Abstract
In vitro cultivation of Plasmodium falciparum is critical for studying the biology of this parasite. However, it is likely that different in vitro cultivation conditions influence various aspects of the parasite’s life cycle. In the present study two P. falciparum isolates were cultivated using the two most common methods, in which AlbuMAX or human serum as additives are used, and the results were compared. The type of cultivation influenced the knob structure of P. falciparum-infected erythrocytes (IEs). IEs cultivated with AlbuMAX had fewer knobs than those cultivated with human serum. Furthermore, knob size varied between isolates and is also depended on the culture medium. In addition, there was a greater reduction in the cytoadhesion of IEs to various endothelial receptors in the presence of AlbuMAX than in the presence of human serum. Surprisingly, cytoadhesion did not correlate with the presence or absence of knobs. Greater numbers of the variant surface antigen families RIFIN, STEVOR, and PfMC-2TM were found at the IE membrane when cultivated in the presence of AlbuMAX. Moreover, the type of cultivation had a marked influence on the transcriptome profile. Compared with cultivation with human serum, cultivation with AlbuMAX increased the expression of approximately 500–870 genes.
Highlights
Molecules involved in these receptor-ligand interactions belong to the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family
1) the physical appearance of infected erythrocytes (IEs) was analysed by transmission electron microscopy and atomic force microscopy, 2) the cytoadhesion of IEs to seven different human endothelial receptors was investigated using transgenic Chinese hamster ovary (CHO) cells expressing the respective receptors on their surface, 3) the localization of members of the variant surface antigen families RIFIN, STEVOR and PfMC-2TM was visualized by immunofluorescence microscopy using family specific, cross-reactive antibodies, and 4) the transcriptome of ring-stage FCR3 parasites was analyzed by mRNA sequencing
Laboratory isolates 3D7 and FCR3 were cultivated in the presence of 0.5% AlbuMAX or 10% human serum for at least three months (Fig. 1)
Summary
Molecules involved in these receptor-ligand interactions belong to the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family. Ex vivo-isolated IEs had a higher knob density than erythrocytes infected by in vitro-cultured parasites[18]. Low knob density on long-term in vitro-cultivated parasites[19] and a complete loss of knobs after prolonged in vitro culture[20] have been reported; some of the knobless isolates showed strong cytoadherence[16,21]. Supplementation with AlbuMAX improves growth profiles and has several additional advantages over human serum[26] It is unclear whether cultivating P. falciparum in AlbuMAX-supplemented medium has an effect on knob formation/size, cytoadhesion to human endothelial receptors, localization of variant surface antigens and, last but not least, the transcript profile ( that of var genes). We cultivated two P. falciparum isolates (3D7 and FCR3) either in the presence of AlbuMAX or human serum and subjected them to a comprehensive comparative analysis. 1) the physical appearance of IEs was analysed by transmission electron microscopy and atomic force microscopy, 2) the cytoadhesion of IEs to seven different human endothelial receptors was investigated using transgenic CHO cells expressing the respective receptors on their surface, 3) the localization of members of the variant surface antigen families RIFIN, STEVOR and PfMC-2TM was visualized by immunofluorescence microscopy using family specific, cross-reactive antibodies, and 4) the transcriptome of ring-stage FCR3 parasites was analyzed by mRNA sequencing
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