School of Medicine, Deakin University, Geelong 3216, Australia.
Institute for Mental and Physical Health and Clinical Translation (IMPACT), Deakin University, Geelong 3216, Australia.
Biomolecules. 2024 Jun 30;14(7):781. doi: 10.3390/biom14070781.
Parasite-derived new permeation pathways (NPPs) expressed at the red blood cell (RBC) membrane enable parasites to take up nutrients from the plasma to facilitate their survival. Thus, NPPs represent a potential novel therapeutic target for malaria. The putative channel component of the NPP in the human malaria parasite is encoded by mutually exclusively expressed genes. Complicating the study of the essentiality of these genes to the NPP is the addition of three paralogs whose contribution to the channel is uncertain. Rodent malaria contains only two genes, and thus studies of genes could significantly aid in dissecting their overall contribution to NPP activity. Previous methods for determining NPP activity in a rodent model have utilised flux-based assays of radioisotope-labelled substrates or patch clamping. This study aimed to ratify a streamlined haemolysis assay capable of assessing the functionality of NPPs. Several isotonic lysis solutions were tested for their ability to preferentially lyse infected RBCs (iRBCs), leaving uninfected RBCs (uRBCs) intact. The osmotic lysis assay was optimised and validated in the presence of NPP inhibitors to demonstrate the uptake of the lysis solution via the NPPs. Guanidinium chloride proved to be the most efficient reagent to use in an osmotic lysis assay to establish NPP functionality. Furthermore, following treatment with guanidinium chloride, ring-stage parasites could develop into trophozoites and schizonts, potentially enabling use of guanidinium chloride for parasite synchronisation. This haemolysis assay will be useful for further investigation of NPPs in and could assist in validating its protein constituents.
寄生虫在红细胞（RBC）膜上表达的寄生虫衍生的新渗透途径（NPPs）使寄生虫能够从血浆中吸收营养物质，以促进其生存。因此，NPPs 代表了疟疾的一个潜在的新治疗靶点。NPP 在人类疟原虫中假定的通道成分由相互排斥表达的基因编码。这些基因对 NPP 的必需性的研究变得复杂，因为添加了三个不确定对 通道有贡献的 基因的 。啮齿动物疟疾仅包含两个 基因，因此对 基因的研究可以显著帮助解析它们对 NPP 活性的整体贡献。以前在啮齿动物模型中测定 NPP 活性的方法利用放射性同位素标记底物的通量测定或膜片钳技术。本研究旨在验证一种简化的溶血测定法，该方法能够评估 NPPs 的功能。测试了几种等渗裂解溶液，以确定它们是否能够优先裂解感染的红细胞（iRBCs），而使未感染的红细胞（uRBCs）保持完整。在存在 NPP 抑制剂的情况下，对渗透裂解测定法进行了优化和验证，以证明通过 NPP 吸收裂解溶液。胍证明是在渗透裂解测定中使用最有效的试剂，以建立 NPP 的功能。此外，在用胍处理后，环状阶段的寄生虫可以发育成滋养体和裂殖体，这可能使胍能够用于寄生虫同步化。这种溶血测定法将有助于进一步研究 和 中的 NPPs，并有助于验证其蛋白质成分。
