DARC extracellular domain remodeling in maturating reticulocytes explains Plasmodium vivax tropism

Abstract

Plasmodium vivax is the most prevalent parasite species that causes malaria in humans and exclusively infects reticulocytes. Reticulocyte infection is facilitated by P vivax Duffy binding protein (DBP), which utilizes DARC (Duffy antigen receptor for chemokines) as an entry point. However, the selective tropism of P vivax for transferrin receptor (CD71)-positive reticulocytes remained unexplained, given the constitutive expression of DARC during reticulocyte maturation. CD71/RNA double staining of reticulocytes enriched from adult peripheral blood reveals 4 distinct reticulocyte populations: CD71^high/RNA^high (∼0.016%), CD71^low/RNA^high (∼0.059%), CD71^neg/RNA^high (∼0.37%), CD71^neg/RNA^low (∼0.55%), and erythrocytes CD71^neg/RNA^neg (∼99%). We hypothesized that selective association of DBP with a small population of immature reticulocytes could explain the preference of P vivax for reticulocytes. Binding of specific monoclonal anti-DARC antibodies and recombinant DBP to CD71^high/RNA^high reticulocytes was significantly higher compared with other reticulocyte populations and erythrocytes. Interestingly, the total DARC protein throughout reticulocyte maturation was constant. The data suggest that selective exposure of the DBP binding site within DARC is key to the preferential binding of DBP to immature reticulocytes, which is the potential mechanism underlying the preferential infection of a reticulocyte subset by P vivax.

Figure 1.

Total protein expression of DARC does not change significantly during reticulocyte maturation. (A) Reticulocytes were purified from peripheral blood as described in “Study design” and stained with CD71 and TO (RNA). The flow cytometry dot plot shows 4 subpopulations of circulating reticulocytes in adult peripheral blood: CD71^high/TO^high (R1), CD71^low/TO^high (R2), CD71^neg/TO^high (R3), CD71^neg/TO^low (R4), and erythrocytes (E). (B-C) Quantification of the dot plot shown in panel A, after reticulocyte enrichment (B; N = 13), and normalized to the actual frequencies in adult peripheral blood (C; N = 13). (D) Reticulocyte subpopulations defined in panel A were sorted, and total protein lysate was subjected to SDS-polyacrylamide gel electrophoresis and western blotting. Blots were stained with anti-band 3 (top) and anti-DARC (clone 2C3; bottom). Note that DARC (calculated peptide mass, 35 kDa) presents as a diffuse pattern of bands as previously described. *a-Specific bands (supplemental Figure 1A).^,

Figure 2.

DARC-specific antibodies and DBP display significantly higher binding to CD71^high/TO^highreticulocytes compared with other reticulocyte subsets or erythrocytes. (A) Schematic representation of 7-transmembrane spanning topology of DARC. N-terminal extracellular domain contains binding site for 2C3 and Fy6 antibodies, which overlap with DBP domain, indicated in gray. The Fy3 binding domain is localized at the third extracellular loop outside the DBP binding pocket. (B) Fold change representation of binding of DARC-specific antibodies to the 4 reticulocyte subpopulations and erythrocytes, as defined in Figure 1A. Enriched fraction of reticulocytes was probed with anti-Fya, anti-Fyb, Fy3, 2C3, and Fy6 antibodies followed by staining with anti-CD71 and TO. Figure is depicted as a fold change normalized to erythrocytes (anti-Fy6; anti-2C3; anti-Fya; anti-Fyb; anti-Fy3; n ≥ 6). (C) Fold change representation CD235a (glycophorin A [GPA]) antibody binding to the different reticulocyte populations and erythrocytes (N = 3). (D) Enriched fraction of reticulocytes was incubated with biotinylated recombinant DBP and counterstained with anti-CD71 and TO. A representable histogram semioffset is depicted, and a quantification of the mean fluorescent intensity (MFI) of DBP-binding (N = 6). Student t test (paired) was used to calculate statistical significance (*P < .05; **P < .01; ***P < .001; ****P < .0001). (E) ImageStream images depicting erythrocytes (top 2 panels), TO^high/lowCD71^neg (middle), and TO^highCD71^high (bottom) stained with RNA (TO, green), CD71 (red), and DBP (purple) as well as normal light. (F) Binding of CD71⁺ reticulocytes on SRP-streptavidine biosensor. CD71⁺ reticulocytes were isolated as indicated in “Study design” and flowed over biosensor coated with DBP (1000 nM) and anti-GPA (n > 100). The response and sedimentation signals were measured and represented as normalized total response/sedimentation response (T/S). Columns and bars represent means and standard deviation, respectively. One-way analysis of variance was used to calculate statistical significance (*P < .05; **P < .01; ***P < .001; ****P < .0001). BSA, bovine serum albumin; IgG, immunoglobulin G.