Use of magnetically purified Plasmodium falciparum parasites improves the accuracy of erythrocyte invasion assays

Abstract

Merozoite invasion of erythrocytes is a crucial step for the asexual cycle of Plasmodium falciparum. Multiple invasion pathways, which involve different ligand-receptor interactions, have been identified in P. falciparum by examining the entry of purified parasite into erythrocytes with different surface receptors, either mutant or under different enzyme treatments. The most critical step for a successful invasion assay is the isolation of erythrocytes infected with viable schizonts. Here, we applied a magnetic column to purify the schizonts for the erythrocyte invasion assay. Comparing to Percoll-sorbitol purification method, this modified approach showed great improvement on reproducibility and reliability of invasion assay, particularly for short-term, culture-adapted parasite isolates. The magnetic purification method is an excellent alternative for parasite isolates that do not tolerate or with unknown sensitivity to Percoll-sorbitol exposure.

Figure 1. Erythrocyte invasion rates of four long-term, culture adapted P. falciparum lines

Four different P. falciparum strains (Dd2, MTS1, M5, and 418) adapted to long-term culture were simultaneously purified by either the Percoll-sorbitol (Percoll) or the magnetic column (MACS) method. Purified parasitized erythrocytes were added to untreated or enzyme-treated target erythrocytes and then cultured to allow time for merozoites to invade erythrocytes and develop to mature stages. Invasion rates were obtained by normalizing the parasitemias of enzyme-treated erythrocytes to those of untreated erythrocytes.

Figure 2. Erythrocyte invasion rates of five short-term, culture-adapted P. falciparum isolates

Five parasite isolates (CP191, CP195, CP271, CP286, CP315) were obtained from Cambodian patients with malaria. After 4-5 weeks of in vitro culture, parasitized erythrocytes were purified by either the Percoll-sorbitol (Percoll) or magnetic column (MACS) method. Invasion assays were performed as outlined in the legend to Figure 1, and the parasitemias were measured and compared.