Institute of Infection, Immunity and Inflammation, Wellcome Centre for Integrative Parasitology, University of Glasgow, Glasgow, Scotland, United Kingdom.
Institute of Infection, Immunity and Inflammation, Wellcome Centre for Integrative Parasitology, University of Glasgow, Glasgow, Scotland, United Kingdom
Antimicrob Agents Chemother. 2020 Jun 23;64(7). doi: 10.1128/AAC.02484-19.
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 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 sensitive lines 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 conditions.
由于东南亚地区青蒿素（目前疟疾治疗的一线药物）耐药性的出现，迫切需要确定遗传决定因素，并了解这种耐药性的分子机制。这些见解可能会导致前瞻性干预措施来遏制耐药性，并防止其最终传播到疟疾流行的其他地区。东南亚青蒿素敏感性降低主要与 Kelch-13 基因的突变有关，该基因目前被广泛认为是青蒿素耐药性的分子标志物。然而，先前已经有研究表明，在疟疾的啮齿动物模型中，泛素水解酶 UBP-1 的两个突变与青蒿素敏感性降低有关，并且在非洲和东南亚已经报道了一些与青蒿素治疗失败相关的 UBP-1 突变变体病例。在这项研究中，我们采用了 CRISPR-Cas9 基因组编辑和预先药物压力测试来检测 UBP-1 中与青蒿素敏感性相关的突变在敏感系中的作用。通过这些方法，我们表明 V2721F UBP-1 突变导致青蒿素敏感性降低，而 V2752F 突变导致氯喹（CQ）耐药性，并对青蒿素的耐受性产生中度影响。V2752F 突变的遗传逆转恢复了这些突变系中的氯喹敏感性，而同时引入这两个突变是不可能的，并且似乎是致命的。有趣的是，这些突变携带有害的生长缺陷，这可能解释了它们在自然感染环境中缺乏扩张的原因。我们的工作为 UBP-1 在调节寄生虫对青蒿素和氯喹的反应方面提供了独立的实验证据。
