Abstract
For intracellular pathogens, the host cell provides needed protection and nutrients. A major challenge of intracellular parasite research is collection of high parasite numbers separated from host contamination. This situation is exemplified by the malaria parasite, which spends a substantial part of its life cycle inside erythrocytes as rings, trophozoites, and schizonts, before egress and reinvasion. Erythrocytic Plasmodium parasite forms refractory to enrichment remain understudied due to high host contamination relative to low parasite numbers. Here, we present a method for separating all stages of Plasmodium-infected erythrocytes through lysis and removal of uninfected erythrocytes. The Streptolysin O-Percoll (SLOPE) method is effective on previously inaccessible forms, including circulating rings from malaria-infected patients and artemisinin-induced quiescent parasites. SLOPE can be used on multiple parasite species, under multiple media formulations, and lacks measurable impacts on parasite viability. We demonstrate erythrocyte membrane cholesterol levels modulate the preferential lysis of uninfected host cells by SLO, and therefore modulate the effectiveness of SLOPE. Targeted metabolomics of SLOPE-enriched ring stage samples confirms parasite-derived metabolites are increased and contaminating host material is reduced compared to non-enriched samples. Due to consumption of cholesterol by other intracellular bacteria and protozoa, SLOPE holds potential for improving research on organisms beyond Plasmodium.
Highlights
For intracellular pathogens, the host cell provides needed protection and nutrients
This is due in part to the larger biomass of these stages and to the existence of effective enrichment methods[7]; erythrocytes infected with late stage parasites can be separated from uninfected erythrocytes by density gradient centrifugation or by using the paramagnetic properties of hemozoin, a byproduct of parasite maturation
This limitation is acute when dealing with material directly from malaria patients as only ring forms of P. falciparum are collected during blood draws[19], and there is a high ratio of uninfected host cells to parasite-infected cells[20]
Summary
The host cell provides needed protection and nutrients. A major challenge of intracellular parasite research is collection of high parasite numbers separated from host contamination. Recent proteomics and metabolomics studies of this early erythrocytic P. falciparum show the heavy influence of host metabolites in non-enriched preparations, which contributes to variability between samples and obscures parasite phenotypes[15,17,18] This limitation is acute when dealing with material directly from malaria patients as only ring forms of P. falciparum are collected during blood draws[19], and there is a high ratio of uninfected host cells to parasite-infected cells (typically 100:
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