Plasmodium falciparum: multifaceted resistance to artemisinins
- Lucie Paloque1, 2,
- Arba P. Ramadani1, 2, 3,
- Odile Mercereau-Puijalon4,
- Jean-Michel Augereau†1, 2 and
- Françoise Benoit-Vical†1, 2Email author
© Paloque et al. 2016
Received: 19 December 2015
Accepted: 1 March 2016
Published: 9 March 2016
Plasmodium falciparum resistance to artemisinins, the most potent and fastest acting anti-malarials, threatens malaria elimination strategies. Artemisinin resistance is due to mutation of the PfK13 propeller domain and involves an unconventional mechanism based on a quiescence state leading to parasite recrudescence as soon as drug pressure is removed. The enhanced P. falciparum quiescence capacity of artemisinin-resistant parasites results from an increased ability to manage oxidative damage and an altered cell cycle gene regulation within a complex network involving the unfolded protein response, the PI3K/PI3P/AKT pathway, the PfPK4/eIF2α cascade and yet unidentified transcription factor(s), with minimal energetic requirements and fatty acid metabolism maintained in the mitochondrion and apicoplast. The detailed study of these mechanisms offers a way forward for identifying future intervention targets to fend off established artemisinin resistance.
Artemisinin (ART) and its derivatives, introduced in 1980s, are the most potent and fastest acting anti-malarials, producing rapid clearance of parasitaemia and rapid resolution of symptoms. The discovery of artemisinin by Professor Youyou Tu has been rewarded the Nobel Prize for Physiology or Medicine 2015, recognizing that it has radically improved treatment against malaria . Plasmodium falciparum artemisinin resistance was first detected in 2006 , after decreased clearance rates were observed in patients receiving an artemisinin therapy in clinical studies performed in 2006. It is nowadays widespread across southeast Asia [2, 3]. The delayed clearance of artemisinin-resistant infections exposes larger numbers of parasites to anti-malarial drugs, potentially driving selection to higher-grade artemisinin or to the partner drug. In Asia, treatment failures of artemisinin-based combination therapy (ACT) have recently emerged, including resistance to piperaquine . This represents a serious threat for malaria eradication, widespread artemisinin resistance being predicted to cause in excess of 116,000 deaths annually, with medical costs and productivity losses evaluated as 146 million US$ and 385 million US$ per year, respectively . The resistance to artemisinins is due to mutation of the PfK13 propeller domain endowing the parasites with an increased ability to enter a quiescent state. Piecemeal evidence associates artemisinin resistance with increased unfolded protein response (UPR), dysregulation of the pre-replication phase and the PI3K/PI3P/AKT pathway. Yet, an overall picture about how these modifications result in quiescence-associated artemisinin resistance is lacking. A conceptual framework for the cellular networks involved from oxidative stress to quiescence and parasite survival is proposed here, opening novel avenues for future research.
Rapid overview of antiplasmodial drugs
Currently used anti-malarials belong to different chemical series and all Plasmodium falciparum stages can be targeted (asexual red blood cell stages, gametocytes, hepatic stages) by one or another anti-malarial. Each antiplasmodial drug series has its own specific mode(s) of action. In the food vacuole, haemozoin synthesis, corresponding to detoxification of the waste from haemoglobin by the parasite, is specially affected by quinolines  and artemisinins. Artemisinin and its derivatives are also responsible for alkylation of proteins and haem leading to oxidative damages . At the mitochondrial level, atovaquone, targeting the cytochrome bc 1 complex, inhibits the parasite electron transport chain and thus the dihydro-orotate dehydrogenase (DHODH) activity linked to the respiratory chain and involved in pyrimidine nucleotide biosynthesis . In the cytosol, inhibition of dihydrofolate reductase (DHFR) by proguanil or pyrimethamine or of dihydropteroate synthase (DHPS) by sulfadoxine , blocks the biosynthesis of folate involved in DNA and RNA synthesis. Unfortunately, most anti-malarial drugs have lost their efficacy as resistance has emerged and spread.
