Guanidinium Chloride-Induced Haemolysis Assay to Measure New Permeation Pathway Functionality in Rodent Malaria .

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.