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

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.恶性疟的有效防控高度依赖青蒿素联合疗法。有报道称，青蒿素类药物（artemisinins, ARTs）耐药性已出现且患病率持续上升，这一消息令人警觉。近期研究表明，青蒿素类耐药性与K13螺旋桨蛋白（K13 propeller protein）的突变密切相关。本研究针对采自柬埔寨拜林地区的恶性疟原虫（Plasmodium falciparum）K13野生型与突变型分离株的药物反应开展了详细的动力学分析。本研究证实，青蒿素类药物处理可诱导疟原虫生长阻滞，并促使泛素化蛋白积累，提示细胞启动了依赖泛素-蛋白酶体系统（ubiquitin/proteasome system）的应激反应。研究发现，耐药疟原虫的泛素化蛋白水平更低，且细胞死亡启动延迟，表明其细胞应激反应得以增强。本研究证实，抑制蛋白酶体可靶向调控该应激反应。据此，临床使用的蛋白酶体抑制剂可显著增强青蒿素类药物对敏感及耐药疟原虫的活性，包括携带K13突变型与野生型的同基因株（isogenic lines）。在体内实验中，该协同效应在伯氏疟原虫（Plasmodium berghei）模型中同样得到验证。本研究构建了详尽的疟原虫反应模型，首次实现了通过体外青蒿素敏感性实验推断体内疟原虫清除动力学特征。研究证实，当前耐药性临床标志物——延迟性疟原虫清除——实为药物疗效的间接检测指标，原因在于血液循环中仍存在形态未发生改变的失活疟原虫；据此，本研究提出了直接检测疟原虫活力的替代方案。本模型预测，将当前为期3天的青蒿素类药物疗程延长至4天，或拆分给药剂量，可有效清除耐药疟原虫感染。本研究为优化青蒿素类药物耐药性的现场检测方法，以及针对青蒿素类耐药地区的治疗策略提供了理论依据。