First reported resistance
Main resistance mechanisms Refs
Disruption of drug accumulation inside the food vacuole by reduced propensity of the drug transporter PfMDR1 to bind to and transfer the anti-malarial
Drug extrusion on from digestive vacuole by mutated drug transporter PfCRT due to higher lipophilicity and negativity of the transporter allowing ionized chloroquine efflux
Modification of the drug target: reduced inhibition of enzymatic activity by the drug
Reduction of parasite susceptibility to mefloquine by amplification of Pfmdr1 copy number
Modification of the drug target by disruption of Cytochrome bc1 complex
Among all mechanisms of resistance described in various organisms, from bacteria, parasites, fungi to human cancer cells, two main mechanisms drive Plasmodium resistance to almost all anti-malarial drugs: (i) reduced drug availability at its site of action, essentially due to mutations in transporter genes; and, (ii) modification of the drug target by mutations in corresponding genes (Table 1). Artemisinin resistance results from a different cellular process, quiescence, which is detailed below. The quiescence-based cellular mechanisms comply with the WHO definition of P. falciparum drug resistance  (see also WHO global report on drug resistance 2010), as artemisinin-resistant parasites survival exposure to therapeutic, lethal concentrations of artemisinin derivatives.
Recommendations for the use of dual and triple therapy
Parasite resistance to drug monotherapy prompted the WHO to recommend dual or triple therapy, which combines molecules with independent modes of action or distinct target enzymes. Drug combination is usually more effective and in the event of resistance to one component, the second one kills residual resistant parasites. However, some combination therapy, such as sulfadoxine-pyrimethamine plus chloroquine or amodiaquine, must be avoided due to the high levels of resistance established to these drugs already extensively used in monotherapy. For the same reason and because of the rapid acquisition of atovaquone resistance, atovaquone-proguanil is not recommended [2, 11, 12]. Clinical resistance to artemisinin was first detected in Cambodia in 2006 and reported in 2008 . In 2009, Noedl et al. reported artemisinin resistance of uncomplicated falciparum malaria cases in Thailand and Cambodia and correlated delayed clearance with increased in vitro artemisinin IC50 values compared to the artemisinin-sensitive clone W2 . Dondorp et al.  documented reduced in vivo susceptibility to artesunate in Pailin (western Cambodia) compared to Wang Pha (northwestern Thailand), characterized by a slow parasite clearance time (72–84 h in Pailin and 48–54 h in Wang Pha) and higher failure rates (recrudescence) after artesunate monotherapy (in Pailin 30 % of patients showed recrudescence compared to 10 % in Wang Pha). These altered clinical characteristics were not associated with reduced in vitro susceptibility, contrasting with observations by Noedl et al. . Resistance to artesunate monotherapy first reported in western Cambodia and along the Thailand-Myanmar border, resulted in lengthening of the parasite clearance half-lives from 2.6 h in 2001 to 3.7 h in 2010 . Artemisinin resistance is now also established in Myanmar, Vietnam, Lao PDR, and China .
In order to avoid the spread of P. falciparum resistance, monotherapy with artemisinin or its derivatives should be banned . ACT has been recommended by the WHO as first-line treatment for uncomplicated malaria since 2001 and is nowadays widely used (Additional file 1). However, in December 2014 eight countries, mainly in Africa (Angola, Cabo Verde, Colombia, Equatorial Guinea, Gambia, Sao Tome and Principe, Somalia, Swaziland) still allowed artemisinin-based monotherapy .
Treatment failures with ACT have only been observed in Asia where, in addition to artemisinin resistance, parasites were also resistant to companion drug, e.g. mefloquine along the Thai-Myanmar border  or piperaquine in Cambodia [20–22]. In such cases, the ACT used is replaced by another one and the therapeutic efficacy of the replacement ACT must be monitored every 2 years as recommended by the WHO  for timely adaptation of the treatment policy. The threshold of 10 % cases with detectable parasites on day 3 after ACT treatment used by the WHO to define areas of artemisinin resistance  was established in Asia, a region of low/moderate transmission. In Africa, where transmission is usually much higher, artemisinin resistance has not yet been reported. However, a recent study suggested that a threshold of 5 % cases on day 3 parasite positivity is more suited to artemisinin resistance monitoring in Africa due to higher levels of acquired immunity against Plasmodium in African populations contributing to faster parasite clearance .
Phenotypic and genotypic basis of artemisinin resistance
It is striking to note that from chloroquine resistance in 1957 to artemisinin resistance in 2006 , all anti-malarial drug resistance plaguing Asia was first reported at the Thai-Cambodian border and more especially in Pailin Province. Poor access to medicines, generating traffic of counterfeit drugs, with sub-clinical quantities, use of monotherapy, combined with an intense and extensive migrant labour system, are potential contributors [24–26]. No correlation was found between an increased mutation rate of the parasite genome and drug resistance in these regions, invalidating the hypothesis of the existence of a ‘hypermutator’ parasite . Resistance in this region has a typical multi-resistance profile, with resistance mutations in Pfcrt, Pfdhps, Pfdhfr, Pfmdr1 fixed in the parasite populations [28, 29].
