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

Abstract

The dominant cause of malaria in Malaysia is now Plasmodium knowlesi, a zoonotic parasite of cynomolgus macaque monkeys found throughout South East Asia. Comparative genomic analysis of parasites adapted to in vitro growth in either cynomolgus or human RBCs identified a genomic deletion that includes the gene encoding normocyte-binding protein Xa (NBPXa) in parasites growing in cynomolgus RBCs but not in human RBCs. Experimental deletion of the NBPXa gene in parasites adapted to growth in human RBCs (which retain the ability to grow in cynomolgus RBCs) restricted them to cynomolgus RBCs, demonstrating that this gene is selectively required for parasite multiplication and growth in human RBCs. NBPXa-null parasites could bind to human RBCs, but invasion of these cells was severely impaired. Therefore, NBPXa is identified as a key mediator of P. knowlesi human infection and may be a target for vaccine development against this emerging pathogen.

Rapid and iterative genome editing in the zoonotic malaria parasitePlasmodium knowlesi: New tools forP. vivaxresearch

“…The loss of the ~44 kb of chromosome 14 is also present in parasites that have been transfected simultaneously with pCas/sg_ p230p , suggesting that the 44 kb deletion occurred in the A1-H.1 parental parasite line prior to transfection and was not an artefact caused by targeting DBPα. Similar spontaneous deletions have been reported previously, including ~ 66 kb loss at the other end of chromosome 14 in the P. knowlesi A1-C line maintained in cynomolgus macaque blood that included the invasion ligand NBPXa (33), and a deletion of DBPγ in the PkYH1 line at the end of chromosome 13 (16). Furthermore, the PAM site of the DBPα targeting guide sequence is absent in DBPβ (S4C Figure) which makes it unlikely that the disruption of DBPβ was induced by Cas9 during DBPα targeting.…”

“…P. knowlesi readily accepts linearised plasmids for homologous recombination (20, 25, 33), so we next tested whether we could use a PCR-based approach for scalable generation of repair templates. As no selectable marker is used within the repair template, this could be easily produced by using PCR to fuse 5’ and 3’ HRs with the region containing the desired insertion, dispensing with the need for a plasmid back-bone.…”

Molecular interactions governing host-specificity of blood stage malaria parasites

“…Moon et al . [48] have since demonstrated that although deletion of the RBL ligand Pk NBPXa facilitated long-term culture of P. knowlesi in cynomologous macaque blood, doing so restricted replication in human cells. This observation suggests that Pk NBPXa may be an essential determinant of human erythrocyte invasion by P. knowlesi .…”

Proteases as antimalarial targets: strategies for genetic, chemical, and therapeutic validation

“…In theory, cKO/cKD P. falciparum lines could be used in the SCID model to test the essentiality of a target within a living host. Finally, Plasmodium knowlesi is more closely related to Plasmodium vivax, Plasmodium malariae or Plasmodium ovale than P. falciparum, and its recent adaptation to grow in human erythrocytes [134] makes it an excellent in vitro model to predict the efficacy of lead inhibitors against these other malaria-causing species.…”