The reticulocyte binding-like proteins of P. knowlesi locate to the micronemes of merozoites and define two new members of this invasion ligand family

Abstract

Members of the Reticulocyte Binding Protein-like (RBL) family are merozoite-expressed proteins hypothesized to be essential for effective invasion of host erythrocytes. Proteins of the RBL family were first defined as merozoite invasion ligands in Plasmodium vivax, and subsequently in P. falciparum and other malaria parasite species. Comparative studies are providing insights regarding the complexity and evolution of this family and the existence of possible functionally alternative members. Here, we report the experimental and bioinformatic characterization of two new rbl genes in the simian malaria parasite species P. knowlesi. Experimental analyses confirm that a P. knowlesi gene fragment orthologous to P. vivax reticulocyte binding protein-1 (pvrbp1) represents a highly degenerated pseudogene in the H strain as well as two other P. knowlesi strains. Our data also confirms that a gene orthologous to pvrbp2 is not present in the P. knowlesi genome. However, two very diverse but related functional rbl genes are present and are reported here as P. knowlesi normocyte binding protein Xa and Xb (pknbpxa and pknbpxb). Analysis of these two rbl genes in Southern hybridizations and BLAST searches established their relationship to newly identified members of the RBL family in P. vivax and other species of simian malaria. Rabbit antisera specific for recombinant PkNBPXa and PkNBPXb confirmed expression of the prospective high molecular

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

“…S1). In A1-O and A1-H lines, this region encompasses 12 genes (Table S2), including PKNH_1472300 which encodes NBPXa (15). This observation was of particular interest, because adaptation to human RBCs appeared to result from improved invasion efficiency rather than from improved intracellular growth (21).…”

“…The adhesin repertoire is smaller in P. knowlesi than in many malaria parasites (9,15,18), and the loss of either DBPa (6,19) or NBPXa creates a critical block in the invasion of human RBCs, perhaps because the paralogues cannot bind human RBCs. 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).…”

“…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.…”

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

“…Rhesus erythrocytes were treated with neuraminidase, trypsin, or chymotrypsin, and eluates from EBAs using these cells were probed in immunoblot assays with a rabbit anti-PkNBPXb specific antibody [20]. Protein bands >250 kDa, corresponding to the full-length protein were observed in all samples that were incubated with the culture supernatant (Figure 2B).…”

“…A future focus on studying PkNBPXa adhesion will be important because, in contrast to PkNBPXb, PkNBPXa not only binds to rhesus erythrocytes in EBA assays [20], but also human erythrocytes (Figure 5). This study attempted to identify a binding domain in PkNBPXa.…”