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

Abstract

Parasite-derived new permeation pathways (NPPs) expressed at the red blood cell (RBC) membrane enable Plasmodium 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 P. falciparum is encoded by mutually exclusively expressed clag3.1/3.2 genes. Complicating the study of the essentiality of these genes to the NPP is the addition of three clag paralogs whose contribution to the P. falciparum channel is uncertain. Rodent malaria P. berghei contains only two clag genes, and thus studies of P. berghei clag 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 P. berghei 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 P. berghei and could assist in validating its protein constituents.

Keywords: Plasmodium berghei; Plasmodium surface anion channel; guanidinium chloride; new permeation pathway; osmotic lysis.

Figure 1

Osmotic lysis of P. falciparum-infected RBCs. Ring-stage parasites and uRBCs do not possess NPPs and thus remain intact following sorbitol lysis assay, as the sorbitol solution is unable to cross RBC membrane via endogenous channels. Late-stage parasites (trophozoite and schizont) possessing NPPs import an isotonic sorbitol solution into the cell, and this is accompanied by the osmotic influx of water. The iRBCs subsequently swell and burst, releasing haemoglobin and other cellular contents into the solution.

Figure 2

Sorbitol-mediated lysis of uninfected and P. berghei-infected rodent RBCs. Lysis assays (n = 3) were performed by incubating iRBCs (10% parasitemia) and uRBCs (negative control) in 280 mM sorbitol buffered in 20 mM HEPES, pH 7.4, at the indicated temperatures for 10 min. The percentage lysis was calculated relative to lysis with saponin (100% lysis). No significant (ns) difference was observed between the infected and uninfected samples at any of the indicated temperatures.

Figure 3

Lysis of uninfected and P. berghei-infected rodent RBCs using different isotonic solutions at varying temperatures. Lysis assays (n = 3) were performed on iRBCs (10% parasitemia) and uRBCs (negative control) using either guanidinium chloride (a), L-isoleucine (b), L-alanine (c), or D-alanine (d) at the specified temperatures, and the percentage of RBC lysis after 10 min was calculated relative to lysis with saponin (100% lysis) (*** p ≤ 0.001, **** p ≤ 0.0001).

Figure 4

Lysis of P. berghei-infected and uninfected rodent RBCs at varying incubation times. Lysis assays (n = 3) were conducted on iRBC (10% parasitemia) and uRBC samples (negative control) using either guanidinium chloride (a), L-isoleucine (b), or L-alanine (c). The assays were carried out at 37 °C for the specified times and the percentage lysis was calculated relative to saponin (100% lysis) (** p ≤ 0.01, **** p ≤ 0.0001).

Figure 5

Lysis of P. berghei- and P. falciparum-infected and uninfected RBCs using NPP inhibitors furosemide and NPPB. Lysis assays (n = 3) were conducted on iRBCs (10% parasitemia) and uRBCs (negative control) from P. falciparum (a,b) and P. berghei (c,d) using either sorbitol (a), guanidinium chloride (b,c), or L-alanine (d). The assays were carried out at 37 °C for 10 min in the presence and absence of 200 μM furosemide, 200 μM 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) or DMSO as a negative (vehicle) control, and the percentage lysis was calculated relative to saponin (100% lysis) (** p ≤ 0.01, **** p ≤ 0.0001).

Figure 6

Lysis of synchronous populations of P. berghei-infected and uninfected RBC. Lysis assays (n = 4) were conducted on iRBC (average parasitemia 1.65%, ±0.32%) and uRBC samples (negative control) using guanidinium chloride. The assays were carried out at 37 °C for 10 min and the percentage lysis was calculated relative to saponin (100% lysis). Populations of ring and trophozoite parasites were used in the assay, where trophozoites were matured from ring stages in vitro (**** p ≤ 0.0001).

Figure 7

Survival assay following guanidinium chloride lysis assay. (a) Ring-stage P. berghei parasites 0-4 h prior to resuspension in guanidinium chloride. (b) Ring-stage parasites following resuspension in guanidinium chloride. (c) Trophozoite-stage parasites ~16 h later. (d) Schizont-stage parasites ~24 h following guanidinium chloride lysis assay (n = 3).