. 2014 Jul 9;16(1):81-93.
STEVOR Is a Plasmodium Falciparum Erythrocyte Binding Protein That Mediates Merozoite Invasion and Rosetting
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STEVOR Is a Plasmodium Falciparum Erythrocyte Binding Protein That Mediates Merozoite Invasion and Rosetting
Cell Host Microbe
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Variant surface antigens play an important role in Plasmodium falciparum malaria pathogenesis and in immune evasion by the parasite. Although most work to date has focused on P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1), two other multigene families encoding STEVOR and RIFIN are expressed in invasive merozoites and on the infected erythrocyte surface. However, their role during parasite infection remains to be clarified. Here we report that STEVOR functions as an erythrocyte-binding protein that recognizes Glycophorin C (GPC) on the red blood cell (RBC) surface and that its binding correlates with the level of GPC on the RBC surface. STEVOR expression on the RBC leads to PfEMP1-independent binding of infected RBCs to uninfected RBCs (rosette formation), while antibodies targeting STEVOR in the merozoite can effectively inhibit invasion. Our results suggest a PfEMP1-independent role for STEVOR in enabling infected erythrocytes at the schizont stage to form rosettes and in promoting merozoite invasion.
Copyright © 2014 Elsevier Inc. All rights reserved.
Conflict of interest statement
Conflict of interest
All authors declare that they have no competing interest.
In vitro inhibition of P. falciparum merozoite invasion by anti-STEVOR antibodies
Invasion of 5A (
A), 3.2C ( B), 5.2A ( C) 5B and A4 ( D) P. falciparum parasite clones in the presence of different dilutions of anti-S1 and -S2 sera. Rabbit anti-PfMSP1.19 antibody (1:10 dilution) and pre-immune serum (PI, 1:10) were used as positive and negative controls respectively. Data are presented as the percentage of inhibition normalized to control without antiserum and represent the average of three independent experiments. Error bars denote two Standard Deviations (SD). E- Specificity- and concentration-dependent inhibition of 5A merozoite invasion into RBCs by IgG purified from rabbit anti-S1. Purified Transferrin protein that co-precipitated with the IgG fraction (see also Figure S1A) did not inhibit merozoite invasion. F- Dual life IFA staining of 5A-merozoite with anti-S2 (green) and anti-MSP1.19 (red) showing constant expression and location of STEVOR on the merozoite surface at different steps of the invasion process (free merozoite, attachment, reorientation and junction). Fixed IFA confirming STEVOR location on the merozoite surface in cytochalasin D-junction arrested 5A merozoites (junction). The fluorescent images (individual stains and merged images) and the bright field (BF) are shown. The dotted white lines delineate the uninfected RBC membrane.
Figure 2. Erythrocyte binding of COS7 cells expressing different STEVOR regions
A- Chimeric constructs for the expression of different STEVOR regions on the surface of COS-7 cells (see details in supplemental methods). The generation of ST1Full and ST2Full was unsuccessful and therefore removed from the figure. The 1TM model of STEVOR is depicted here based on recent findings (Joannin et al., 2011; Niang et al., 2009). Dashed line delineates the regions served for generation of anti-S1 and -S2. B- Erythrocyte binding of COS-7 cells expressing the SC and HVR regions of the three stevor genes. The P. vivax Duffy Binding Protein II (PvDBPII) and the empty pDisplay vector (pDispl) and/or non-transfected COS-7 (NT) were used as controls. Binding of the ST3Full is also shown. C- Binding of the three SC regions to untreated and enzyme-treated erythrocytes showing significant reduction of binding following trypsin and neuraminidase treatments. D- Pattern of inhibition of the binding of the three SC regions by anti-STEVOR antibodies. Preimmune serum (PI) at 1:100 dilution was used as control. Error bars represent standard deviations (SD) of 3 independent experiments. Effects of anti-S1 and anti-S2 were statistically compared to control (no antibody), significant differences are shown by asterisk (* indicates p<0.05, ** indicates P<0.01).