Resistance to artemisinins was selected from such multi-resistant parasites circulating in western Cambodian provinces and was shown to be an inheritable genetic trait of the parasites [16, 30]. The mechanism driving artemisinin resistance was discovered using an experimental model, the F32-ART line -a highly artemisinin-resistant line- established in vitro after 5 years of exposure to escalating concentrations of artemisinin . Artemisinin resistance of F32-ART is mediated by a resistance mechanism quite distinct from those previously described [15, 31–33] (Table 1), since these parasites score susceptible using the standard in vitro susceptibility assays [31, 33–35]. Plasmodium resistance to artemisinins is due to the enhanced number of young ring forms to enter into a quiescence state upon exposure to artemisinins, and quickly resume growth once the artemisinins are removed. This capacity is conferred by mutations of a gene called Pfk13 [15, 31, 33]. This gene is now currently monitored to follow artemisinin resistance spread according to WHO recommendations [2, 18, 36].
Plasmodium falciparum resistance to artemisinin is quiescence-based
The first hypothesis of artemisinin resistance based on a partial cytostatic effect of the drug was evoked thanks to a mathematical model in 2000 . The classical definition of quiescence is ‘a reversible absence of proliferation’, i.e., a non-dividing cell which eventually restarts its cell cycle when conditions become appropriate. Various quiescent states may depend on the cell history; alternatively, quiescence can be the convergence of an adaptive process to cope with an adverse environment and an active preparation to efficiently resume proliferation . The development cycle of P. falciparum presents normal arrest at the sporozoite stage in the mosquito salivary glands or at the gametocyte stage in red blood cells. In vitro, P. falciparum cell cycle progression can be strongly delayed at the trophozoite stage in response to isoleucine starvation . A fraction of P. vivax and Plasmodium ovale sporozoites is able to stop its cell cycle during the hepatic phase to enter into a particular state, named hypnozoites, another form of dormant or quiescent parasites, and responsible for relapses, all under an epigenetic control of gene expression by histone modification enzymes .
Quiescence and artemisinin-resistant parasite stages
Data obtained with the highly artemisinin-resistant F32-ART line and P. falciparum isolates showed that artemisinin pressure induces developmental arrest of a sub-population of very young (0–3 h) ring stages which enter a quiescent state, while killing all other stages [31, 41]. Quiescence has also been correlated with K13-mediated artemisinin resistance in field isolates .
Quiescent parasites are difficult to identify under the microscope but staining mitochondrial activity with fluorescent dyes, unambiguously labels live quiescent cells in artemisinin-treated cultures [31, 42, 43]. Cell cycle arrest leads to overestimations of the parasite clearance time in patients , and a defect of H3-hypoxanthine incorporation in standard chemosensitivity assay leads to an overestimation of the effectiveness (IC50) of anti-malarials [31, 42]. As a result, classical susceptibility assays based on parasite proliferation are not suitable to differentiate parasites that are artemisinin-resistant from those that are artemisinin-sensitive. Recrudescence assays and ring stage survival assay (RSA0–3h) using highly synchronized P. falciparum parasites in culture, based on a 6-h dihydroartemisinin exposure in vitro, followed by culture in drug-free conditions until microscopic read-out at 72 h, are able to detect and quantify artemisinin resistance [31, 35, 41]. Quiescence takes place at a moment of the parasite cycle (young ring stage) where artemisinin is less efficient . Indeed, artemisinin toxicity is potentiated by products of haemoglobin digestion inside the food vacuole, which begins in the mid-ring stage. Haemoglobin degradation products, such as haem and ferrous iron, react with artemisinin to cause oxidative stress and irreversible damages . The low levels of haemoglobin endocytosis and therefore its absence or low digestion level in very young rings, could explain their reduced sensitivity. At this stage, which is approximately 14 h longer in resistant parasites than in wild-type parasites , a lower (moderate) oxidative stress is likely more manageable by the parasite, and particularly so by PfK13 mutants, while at the trophozoite stage, artemisinin-dependent damage exceeds the parasite’s response abilities . The ring stage arrested development allows parasites to survive during the relatively short period of elevated artemisinin concentration (the elimination half-life in humans varies from one to 11 h depending on the derivative) [31, 41, 47]. Why only a small sub-set population (<1 %) of resistant parasites  is able to enter a quiescence stage despite their common genetic pattern remains to be elucidated.
