Parasitology Unit, Infectious Disease Research Centre, Institute for Medical Research, National Institute of Health, Shah Alam, Malaysia.
Department of Chemical Engineering & Sustainability, Faculty of Engineering, International Islamic University Malaysia, Kuala Lumpur, Malaysia.
PLoS One. 2024 Aug 15;19(8):e0306975. doi: 10.1371/journal.pone.0306975. eCollection 2024.
Malaria, an ancient mosquito-borne illness caused by Plasmodium parasites, is mostly treated with Artemisinin Combination Therapy (ACT). However, Single Nucleotide Polymorphisms (SNPs) mutations in the P. falciparum Kelch 13 (PfK13) protein have been associated with artemisinin resistance (ART-R). Therefore, this study aims to generate PfK13 recombinant proteins incorporating of two specific SNPs mutations, PfK13-V494I and PfK13-N537I, and subsequently analyze their binding interactions with artemisinin (ART). The recombinant proteins of PfK13 mutations and the Wild Type (WT) variant were expressed utilizing a standard protein expression protocol with modifications and subsequently purified via IMAC and confirmed with SDS-PAGE analysis and Orbitrap tandem mass spectrometry. The binding interactions between PfK13-V494I and PfK13-N537I propeller domain proteins ART were assessed through Isothermal Titration Calorimetry (ITC) and subsequently validated using fluorescence spectrometry. The protein concentrations obtained were 0.3 mg/ml for PfK13-WT, 0.18 mg/ml for PfK13-V494I, and 0.28 mg/ml for PfK13-N537I. Results obtained for binding interaction revealed an increased fluorescence intensity in the mutants PfK13-N537I (83 a.u.) and PfK13-V494I (143 a.u.) compared to PfK13-WT (33 a.u.), indicating increased exposure of surface proteins because of the looser binding between PfK13 protein mutants with ART. This shows that the PfK13 mutations may induce alterations in the binding interaction with ART, potentially leading to reduced effectiveness of ART and ultimately contributing to ART-R. However, this study only elucidated one facet of the contributing factors that could serve as potential indicators for ART-R and further investigation should be pursued in the future to comprehensively explore this complex mechanism of ART-R.
疟疾是一种古老的蚊媒病，由疟原虫寄生虫引起，主要用青蒿素联合疗法（ACT）治疗。然而，疟原虫 Kelch 蛋白 13（PfK13）中的单核苷酸多态性（SNPs）突变与青蒿素耐药性（ART-R）有关。因此，本研究旨在生成包含两个特定 SNP 突变（PfK13-V494I 和 PfK13-N537I）的 PfK13 重组蛋白，并随后分析它们与青蒿素（ART）的结合相互作用。使用改良的标准蛋白表达方案表达 PfK13 突变和野生型（WT）变体的重组蛋白，随后通过 IMAC 进行纯化，并通过 SDS-PAGE 分析和 Orbitrap 串联质谱进行确认。通过等温滴定量热法（ITC）评估 PfK13-V494I 和 PfK13-N537I 推进器结构域蛋白与 ART 的结合相互作用，随后使用荧光光谱法进行验证。获得的蛋白浓度为 PfK13-WT 为 0.3 mg/ml，PfK13-V494I 为 0.18 mg/ml，PfK13-N537I 为 0.28 mg/ml。结合相互作用的结果表明，与 PfK13-WT（33 a.u.）相比，突变体 PfK13-N537I（83 a.u.）和 PfK13-V494I（143 a.u.）的荧光强度增加，表明 PfK13 蛋白突变体与 ART 的结合变松，导致表面蛋白暴露增加。这表明 PfK13 突变可能导致与 ART 的结合相互作用发生改变，从而可能降低 ART 的有效性，并最终导致 ART-R。然而，本研究仅阐明了导致 ART-R 的因素之一，它可能成为 ART-R 的潜在指标，未来应进一步研究以全面探讨 ART-R 的复杂机制。
