The online version of this article (doi:10.1186/1475-2875-11-228) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
AAS designed the study, performed experiments, analysed and interpreted results, and wrote the manuscript. TMT contributed to the research design, discussion of data and manuscript writing. EM generated reagents, contributed to experimental design, interpreted results and contributed to manuscript writing. JWB generated reagents, contributed to data analysis, concept discussions and manuscript writing. MRG contributed to study design, analysis, interpretation of results, and manuscript writing. All authors read and approved the final manuscript.
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. 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).
Overlapping 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.
An 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.
The 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.
Additional file 1: Primer sequences for regions cloned into pDisplay vector. (PDF 96 KB)12936_2012_2155_MOESM1_ESM.pdf
Additional file 2: Primer sequences for site-directed mutagenesis of PkNBPXb-II. (PDF 96 KB)12936_2012_2155_MOESM2_ESM.pdf
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- Two functional reticulocyte binding-like (RBL) invasion ligands of zoonotic Plasmodium knowlesi exhibit differential adhesion to monkey and human erythrocytes
Amma A Semenya
Tuan M Tran
Esmeralda VS Meyer
John W Barnwell
Mary R Galinski
- BioMed Central
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