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

01 May 2017
Robert W. Moon, Hazem Sharaf, Claire H. Hastings, Yung Shwen Ho, Mridul B. Nair, Zineb Rchiad, Ellen Knuepfer, Abhinay Ramaprasad, Franziska Mohring, Amirah Amir, Noor A. Yusuf, Joanna Hall, Neil Almond, Yee Ling Lau, Arnab Pain, Michael J. Blackman and Anthony A. Holder. - See more at: http://malaria.lshtm.ac.uk/publications#sthash.t7CffgVa.dpuf
End of life care

A causative agent of severe zoonotic disease in South East Asia is Plasmodium knowlesi, a malaria parasite of the cynomolgus macaque, Macaca fascicularis (1, 2). In countries such as Malaysia where other malaria parasites are being controlled effectively, P. knowlesi infections are the dominant cause of human malaria (3, 4). In all malaria parasite species, disease is caused by cyclic parasite multiplication within RBCs, releasing invasive merozoites that invade new RBCs. Parasite adhesins transported onto the merozoite surface bind specific host cell receptors to facilitate RBC invasion (reviewed in ref. 5). The first adhesin–RBC interaction was identified in P. knowlesi and Plasmodium vivax, in which it was shown that parasites must bind to the Duffy antigen/chemokine receptor (DARC) to invade human RBCs (6). These adhesins are determinants of parasite virulence (7), host cell tropism (8), and potential vaccine candidates (9), and understanding the role of these adhesins may be critical for identifying the causes of increased risk of human infection and for the development of novel interventions.

Reticulocyte-binding proteins (RBPs) were first described in P. vivax (10) and are the prototypical examples of the reticulocyte binding-like/reticulocyte-binding homolog (RBL/RH) proteins also characterized in other malaria parasite species including Plasmodium cynomolgi (11) and Plasmodium yoelii (12). Plasmodium falciparum normocyte-binding proteins (NBPs) (13, 14) are members of this family that includes four conventional RBL/RH proteins and one unconventional short RBL/RH protein (RH5) in this species (reviewed in ref. 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/erythrocyte-binding ligand (DBP/EBL) family of proteins play key roles during merozoite invasion of RBCs (reviewed in refs. 5 and 9). However, ascribing specific roles to these proteins has been hampered by functional redundancy, and only RH5 may be essential for P. falciparum invasion (17). In P. knowlesi, DBPα is the parasite adhesin binding DARC, and two paralogues, DBPβ and DBPγ, also have been identified (18), but thus far only DBPα has been shown to be required for RBC invasion (19).

P. knowlesi can be grown readily in both cynomolgus and rhesus (Macaca mulatta) macaque RBCs in vitro (20⇓–22) but requires extended adaptation for growth in human RBCs in vitro, displaying insufficient invasion efficiency to support continuous culture in human RBCs. In previous work we overcame this impediment by extended adaptation in a mixture of human and cynomolgus RBCs, producing a parasite line that can be maintained in human RBCs alone (21). This previous analysis suggested that improved parasite invasion of human RBCs was essential for the maintenance of parasite growth in vitro.

Here, we undertook a comparative analysis of the genomes of P. knowlesi lines adapted to human or cynomolgus RBCs in culture, in parallel with analysis of the preadapted parasites. The comparison highlighted a crucial role for NBPXa in the invasion of human RBCs. Using the unique capacity to grow this parasite in two host cell types, we were able to disrupt the gene encoding NBPXa and show that it is required for invasion of human but not of cynomolgus macaque RBCs. Human-adapted P. knowlesi parasites provide a robust experimental platform to unravel the mechanisms of host cell invasion in both P. knowlesi and the related pathogen P. vivax.