Response of non-mutated PfK13 parasites to artemisinin exposure
It appears that all parasite lines are able to enter quiescence or dormancy-mediated processes . In vitro, this is monitored by the capacity to proliferate after drug removal, i.e., to recrudesce. Artemisinin-resistance is characterized by a short time to recrudescence as demonstrated with the F32-ART (artemisinin-resistant) and F32-TEM (sensitive) sibling laboratory lines. After exposure to up to 18 µM of artemisinin, F32-TEM was able to resume growth and reach 5 % of parasitaemia 17 days after artemisinin removal compared to 10 days for F32-ART . Other studies reported that several artemisinin-sensitive P. falciparum strains restarted their cell cycle from 4 to 25 days after the drug removal [48, 49]. Thus, some wild-type parasites are able to survive artemisinin exposure but artemisinin-resistant strains restart faster with more quiescent parasites and/or quicker awakening [31, 35].
Involvement of the Pfk13 locus in Plasmodium falciparum resistance to artemisinins
Whole genome-sequencing of F32-ART and its sibling-sensitive line F32-TEM as well as of artemisinin-resistant P. falciparum isolates from Cambodia, allowed artemisinin resistance to be associated with mutations of the K13 protein (accession number PF3D7_1343700), belonging to the kelch super-family of proteins. All mutations found in the Pfk13 gene and correlated with artemisinin resistance are non-synonymous, and are present after position 440 in the propeller domain (K13-propeller) [33, 36, 50]. Just one of these mutations is sufficient to confer artemisinin-resistance . There is a strong genetic correlation in Asia between K13-propeller mutation and a slow parasite clearance time (half-life longer than 5 h) [3, 28, 33, 36, 51–53]. In Myanmar, among the K13 mutations observed, C580Y and M476I (the mutation acquired by F32-ART parasites) were associated with delayed clearance-time . In Southern China, at the Myanmar border, the single F446I mutation predominates and is associated with delayed clearance . The current picture is that artemisinin-resistant parasites circulating in different geographic areas of Southeast Asia stem from several independent emergences of unique mutations in the K13-propeller domain [28, 36, 53] where the parasite genetic background could play a role in the propensity of K13 mutants to emerge. Three prevalent K13-propeller mutations -C580Y, R539T and Y493H- were shown to correlate strongly with increased parasite clearance in vivo and with increased survival rates in the in vitro RSA0–3h [33, 55, 56]. Gene editing showed that introduction of the wild-type allele into resistant parasites procured a sensitive, very low RSA0–3h survival rate (0.3–0.7 %) whereas the parent isolates harbouring a K13 mutation (R539T, I543T or C580Y) displayed 40–49 % RSA0–3h survival. Conversely C580Y introduced into wild-type artemisinin-sensitive Cambodian clinical isolates and reference lines conferred varying degrees of in vitro resistance, suggesting additional contribution from the genetic background . Importantly, Asian K13 mutations are generally not found in Africa where numerous additional but rare Pfk13 alleles were identified [36, 57–59]. An exception is the SNP A578S, observed in many African countries [58–60] as well as Bangladesh  and Thailand , however this mutation is not associated with artemisinin resistance .
Apart from one study in a limited number of African children with severe malaria , none of the K13 mutations has been associated with clinical artemisinin resistance for the time being, despite evidence that introducing the C580Y mutation generates artemisinin resistance in vitro in the NF54 parasite strain considered to be of African origin . Genetic determinants of artemisinin resistance have been reviewed recently by Fairhurst . A search of mutations in the k13 P. vivax orthologue showed reduced polymorphism compared to Pfk13 and the V552I mutation identified cannot be currently associated with any P. vivax drug resistance .
Biochemical consequences of a mutant K13 propeller
In C580Y mutant parasites, PfPI3K was no longer ubiquitinylated, resulting in 1.5–twofold increased basal PI3P levels. This may reflect disrupted interactions between PfPI3K and PfK13 or disrupted interactions within the PfK13 associated protein complex (Fig. 2). Elevated basal phosphatidylinositol 3 phosphate (PI3P) levels appear central to artemisinin resistance, as K13 wild-type parasites became resistant upon elevation of PI3P levels induced by transgenic expression of human VPS34. Similarly transgenic elevation of PfAKT (also known as protein kinase B) in a wild-type parasite confers in vitro artemisinin resistance in connection with increased levels of PI3P by a feedback mechanism supposed . As PfPI3K enzymatic activity is inhibited by artemisinins, the levels of PI3P likely drop rapidly upon exposure to artemisinins in sensitive parasites, whereas the PI3P level is already elevated in resistant parasites . Additional downstream effectors likely come into play to orchestrate the quiescence response to artemisinin and withstand its toxicity.
