Exposure of the Plasmodium falciparum clonally variant STEVOR proteins on the merozoite surface

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Abstract

Background: Plasmodium falciparum merozoites are free invasive forms that invade host erythrocytes in iterative cycles in the presence of different arms of the immune system. Variant antigens are known to play a role in immune evasion and several gene families coding for variant antigens have been identified in P. falciparum. However, none of them have been reported to be expressed on the surface of merozoites. Methods. Flow cytometry, immunofluorescence microscopy, and immunoblotting assays were performed to assess surface exposure, membrane association and stage specific expression of the STEVOR family of variants proteins, respectively. Results: Using a polyclonal antibody (anti-PFL2610w) with a broad specificity towards different STEVOR variants, the STEVOR proteins were identified on the surface of non-permeabilized/non-fixed merozoites in flow cytometry assays. Anti-PFL2610w antibody showed that several STEVORs were expressed in the trophozoite stage of the parasite but only one variant was integrated into the merozoite membrane. Moreover, this antibody failed to identify STEVORs on the surface of the parent schizont infected erythrocytes (IE) although they were readily identified when schizont IE were permeabilized. Conclusions: These data suggest for a role for STEVOR in immune evasion by P. falciparum merozoites to allow successful invasion of erythrocytes. Additionally, the expression of STEVORs in the schizont stage may only represent a step in the biogenesis process of the merozoite surface coat. © 2011 Khattab and Meri; licensee BioMed Central Ltd.

Figures

  • Figure 1 Domain architecture of the STEVOR protein family. The recombinantly expressed STEVOR used to produce the antiPFL2610w antibody is delineated by the arrows. Abbreviations: signal peptide (SP), conserved region (CR), hypervariable region (HR), transmembrane domain (TM) and cytoplasmic tail (CT).
  • Figure 2 Recognition of STEVORs in non-permeabilized and saponin-permeabilized schizont infected erythrocytes (IE). (A, lower panel) Flow cytometry histogram of non-permeabilized schizont IE recognized by the pre-immune serum (dot line) and by the anti-PFL2610w antibody (dot-dash line). (A, upper panel) Fluorescence microscopy image of the schizont IE that were recognized by anti-PFL2610w antibody in flow cytometry. (B, lower panel) Flow cytometry histogram of permeabilized schizont IE recognized by the anti-PFL2610w antibody (solid line). (B, upper panel) fluorescence microscopy image of the permeabilized schizont IE recognized by anti-PFL2610w antibody in flow cytometry.
  • Figure 3 Colocalization of STEVORs with MSP-1 at the free merozoite membranes. Fluorescence staining using anti-PFL2610w and anti-MSP-1 antibodies was analysed in free merozoites. (A) Bright field, (B) Alexa 488 stained STEVORs, (C) Alexa 594 stained MSP-1, (D) DAPI stained parasite nuclei, (E) the overlay of STEVORs and MSP-1 (overlay 1) and (F) the overlay of STEVORs, MSP-1 and nuclei (overlay 2) images are shown.
  • Figure 4 Detection of STEVORs on the surface of merozoites by flow cytometry. (A) Merozoite preparation stained with Giemsa. (B and C, upper panels) Dot-plots of forward scatter (FSC) to side scatter (SSC), merozoite populations are surrounded by the R1 gate. (B and C, lower panel) Dot-plots of Ethidium bromide- to Alexa 488-derived fluorescence of the gated populations. (C, lower panel) double-positive merozoite population demonstrating surface exposure of STEVORs at the upper right quadrant.
  • Figure 5 Stage-specific expression of STEVORs. (A) Western blot analysis of total protein from saponin permeabilized erythrocytes (nE), ring IE (R), trophozoite IE (T), and merozoites (M) probed with anti-PFL2610w. (B) A blot similar to (A) probed with antiGlycophorin A antibody to monitor for the presence of erythrocyte membranes. Migration of molecular mass markers is indicated on the left. Estimations of the molecular masses of the identified STEVORs are indicated on the right. Abbreviations: Glycophorin A (GPA), Glycophorin B (GPB).
  • Table 1 Quantification of STEVOR bands identified by the anti-PFL2610w in Western blot analysis

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APA

Khattab, A., & Meri, S. (2011). Exposure of the Plasmodium falciparum clonally variant STEVOR proteins on the merozoite surface. Malaria Journal, 10. https://doi.org/10.1186/1475-2875-10-58

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