Erythrocyte-binding assays reveal higher binding of Plasmodium knowlesi Duffy binding protein to human Fya+/b+ erythrocytes than to Fya+/b- erythrocytes

Abstract

Background: The merozoite of the zoonotic Plasmodium knowlesi invades human erythrocytes via the binding of its Duffy binding protein (PkDBPαII) to the Duffy antigen on the eythrocytes. The Duffy antigen has two immunologically distinct forms, Fy^a and Fy^b. In this study, the erythrocyte-binding assay was used to quantitatively determine and compare the binding level of PkDBPαII to Fy^a+/b+ and Fy^a+/b- human erythrocytes.

Results: In the erythrocyte-binding assay, binding level was determined by scoring the number of rosettes that were formed by erythrocytes surrounding transfected mammalian COS-7 cells which expressed PkDBPαII. The assay result revealed a significant difference in the binding level. The number of rosettes scored for Fy^a+/b+ was 1.64-fold higher than that of Fy^a+/b- (155.50 ± 34.32 and 94.75 ± 23.16 rosettes, respectively; t₍₆₎ = -2.935, P = 0.026).

Conclusions: The erythrocyte-binding assay provided a simple approach to quantitatively determine the binding level of PkDBPαII to the erythrocyte Duffy antigen. Using this assay, PkDBPαII was found to display higher binding to Fy^a+/b+ erythrocytes than to Fy^a+/b- erythrocytes.

Keywords: Duffy antigen; Duffy binding protein; Erythrocyte-binding assay; Plasmodium knowlesi.

Fig. 1

Erythrocyte-binding assay to determine binding activity of PkDBPαII to erythrocytes. a Rosette formation (red arrow) on PkDBPαII-pDisplay™ transfected COS-7 cell, with more than 50% of the cell surface covered by adherent erythrocytes. b Nuclei of COS-7 cells are stained blue with Hoechst dye. c Transfected COS-7 cells show green fluorescence indicating expression of GFP fluorescence tag and PkDBPαII. d Merged images of a, b and c showing the location of rosette, transfected cells and their nuclei