Abstract
Avoidance of antibody-mediated immune recognition allows parasites to establish chronic infections and enhances opportunities for transmission. The human malaria parasite Plasmodium falciparum possesses a number of multi-copy gene families, including var, rif, stevor and pfmc-2tm, which encode variant antigens believed to be expressed on the surfaces of infected erythrocytes. However, most studies of these antigens are based on in vitro analyses of culture-adapted isolates, most commonly the laboratory strain 3D7, and thus may not be representative of the unique challenges encountered by P. falciparum in the human host. To investigate the expression of the var, rif-A, rif-B, stevor and pfmc-2tm family genes under conditions that mimic more closely the natural course of infection, ex vivo clinical P. falciparum isolates were analyzed using a novel quantitative real-time PCR approach. Expression patterns in the clinical isolates at various time points during the first intraerythrocytic developmental cycle in vitro were compared to those of strain 3D7. In the clinical isolates, in contrast to strain 3D7, there was a peak of expression of the multi-copy gene families rif-A, stevor and pfmc-2tm at the young ring stage, in addition to the already known expression peak in trophozoites. Furthermore, most of the variant surface antigen families were overexpressed in the clinical isolates relative to 3D7, with the exception of the pfmc-2tm family, expression of which was higher in 3D7 parasites. Immunofluorescence analyses performed in parallel revealed two stage-dependent localization patterns of RIFIN, STEVOR and PfMC-2TM. Proteins were exported into the infected erythrocyte at the young trophozoite stage, whereas they remained inside the parasite membrane during schizont stage and were subsequently observed in different compartments in the merozoite. These results reveal a complex pattern of expression of P. falciparum multi-copy gene families during clinical progression and are suggestive of diverse functional roles of the respective proteins.
Highlights
During erythrocyte schizogony, the human malaria parasite Plasmodium falciparum modifies the host cell surface, and these alterations have been linked to immune evasion and falciparum malaria pathology
Quantitative analysis of overall transcription of the multi-copy gene families in different P. falciparum clinical isolates was carried out using degenerate primer pairs designed to amplify semiconserved regions of the var [16,41], rif-A, rif-B, stevor and pfmc-2tm genes
PCR was carried out using genomic DNA from the clinical isolates as a template in order to control for primer pair performance in different P. falciparum isolates
Summary
The human malaria parasite Plasmodium falciparum modifies the host cell surface, and these alterations have been linked to immune evasion and falciparum malaria pathology. VSAs displayed on the surface of infected erythrocytes (IEs) mediate binding to endothelial cells, thereby enabling the parasite to avoid passage through the spleen, recognition and subsequent killing [2,3,4] These parasite-encoded membrane proteins are exposed to the host immune system. 143 rif (repetitive interspersed family), 32 stevor (subtelomeric variable open reading frame) and 13 pfmc-2tm (P. falciparum Maurer’s clefts 2 transmembrane) genes have been identified in the 3D7 reference genome These genes are expressed during red blood cell schizogony and in other developmental stages of P. falciparum, including sexual blood stages, as well as in infective sporozoites, which indicates that these genes are functionally important during most phases of the P. falciparum life cycle [15,16,17,18,19,20,21,22,23,24]. STEVOR proteins can be observed at either the apical tip or the parasite surface during the invasive stage, depending on the antisera and parasite strain used for detection [22,23,24]
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