This model does not exclude the existence of more than one uTF. Moreover, the PfPK4/PfPI3K downstream regulation cascades remain to be elucidated and experimentally demonstrated (Fig. 2). The involvement of the PfAKT pathway in artemisinin resistance, like PI3P involvement, still remains to be detailed. Moreover, the exact kinetics of events once the parasites are exposed to artemisinin oxidative and alkylating damage needs to be clarified, in particular as PI3K is inhibited by artemisinins  and should no longer contribute to refuelling PI3P levels.
Artemisinin resistance possibly includes a combination of the increased degradation of misfolded proteins/repair of damaged proteins especially those involved in the cell cycle progression and the ‘PfPK4/PfPI3K’ mediated response to the artemisinins’ oxidative stress. This cellular response of K13 mutant parasites to artemisinin stress could lead to a sub-set of ring stage parasites to enter into a quiescence state (Fig. 2). Why only a sub-set and a variable fraction of ring stages is involved needs to be understood. It may reflect a finely tuned balance between different cellular effectors that is unequally distributed between individual infected cells.
Artemisinin-induced quiescence and Plasmodium metabolism
Thus, the quiescent state induced at the young ring stage by artemisinin in sensitive and resistant parasites likely seems to involve the same cellular mechanisms, but the PfK13 mutation shifts the intra-population distribution so as to allow more parasites to become quiescent in the case of resistant parasites, and to quickly resume growth after the drug removal in vitro or drug elimination in patients.
It is increasingly clear that the P. falciparum resistance to artemisinin and its derivatives is not due to efflux modulation or target modifications as described for other anti-malarials, but is based on increased capacity of PfK13-mutant parasites to manage oxidative damage thanks to greater UPR mobilization. The over-expression of UPR target genes should impact three key points: (an) unidentified transcription factor(s) (uTF) regulating transcription of UPR/oxidative response genes, the PI3 K/PI3P/AKT pathway activity and the PfPK4/eIF2α cascade. This would allow parasite entrance into a quiescence state with minimal energy metabolism provided by the apicoplast and the mitochondrion, maintained by alternative tricarboxylic acid cycle and FASII metabolism, until drug removal/excretion when parasites can resume growth. Recently, the artemisinin-resistant F32-ART line, selected by long-term drug pressure with solely artemisinin, was shown to display an extended age range of stages surviving artemisinin treatment extending to older ring stages (13–16 h) and even trophozoite stages. These parasites were also able to survive lethal doses of diverse classes of anti-malarial drugs, including molecules used as partners in currently recommended ACT, in the absence of the ‘classical’ mutations of the target genes for these drugs. Thus, long-term in vitro artemisinin exposure selects a novel multidrug tolerant phenotype, which could represent a major threat to anti-malarial drug policy in the field . This threat is exacerbated by the fact that artemisinin-resistant parasites seem able to infect and be transmitted by a large panel of Anopheles species, including the major African species Anopheles gambiae . This is yet a another reason to urgently clarify the cellular network of ART resistance in order to identify new therapeutic modalities (to protect the molecules currently used), and/or novel drug development, in order to avoid entrance into a quiescent state, target quiescent parasites and promote restart of the cell cycle, rescuing drug susceptibility of parasites.
JMA and FBV designed the review; all authors contributed to acquisition, analysis and interpretation of data. LP, APR, OMP, JMA, and FBV have been involved in drafting and revising the manuscript critically. All authors read and approved the final manuscript.
OM-P and FB-V are co-inventors on the pending patents #US61/904651 and US62/062439, filed by Institut Pasteur. These patents cover the use of K13 mutations as a molecular marker of P. falciparum ART resistance.
This study was supported in part by the French Agence Nationale de la Recherche (Grant ANR-13-BSV3-0018-01) and by the French Government programme Investissement d’Avenir, Laboratoire d’Excellence “Integrative Biology of Emerging Infectious Diseases” (Grant ANR-10-LABX-62-IBEID).
The authors declare that they have no competing interests.
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