Transient temperature fluctuations severely decrease P. falciparum susceptibility to artemisinin in vitro

Henrici, Ryan C.; Schalkwyk, Donelly A. van; Sutherland, Colin J.

doi:10.1016/j.ijpddr.2018.12.003

Cited by 15 publications

(13 citation statements)

References 25 publications

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“…In P. falciparum, ART resistance is confined to early ring stage parasites, which has been translated in laboratory conditions to increased survival in ring stage survival assays (RSAs) (6). Mutations in Pfkelch13 and PfCoronin, as well as transient (hypothermic-hyperthermic) temperatures, have been shown to enhance ring stage parasite survival in the RSAs (10,22,23). More recently, characterization of Kelch-13 interacting factors has revealed that disruption of proteins that colocalize with Kelch-13, such as the parasite endocytosis proteins ESP15, UBP-1, and others of unknown function, modulate susceptibility to ARTs (24).…”

Section: Discussionmentioning

confidence: 99%

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

Simwela

Hughes

Roberts

et al. 2020

Antimicrob Agents Chemother

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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.

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Section: Discussionmentioning

confidence: 99%

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

Simwela

Hughes

Roberts

et al. 2020

Antimicrob Agents Chemother

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“…Resistance to artemisinins is unlike that of any other class of antimalarial compounds, being confined to the first few hours of intraerythrocytic development. We and others have now demonstrated this phenomenon with mutations in pfk13 (22), pfcoronin (17), pfap2, and pfubp1 as well as with transient pulses of low temperature (16), suggesting that artemisinin resistance has potentially many genetic and environmental origins. In all these cases so far, trophozoites and schizonts are fully sensitive to artemisinin.…”

Section: Figmentioning

confidence: 64%

“…We then characterized the susceptibility of these clones to artemisinin using the ring-stage survival assay (RSA) (2,16), an in vitro correlate of in vivo parasite susceptibility in which early-ring-stage parasites are exposed to a brief pulse of 700 nM DHA. Artemisinin susceptibility was also determined for three Cam3.II-derived Cambodian parasite lines (10), and the parental 3D7 line, as validated controls.…”

Section: Resultsmentioning

confidence: 99%

“…The plate was incubated at 37°C for 48 h, and EC 50 estimates were determined as described previously (26). Assays to measure artemisinin survival of synchronized ring-stage parasites within 4 h of erythrocyte invasion (RSA 0 -4 h ) were carried out exactly as described previously (16). Fluorescence-activated cell sorter (FACS) analysis was performed on live cells stained with a 1:10,000 dilution of MitoTracker deep red and a 1:1,000 dilution of SYBR green (Invitrogen-Molecular Probes) in phosphate-buffered saline (PBS) for 20 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…On this basis, we can confidently compare RSA survival estimates for our transgenic mutant lines back to those obtained for the 3D7 parental line. However, as laboratories differ in the duration of the 700 nM DHA pulse used, and in the methodology for survival readout (flow cytometry or microscopy) (16,17), further work on assay standardization is required before interlaboratory comparisons of survival estimates will have adequate validity. This is likely to include simple normalization approaches, based on relative survival compared to agreed standard comparators.…”

Section: Figmentioning

confidence: 99%

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Modification of pfap2μ and pfubp1 Markedly Reduces Ring-Stage Susceptibility of Plasmodium falciparum to Artemisinin In Vitro

Henrici

Schalkwyk

Sutherland

2019

Antimicrob Agents Chemother

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Management of uncomplicated malaria worldwide is threatened by the emergence in Asia of Plasmodium falciparum carrying variants of the pfk13 locus and exhibiting reduced susceptibility to artemisinin. Mutations in two other genes, ubp1 and ap2μ, are associated with artemisinin resistance in rodent malaria and with clinical failure of combination therapy in African malaria patients. Transgenic P. falciparum clones, each carrying orthologues of mutations in pfap2μ and pfubp1 associated with artemisinin resistance in Plasmodium chabaudi, were derived by Cas9 gene editing. Susceptibility to artemisinin and other antimalarial drugs was determined. Following exposure to 700 nM dihydroartemisinin in the ring-stage survival assay, we found strong evidence that transgenic parasites expressing the I592T variant (11% survival), but not the S160N variant (1% survival), of the AP2μ adaptin subunit were significantly less susceptible than the parental wild-type parasite population. The V3275F variant of UBP1, but not the V3306F variant, also displayed reduced susceptibility to dihydroartemisinin (8.5% survival versus 0.5% survival). AP2μ and UBP1 variants did not elicit reduced susceptibility to 48 h of exposure to artemisinin or to other antimalarial drugs. Therefore, variants of the AP2 adaptor complex μ-subunit and of the ubiquitin hydrolase UBP1 reduce in vitro artemisinin susceptibility at the early ring stage in P. falciparum. These findings confirm the existence of multiple pathways to perturbation of either the mode of action of artemisinin, the parasite’s adaptive mechanisms of resistance, or both. The cellular role of UBP1 and AP2μ in Plasmodium parasites should now be elucidated.

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The newly discovered role of endocytosis in artemisinin resistance

Behrens

Schmidt

Spielmann

2021

Medicinal Research Reviews

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Artemisinin and its derivatives (ART) are the cornerstone of malaria treatment as part of artemisinin combination therapy (ACT). However, reduced susceptibility to artemisinin as well as its partner drugs threatens the usefulness of ACTs. Single point mutations in the parasite protein Kelch13 (K13) are necessary and sufficient for the reduced sensitivity of malaria parasites to ART but several alternative mechanisms for this resistance have been proposed.Recent work found that K13 is involved in the endocytosis of host cell cytosol and indicated that this is the process responsible for resistance in parasites with mutated K13.These studies also identified a series of further proteins that act together with K13 in the same pathway, including previously suspected resistance proteins such as UBP1 and AP-2μ. Here, we give a brief overview of artemisinin resistance, present the recent evidence of the role of endocytosis in ART resistance and discuss previous hypotheses in light of this new evidence. We also give an outlook on how the new insights might affect future research.

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Transient temperature fluctuations severely decrease P. falciparum susceptibility to artemisinin in vitro

Cited by 15 publications

References 25 publications

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

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

Modification of pfap2μ and pfubp1 Markedly Reduces Ring-Stage Susceptibility of Plasmodium falciparum to Artemisinin In Vitro

The newly discovered role of endocytosis in artemisinin resistance

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