Targeting the Cell Stress Response of Plasmodium falciparum to Overcome Artemisinin Resistance

Abstract

Successful control of falciparum malaria depends greatly on treatment with artemisinin combination therapies. Thus, reports that resistance to artemisinins (ARTs) has emerged, and that the prevalence of this resistance is increasing, are alarming. ART resistance has recently been linked to mutations in the K13 propeller protein. We undertook a detailed kinetic analysis of the drug responses of K13 wild-type and mutant isolates of Plasmodium falciparum sourced from a region in Cambodia (Pailin). We demonstrate that ART treatment induces growth retardation and an accumulation of ubiquitinated proteins, indicative of a cellular stress response that engages the ubiquitin/proteasome system. We show that resistant parasites exhibit lower levels of ubiquitinated proteins and delayed onset of cell death, indicating an enhanced cell stress response. We found that the stress response can be targeted by inhibiting the proteasome. Accordingly, clinically used proteasome inhibitors strongly synergize ART activity against both sensitive and resistant parasites, including isogenic lines expressing mutant or wild-type K13. Synergy is also observed against Plasmodium berghei in vivo. We developed a detailed model of parasite responses that enables us to infer, for the first time, in vivo parasite clearance profiles from in vitro assessments of ART sensitivity. We provide evidence that the clinical marker of resistance (delayed parasite clearance) is an indirect measure of drug efficacy because of the persistence of unviable parasites with unchanged morphology in the circulation, and we suggest alternative approaches for the direct measurement of viability. Our model predicts that extending current three-day ART treatment courses to four days, or splitting the doses, will efficiently clear resistant parasite infections. This work provides a rationale for improving the detection of ART resistance in the field and for treatment strategies that can be employed in areas with ART resistance.

Host-mediated impairment of parasite maturation during blood-stage Plasmodium infection

“…Other studies suggest that drug treatment is also able to slow or halt (dormancy) the life cycle of P. falciparum parasites, which is thought to be a parasite stress response allowing parasites to survive treatment (33)(34)(35)(36)(37). Hence, it seems likely that slower maturation occurs as a parasite survival mechanism in response to environmental stress, perhaps as a result of the parasite directing resources to mitigate damage caused by some form of stress placed on the parasite, whether by drug or by the host environment.…”

Resisting resistance: is there a solution for malaria?

“…These findings suggest that this mechanism of resistance to vancomycin involves the permanent activation of a stress response in resistant bacteria [84]. A recent study revealed that malaria parasites resistant to artemisinin exhibit an enhanced cell stress response with lower levels of ubiquitinated proteins and delayed onset of cell death compared to artemisinin susceptible parasites, suggesting the involvement of a proteasomeengaging cell stress response [85]. Clinically used proteasome inhibitors strongly synergize artemisinin activity against both sensitive and resistant parasites.…”

Artemisinin resistance, some facts and opinions

“…More recently, a mutation on the K13-Propeller locus has been observed to be associated to DPC in Cambodia [9]; subsequently, additional mutations on the same gene have been observed in other regions in South Asia [10][11][12][13][14][15][16]. This promising marker is currently used in association with DPC to identify ART resistance but both still require a substantial clinical validation.…”

“…This promising marker is currently used in association with DPC to identify ART resistance but both still require a substantial clinical validation. Wide clinical trials to correlate DPC and K13 mutations with confirmed ART resistance are therefore needed to assess the isolated and cumulative performances of the markers [16]. Unfortunately the intrinsic complexity of those markers do not favor the sustainability and the implementation of the large clinical trials that should be required to validate and to assign precise performances to available makers.…”

“…Lack of information on the mechanism of action of ART, however, limits again a rational choice of the partner drug; and possible negative interferences between drugs cannot be excluded [16]. This issue is even more relevant as most of the available antimalarial drugs already displayed manifest resistance [17] and as ART resistance is observed only in presence of a demonstrated resistance to the partner drug [7].…”