Characterisation of erythrocyte binding activity of Plasmodium cynomolgi duffy binding protein haplotypes from Macaca fascicularis in Peninsular Malaysia.

Human infections with the zoonotic Plasmodium cynomolgi are rising across Southeast Asia. This parasite employs two Duffy binding proteins (DBPs), PcDBP1(II) and PcDBP2(II), which interact with the Duffy antigen receptor chemokine (DARC) for erythrocyte invasion. Previous studies on P. cynomolgi DBPs have reported two distinct haplotype groups (groups 1 and 2) in Peninsular Malaysia. In this study, the differential erythrocyte binding activity of PcDBP1(II) and PcDBP2(II) haplotypes was investigated. Four isolates were selected for each DBP: group 1 consisted of a recent field isolate and the old reference isolate Strain B, whereas group 2 included a recent field isolate and the old reference isolate Berok. All isolates were cloned into the pDisplay-AcGFP1 vector and transfected into COS-7 cells. The transfected cells were incubated with human erythrocytes/reticulocytes and macaque erythrocytes at 1 % haematocrit. The binding activity was assessed by counting the number of rosette formations. Result showed a significant difference (P < 0.05) in binding between group 1 isolates (recent vs old) for PcDBP1(II) with macaque erythrocytes, while no differences in binding were seen with PcDBP2(II) isolates. Notably, the recent PcDBP1(II) isolate exhibited significantly higher binding activity compared to the old isolate, indicating that the protein may have evolved over time to enhance parasite invasion into macaque erythrocytes. However, both PcDBPs showed no binding with human erythrocytes or reticulocytes. This study provides the first evidence that variations in PcDBP1(II) and PcDBP2(II) alone do not explain the observed differences in binding activity, suggesting that additional parasite-host interactions may be required for human erythrocyte or reticulocyte invasion.