Identifying novel Plasmodium falciparum erythrocyte invasion receptors using systematic extracellular protein interaction screens

Abstract

The invasion of host erythrocytes by the parasite Plasmodium falciparum initiates the blood stage of infection responsible for the symptoms of malaria. Invasion involves extracellular protein interactions between host erythrocyte receptors and ligands on the merozoite, the invasive form of the parasite. Despite significant research effort, many merozoite surface ligands have no known erythrocyte binding partner, most likely due to the intractable biochemical nature of membrane-tethered receptor proteins and their interactions. The few receptor-ligand pairs that have been described have largely relied on sourcing erythrocytes from patients with rare blood groups, a serendipitous approach that is unsatisfactory for systematically identifying novel receptors. We have recently developed a scalable assay called AVEXIS (for AVidity-based EXtracellular Interaction Screen), designed to circumvent the technical difficulties associated with the identification of extracellular protein interactions, and applied it to identify erythrocyte receptors for orphan P. falciparum merozoite ligands. Using this approach, we have recently identified Basigin (CD147) and Semaphorin-7A (CD108) as receptors for RH5 and MTRAP respectively. In this essay, we review techniques used to identify Plasmodium receptors and discuss how they could be applied in the future to identify novel receptors both for Plasmodium parasites but also other pathogens.

Fig 1

Identifying novel Plasmodium receptors by AVEXIS.A. Schematic representation of erythrocyte invasion by the malarial parasite highlighting the extracellular protein interactions between parasite ligands and erythrocyte receptors.B. Cartoon of interacting cell surface receptors between an erythrocyte receptor (red) and a parasite ligand (blue).C. The AVEXIS assay detects direct interactions between soluble recombinant proteins representing the entire ectodomain of cell surface receptors. Here, the erythrocyte receptors are expressed as the pentamerized β-lactamase-tagged ‘preys’ and the parasite ligands as monomeric biotin-tagged ‘baits’. The baits are captured on a streptavidin-coated solid phase, such as a microtitre plate or glass slide and probed with the preys. A brief wash is performed and captured preys are detected by addition of a β-lactamase substrate, nitrocefin which hydrolyses a yellow substrate into a red product, indicating a positive interaction.D. AVEXIS involves the systematic screening within libraries of both baits and preys in an all-versus-all matrix of direct binding tests.E. An image of a typical screening plate. Each well contains a different bait protein screened with a single prey. A positive interaction was observed in well E2 and control interactions are indicated within the dotted line.

Fig 2

The cell surface receptor repertoire of the human erythrocyte. A schematic structural representation of the human erythrocyte cell surface receptor repertoire drawn approximately to scale together with the receptor abundances, where known, shown as bars on the scale. The figure contrasts large receptors such as CR1, which project 85 nm away from the membrane, but, at 1000 copies per cell, are relatively rare whereas the much smaller glycophorins are vastly more abundant, containing up to a million copies per cell in the case of GYPA.