In vivo studies support the role of trafficking and cytoskeletal-binding motifs in the interaction of MESA with the membrane skeleton of Plasmodium falciparum-infected red blood cells

Abstract

In red blood cells (RBCs) infected with the malaria parasite Plasmodium falciparum, a 19-residue region of the mature parasite-infected erythrocyte surface antigen (MESA) associates with RBC cytoskeleton protein 4.1R; an interaction essential for parasite survival. This region in MESA is adjacent to a host targeting motif found in other malaria parasite proteins exported to the membrane skeleton. To demonstrate function of these motifs in vivo, regions of MESA fused to a reporter were expressed in malaria parasites. Immunochemical analyses confirmed the requirement for both motifs in the trafficking and interaction of MESA with the cytoskeleton and demonstrates their function in vivo.

Fig. 1

(A) Schematic representation of the two-exon structure of MESA showing the locations of the embedded signal peptide, intron splice site, putative Pexel, 4.1R binding site and the GESKET repeats. Below the schematic is the amino acid sequence of the N-terminal portion of MESA as indicated by the dashed lines. The exon 1 sequence is shaded in grey, the embedded signal peptide is shown in lowercase, the putative Pexel is underlined and the 19-residue 4.1R binding site is boxed. (B) Reporter constructs used for transfection of P. falciparum 3D7. The structural features located within the N-terminal sub-fragments of MESA are highlighted as described in part (A) above. The S-antigen sequence (allele from P. falciparum isolate FC27) is shaded in grey. Control plasmid Sag contains S-antigen alone (with its endogenous signal peptide). Plasmid SP contains the first exon of MESA (amino acids 1 to 50), which encodes an embedded hydrophobic signal peptide, fused to the S-antigen reporter. Plasmid Pexel+4.1BS contains the signal peptide, putative Pexel and 4.1R binding site of MESA (amino acids 1 to 204) fused to the S-antigen reporter. Plasmid Δ4.1BS contains the signal peptide and putative Pexel of MESA (amino acids 1 to 95) fused to the S-antigen reporter. (C) Percoll purified transfectant parasite lines were resolved on SDS-PAGE (under denaturing conditions) and transferred to polyvinylidene fluoride (PVDF) membranes (NEN) for immunoblotting as previously described [17]. Polyclonal antiserum raised in rabbits against S-antigen (isolate FC27) was used as the primary antibody (1:1000 dilution). The primary antibody was detected using anti-rabbit immunoglobulin (Ig) conjugated to horseradish peroxidase (Silenus) (1:2000 dilution) and developed as previously described [17]. Lane 1, wild-type 3D7 parasites; lane 2, 3D7_Sag parasites; lane 3, 3D7_SP parasites; lane 4, 3D7_Pexel+4.1BS parasites; lane 5, 3D7_Δ4.1BS parasites.

Fig. 2

(A) Immunofluorescence (IFA) analysis of the transfectant parasite lines using rabbit anti-S-antigen (isolate FC27) (1:500 dilution) and mouse anti-Glycophorin A (GpA) (Caltag Laboratories) (1:100 dilution) as primary antibodies. Reactivity was detected using Alexa Fluor 488-conjugated anti-rabbit Ig and Alexa Fluor 568-conjugated anti-mouse Ig as secondary antibodies (Invitrogen) (1:400 dilution). Smears from asynchronous parasite cultures were examined by confocal microscopy (Olympus FluoView FV1000). (B) Triton X-100 (TX-100) solubilisation to determine association with the RBC cytoskeleton. Asynchronous P. falciparum parasites were cultured to ~6% parasitemia and harvested using 0.15% (w/v) saponin and washed with PBS. The purified parasites were solubilised in cold (4°C) TX-100 and the resulting insoluble material was then solubilised in SDS. The samples include the total parasite material (T), the TX-100 soluble material (S) and the TX-100 insoluble, SDS soluble material (P). The samples were then resolved (under denaturing conditions) on SDS-PAGE and transferred to PVDF membranes (NEN) for immunoblotting as previously described [17]. Polyclonal antiserum raised in rabbits against S-antigen (strain FC27) was used as the primary antibody (1:1000 dilution). The primary antibody was detected using anti-rabbit Ig conjugated to horseradish peroxidase (Silenus) (1:2000 dilution) and developed as previously described [17].