Figure 3. Rosetting enrichment and STEVOR expression of 5A, 3.2C and 5.2A (R+) and (R-) parasites
A- Live wet-preparation images of 5A rosetting parasites in whole field (i) and at single iRBC level (ii-v) viewed under fluorescent and direct light microscope at 100X magnification. Infected RBCs were stained with ethidium bromide. B-C Western blot of schizont extracts from unselected (UNS), R+ and R- 5A (B), 3.2C and 5.2A (C) clones with rabbit anti-S1 or -S2 showing higher STEVOR expression in R+ parasites. Mouse anti-GPC and rabbit anti-actin were used to show equal loading. D- Disruption (reversal) and blockage (reformation) of 5A rosetting by anti-S1 and -S2. Effects of anti-S1 and anti-S2 were statistically compared to control. Asterisk shows significant difference (p<0.05). E- Western blot of 3D7 schizont extract performed with a low percentage gel showing the specific recognition of the lower molecular STEVOR by anti-STEVOR sera (white arrow) and the absence of cross-reactivity with PfEMP1 (black arrows). PfEMP1 was only detected by anti-ATS antibody (black arrows). Data are expressed as percentage of rosettes in 200 counts. Error bars represent SD of two independent experiments.
Figure 4. Functional investigation of STEVOR requirement for rosetting of A4 parasites
A- Generation of pARL-STEVOR1-GFP construct encoding a full length stevor gene (PF10_0395) driven by the cg4 promoter obtained from modification of the pARL-STEVOR 80 vector. B- Western blot validation of the correct GFP fusion protein expression with anti-GFP antibody and anti-GPA antibody (loading control). C- Live IFA showing correct surface trafficking of the GFP fusion protein using anti-S1 antibody. From left to right are shown bright field (BF), nuclei staining (DAPI), GFP, anti-S1 staining and merged images. D- Live wet-preparation images of A4-pARL-STEVOR1-R+ iRBCs viewed under fluorescent and direct light microscope at 100X magnification. Parasite nuclei were stained with DAPI (2 μg/ml); GFP expression is shown as green dots within the iRBCs. E- Disruption of A4-pARL-STEVOR1 rosetting by anti-STEVOR sera showing 62.5% inhibition by anti-S1 compared to 20% for anti-S2. Data represent average of two independent experiments. Error bars denote two standard deviation. F- Microarray analysis of stevor transcript levels of 5A-R+ and 5A-R-. Red and green indicate upregulated and downregulated genes, respectively, in relation to a pool of RNA.
Figure 5. Analysis of STEVOR expression in the 3D7ΔMAHRP1 parasites
A- Detection of STEVOR on live 3D7ΔMAHRP1-R+ -iRBCs surface by anti-S1 and -S2 (top panel). Dual staining of live 3D7ΔMAHRP1-R+ -iRBCs with anti-S2 and anti-GPC antibodies (bottom panel, top row) or anti-SBP1 (bottom panel, middle row) illustrating the external location of both STEVOR and GPC and lack of surface staining of 3D7ΔMAHRP1-R+ iRBCs with anti-SBP1, respectively. Confirmation of SBP1 internal location is revealed by IFA on fixed parasites (bottom panel, bottom row). B- Live wet preparation images showing successful rosetting enrichment of 3D7ΔMAHRP1 line. C- Immunoblotting with rabbit anti-S2 showing increased STEVOR expression in 3D7ΔMAHRP1-R+. Rabbit anti-actin was used to show equal loading. D- Statistically compared inhibition of 3D7ΔMAHRP1 rosetting by anti-S1 and -S2 on rosetting reversal and reformation assays. Significant difference (P<0.05) is shown by an asterisk. E- Isolation and establishment of monovariant 3D7ΔMAHRP1 cultures expressing abundant STEVOR at the cell surface (dark blue subpopulation) by sorting and re-culturing the anti-S1 and -S2 (See also Figure S3E) surface positive iRBCs subpopulations (top panel) and re-evaluation for surface-positive STEVOR (see also Figure S3E) by flow cytometry after 5 weeks of weekly rosetting enrichment. F- Microarray analysis comparing stevor mRNA level in unselected (UNS)-, R- and R+ 3D7ΔMAHRP1-iRBCs; red and green indicate upregulated and downregulated genes respectively in relation to a pool of RNA. G- Rosette reformation of purified 5A-, 3D7ΔMAHRP1- and pARL-STEVOR1-R+ parasites with normal, trypsin-, chymotrypsin- and neuraminidase-treated RBCs revealing the chymotrypsin-resistant phenotype of all tested lines.
