Absence of erythrocyte sequestration and lack of multicopy gene family expression in Plasmodium falciparum from a splenectomized malaria patient

Abstract

Background: To avoid spleen-dependent killing mechanisms parasite-infected erythrocytes (IE) of Plasmodium falciparum malaria patients have the capacity to bind to endothelial receptors. This binding also known as sequestration, is mediated by parasite proteins, which are targeted to the erythrocyte surface. Candidate proteins are those encoded by P. falciparum multicopy gene families, such as var, rif, stevor or PfMC-2TM. However, a direct in vivo proof of IE sequestration and expression of multicopy gene families is still lacking. Here, we report on the analysis of IE from a black African immigrant, who received the diagnosis of a malignant lymphoproliferative disorder and subsequently underwent splenectomy. Three weeks after surgery, the patient experienced clinical falciparum malaria with high parasitemia and circulating developmental parasite stages usually sequestered to the vascular endothelium such as late trophozoites, schizonts or immature gametocytes.

Methodology/principal findings: Initially, when isolated from the patient, the infected erythrocytes were incapable to bind to various endothelial receptors in vitro. Moreover, the parasites failed to express the multicopy gene families var, A-type rif and stevor but expression of B-type rif and PfMC-2TM genes were detected. In the course of in vitro cultivation, the parasites started to express all investigated multicopy gene families and concomitantly developed the ability to adhere to endothelial receptors such as CD36 and ICAM-1, respectively.

Conclusion/significance: This case strongly supports the hypothesis that parasite surface proteins such as PfEMP1, A-type RIFIN or STEVOR are involved in interactions of infected erythrocytes with endothelial receptors mediating sequestration of mature asexual and immature sexual stages of P. falciparum. In contrast, multicopy gene families coding for B-type RIFIN and PfMC-2TM proteins may not be involved in sequestration, as these genes were transcribed in infected but not sequestered erythrocytes.

Figure 1. Plasmodia in peripheral blood of splenectomized patient.

Shown are Giemsa-stained thin blood films containing various parasite stages, including those usually not seen in peripheral blood of P. falciparum malaria, such as late trophozoites (lt), schizonts (s) or immature gametocytes (ig).

Figure 2. Multicopy gene family expression of IE during parasite culture.

(A) Shown are ethidium bromide stained agarose gels of var RT-PCR products of RNA extracted from the ex vivo blood sample as well as from samples of subsequent parasite cultures at time points as indicated. Genomic parasite DNA (gDNA) and water (H₂O), respectively, served as positive and negative controls. To rule out amplification of DNA that might have contaminated the various RNA preparations, all RNA samples were subjected to PCR with (+) and without (−) reverse transcription. (B) RT-PCR products from the multicopy gene families rif, stevor, Pfmc-2TM and from the housekeeping gene seryl-tRNA synthetase from ex vivo parasites and at day 29 of cultivation.

Figure 3. Binding of IE to common endothelial receptors during parasite culture.

IE obtained ex vivo and from the time points day 8, day 15 and day 30 of subsequent parasite cultures were incubated with CHO-745 cells expressing endothelial receptors CD36, ICAM-1, VCAM-1 and P-selectin, respectively. CHO-745 cells expressing GFP were used as negative control. Bound erythrocytes were stained with Giemsa and the number of adherent IE per 100 CHO-745 cells is shown. Assays were performed in triplicates. All data are mean values (± standard deviation).