it has been difficult to confidently associate these UBP-1 mutations with ART and CQ susceptibility in the absence of appropriate reverse genetics approaches. These mutations have been introduced into UBP-1 in P. falciparum, and the V2721F equivalent has been shown to associate with increased DHA ring stage survival assays (RSA) survival with no CQ resistance phenotype, whereas the V2728F orthologue appeared to have no ART or CQ resistance profiles [15]
To experimentally demonstrate that UBP-1 mutations confer selective advantage upon ART pressure, we introduced P. chabaudi UBP-1 candidate mutation (V2697F and V2728F) equivalents into the P. berghei 820 line using a CRISPR-Cas9 system developed and optimized in our lab (Fig. 1A)
Summary
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. ART resistance is thought to be primarily conferred by specific mutations in the Plasmodium falciparum Kelch-13 (PfKelch13) gene, and such mutations are currently almost endemic in most parts of SEA [1, 4, 5] These mutations are associated with delayed parasite clearance rates in vivo and with reduced susceptibility of ring stage parasites in vitro in ring stage survival assays (RSA) [3, 6]. Due to the complexity of the selection procedure with multiple drugs, it has been difficult to confidently associate these UBP-1 mutations with ART and CQ susceptibility in the absence of appropriate reverse genetics approaches These mutations have been introduced into UBP-1 in P. falciparum, and the V2721F equivalent has been shown to associate with increased DHA RSA survival with no CQ resistance phenotype, whereas the V2728F orthologue appeared to have no ART or CQ resistance profiles [15]. We have characterized their relative fitness compared to that of the wild-type nonmutant parasite
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