Two functional reticulocyte binding-like (RBL) invasion ligands of zoonotic Plasmodium knowlesi exhibit differential adhesion to monkey and human erythrocytes

Abstract

BackgroundPlasmodium knowlesi is a monkey malaria species that is becoming a serious public health concern infecting hundreds and perhaps thousands of humans in Southeast Asia. Invasion of erythrocytes by merozoites entails a cascade of molecular interactions. One step involves the adhesion of Plasmodium reticulocyte binding-like (RBL) proteins. Plasmodium knowlesi merozoites express only two RBL invasion ligands, known as Normocyte Binding Proteins (PkNBPXa and PkNBPXb).MethodsOverlapping N-terminal regions of PkNBPXa and PkNBPXb were expressed in COS7 cells and tested for surface expression and adhesion to rhesus monkey erythrocytes. Subsequent tests to study specific receptor ligand interactions included adhesion to a panel of human and non-human primate erythrocytes, enzymatic treatment, and site directed mutagenesis.ResultsAn N-terminal cysteine-rich region of PkNBPXb (PkNBPXb-II) exhibited specific adhesion to rhesus monkey erythrocytes. Mutation of four of five cysteines in PkNBPXb-II interfered with its surface expression on COS7 cells, suggesting disulphide bond conformation is critical for intracellular trafficking. Binding of PkNBPXb-II was abolished when rhesus erythrocytes were pre-treated with chymotrypsin, but not trypsin or neuraminidase. PkNBPXb-II also bound other Old World monkey species and gibbon erythrocytes. However, erythrocytes from other primate species including humans did not bind to PkNBPXb-II or native PkNBPXb. Importantly, unlike PkNBPXb, PkNBPXa bound human erythrocytes, and this binding was independent of the Duffy blood group determinant.ConclusionsThe data reported here begins to clarify the functional domains of the P. knowlesi RBLs. A binding domain has been identified and characterized in PkNBPXb. Notably, this study demonstrates that unlike PkNBPXb, PkNBPXa can bind to human erythrocytes, suggesting that PkNBPXa may function as a ligand to enable the invasion of P. knowlesi merozoites into human cells.

Normocyte-binding protein required for human erythrocyte invasion by the zoonotic malaria parasite Plasmodium knowlesi

“…For example, although PkNBPXa and DBPa proteins bind to both human and macaque RBCs, DBPβ, DBPγ, and NBPXb bind only to macaque RBCs (16,39). This apparent lack of adhesin redundancy for human RBC invasion suggests that P. knowlesi is vulnerable to perturbation of DBPα/NBPXa function.…”

“…However, even the humanadapted P. knowlesi line invades cynomolgus RBCs more efficiently than human RBCs, suggesting that invasion efficiency may remain a bottleneck in human infections. Interestingly, variation in NBPXb, which had been found not to bind to human RBCs, also has been linked to disease severity in humans (7,16). The role of NBPXb and variants of both P. knowlesi NBPs now can be investigated in vitro.…”

“…5). In P. knowlesi two proteins in the RBL family, NBPXa and NBPXb, have been identified (15), and recombinant fragments of these proteins bind RBCs (16). The RBL/RH adhesins and the Duffy-binding protein/erythrocytebinding ligand (DBP/EBL) family of proteins play key roles during merozoite invasion of RBCs (reviewed in refs.…”

Disease Progression in Plasmodium knowlesi Malaria Is Linked to Variation in Invasion Gene Family Members

“…Pknbpxa is located on chromosome 14 and Pknbpxb on chromosome 7[26]. A recent study on an experimental line of P. knowlesi demonstrated binding of Pknbpxa but not Pknbpxb protein products to human erythrocytes implicating Pknbpxa in human erythrocyte invasion [27].…”

Characterizing the genetic diversity of the monkey malaria parasite Plasmodium cynomolgi

“…in a population of knowlesi malaria patients (Ahmed et al , 2014). These two genes are also known to show differential binding between macaque and human erythrocytes (Semenya et al , 2012). Strain-specific CNV has also been found in the antigen gene merozoite surface protein 3 (MSP3) of P. cynomolgi strains B and Berok (Rice et al .…”