Figure 6. STEVOR binding to RBC correlates with the level of GPC on the RBC surface
A- Effects of RBC treatment with 50 μg/ml of monoclonal antibodies against CD36, GPA, CR1 and GPC on rosette reformation of purified 5A-R+ iRBCs. Untreated RBCs (control) or RBCs treated with preimmune serum (PI) were used as controls. Data represent the average of two independent experiments. B and C- Concentration-dependent inhibition of rosetting of purified 5A-R+ and 3D7ΔMAHRP1-R+ iRBCs by soluble GPC ( B) and binding of COS7 expressing ST3C construct ( C). Soluble GPC reduces both the number of ST3C-expressing COS7 cells binding to RBC ( C, top panel graph) as well the size of rosettes formed ( C, bottom panel images) in a concentration-dependent manner compared to control (ST3C-UN). PvDBPII and untransfected (NT) and/or pDisplay vector-transfected (pDispl) were used as positive and negative controls for binding, respectively. D- Impairment (top panel graph) of binding of COS7 expressing ST3C to Gerbich negative (G(-)) RBC compared to normal RBC (nRBC) as illustrated by micrograph images (bottom panel images). PvDBPII and untransfected (NT) and/or pDisplay vector-transfected (pDispl) were used as positive and negative control for binding respectively. Data are presented as average of two independent experiments, each performed in duplicate. E- Pull down assay with recombinant STEVOR (rSTEVOR) and Glycophorin C (rGPC) in the presence (+) or absence (-) of anti-STEVOR or anti-GPC antibodies showing specific interactions of STEVOR and GPC (lanes 1 and 2). F and G- Impact of GPC knock down (KD) on rosetting of 5A-iRBCs ( F) and ST3C binding ( G). Rosetting was greatly reduced when performed with GPC knock down (KD) RBCs following puromycin (Puro, 97% KD compared to pLKO control, see also Figure S5) and Neomycin (Neo, 89% KD compared to pLKO control, Figure S5) selection compared to pLKO. In vitro generated-RBC (cRBC) and donor RBC (RBC) were used as controls. CR1 KD (27% KD compared to pLKO control, see also Figure S5) has no impact on rosetting. For ST3C binding (G), only Puromycin- selected GPC-knock down RBCs were used due to the limited number of available cells. Statistical differences of GPC and CR1-KD in comparison to pLKO control are shown. Data represent the average of three independent experiments. Error bars denote standard deviation.
Figure 7. Targeting of invasion by R+ and R- of 5A parasites
Comparison of invasion efficiency of R+ and R- parasites of 5A (
A), 5.2A ( B) and 3.2C ( C) clones grown in suspension following three consecutive rounds of replication at a 0.1-0.2% starting parasitemia showing growth advantage of R+ over R- parasites. D- Invasion of 5A-, 5.2A- and 3.2C-R+ and -R- parasites in the presence of a high concentration (1:100 dilution) of MSP1.19 inhibitory antibody and non inhibitory pre-immune serum (PI, 1:10 dilution). Data is presented as percentage of inhibition compared to control without serum and represent the average of two independent experiments performed in triplicate each. Statistical comparisons are shown.
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Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Antigens, Protozoan / metabolism*
Erythrocytes / parasitology*
Glycophorins / metabolism*
Plasmodium falciparum / physiology*
Protozoan Proteins / metabolism*
Receptors, Cell Surface / metabolism*
Virulence Factors / metabolism
Duffy antigen binding protein, Plasmodium
STEVOR antigen, Plasmodium falciparum
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