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

Abstract

Background: 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).

Methods: 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.

Results: 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.

Conclusions: 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.

Figure 1

Expression ofpknbpgene segments in COS7 cells and binding profile to rhesus erythrocytes. (A) Three regions spanning the 5′ end of the coding region of PkNBPXa were cloned into pDisplay as overlapping 500 nucleotide gene segments. The numbers listed correspond to the amino acid positions delineating a particular expressed gene segment. Though the recombinant proteins were expressed intracellularly, they never reached the surface of the COS7 cells (data not shown). Eight recombinant segments (Regions I - VIII) spanning the N-terminal half of PkNBPXb were cloned and expressed in COS7 cells. (B) Protein expression for each COS7/PkNBPXb region was evaluated by immunoblot using a specific antibody against the c-myc tag encoded in the C-terminus of the pDisplay vector. (C) Rhesus monkey erythrocytes were found to adhere only to PkNBPXb-II forming a large rosette pattern on the surface of COS7 cells. Panels 1 and 2 show images representing nuclear staining with Hoechst dye, COS7 cell protein expression detected by anti-HA and Alexa Fluor 488-labeled anti-mouse IgG, and erythrocyte rosetting assay results, respectively. Top and bottom panels show the surface expression and binding capability of Region II and Region VIII, respectively. Region VIII showed high expression by immunoblot while displaying no binding affinity to rhesus erythrocytes. Images were captured at room temperature using a Nikon ECLIPSE TE 300 inverted microscope using the Plan Fluor ELWD 40x / 0.45 aperture and 10x magnification eye piece and analysed using the Diagnostic Instrument, Inc. software.

Figure 2

Binding of PkNBPXb-II and native PkNBPXb to enzymatically treated rhesus monkey erythrocytes. (A) PkNBPXb-II cells were tested for relative binding in adhesion (rosetting) assays to rhesus monkey erythrocytes that were either untreated, or treated with chymotrypsin (CT), trypsin (T), or neuraminidase (NA), * p-value <0.05 and *** p-value <0.001. (B) Native PkNBPXb binding was tested in erythrocyte binding assays with rhesus monkey erythrocytes that were untreated or treated with CT, T, or NA. Total supernatants (TS) and comparable eluates from untreated rhesus monkey erythrocytes (R) incubated without supernatants were also applied to the SDS-PAGE gels and transferred to nitrocellulose membranes. The immunoblot was probed with an anti-PkNBPXb antibody [20]. (C) A negative binding control was included using duplicate aliquots of the RBC samples tested in B whereby anti-PkMSP3/140 antibody recognizes the native protein in the supernatant only.

Figure 3

Amino acid alignment of the N-terminal regions of PkNBPXa, PkNBPXb, PvRBP2 and PfNBP2a. Alignment of the first 300–400 amino acids of the N-terminus of PkNBPXa, PkNBPXb, PvRBP2, and PfNBP2a demonstrates conservation of three cysteine residues among species. These three residues in PkNBPXb correspond to Cys²⁵⁴, Cys²⁹⁸, and Cys³³². Two other cysteine residues are also present in the N-terminus of PkNBPXb-II and are Cys¹⁹³ and Cys³²⁶.

Figure 4

Four disulphide bonds in PkNBPXb are important for expression on the surface of COS cells. The N-terminal domain of PkNBPXb contains five cysteine residues (C¹⁹³, C²⁵⁴, C²⁹⁸, C³²⁶, C³³²). Using site directed mutagenesis, all five residues were individually mutated to glycine and all but C¹⁹³ were found to be required for binding to erythrocytes. The glycine residues present in each construct were mutated back to cysteine residues; these constructs were able to express protein on the surface and also able to bind erythrocytes. Surface and internal expression is indicated with + or – signs.

Figure 5

PkNBPXa binds to Duffy negative and Duffy positive human erythrocytes. Native PkNBPXa (A) and PkNBPXb (B) present in parasite supernatants were tested for their ability to bind Duffy positive and Duffy negative human cells by EBA. Culture supernatants (S) containing soluble merozoite proteins were incubated with human Duffy positive (Fy^a+b+) and Duffy negative (Fy^a-b-) erythrocytes, and then the bound proteins were eluted, electrophoresed and transferred to nitrocellulose membranes. PkNBPXa and PkNBPXb specific antibodies were used to probe the membranes. PkNBPXa was detected in the supernatants and eluates from Duffy positive and Duffy negative erythrocytes. PkNBPXb was present in the supernatant but did not bind to either type of human erythrocytes. As a control, erythrocytes were incubated in the absence of supernatants and no PkNBPXa specific bands were detected in those samples (R).