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.
Highlights
Plasmodium knowlesi is a monkey malaria species that is becoming a serious public health concern infecting hundreds and perhaps thousands of humans in Southeast Asia
An N-terminal region of PkNBPXb binds to rhesus monkey erythrocytes Three segments from the beginning of the P. knowlesi nbpxa gene and eight encompassing the 5′ half of the P. knowlesi nbpxb gene were cloned into the pDisplay vector (Figure 1A)
The only other reticulocyte binding-like (RBL) ligand expressed by P. knowlesi merozoites, PkNBPXa, in contrast with PkNBPXb, strongly binds human erythrocytes in addition to monkey host erythrocytes by traditional Erythrocyte binding assays (EBAs)
Summary
Plasmodium knowlesi is a monkey malaria species that is becoming a serious public health concern infecting hundreds and perhaps thousands of humans in Southeast Asia. Plasmodium knowlesi merozoites express only two RBL invasion ligands, known as Normocyte Binding Proteins (PkNBPXa and PkNBPXb). A fifth species, Plasmodium knowlesi, a well-known cause of monkey malaria, has emerged as a potential cause of severe and fatal malaria in humans with more than 700 documented cases [2,3,4,5]; unreported cases are likely to be in the thousands in Southeast Asia. The asexual erythrocytic stage of the Plasmodium life cycle is the cause of all clinical symptomology of malaria, and successful merozoite invasion is essential for the maintenance of a malaria infection and propagation of the parasites. An improved understanding of these processes is important for devising prophylactic and therapeutic strategies against the various species that cause human malaria
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