High-throughput screens to identify novel interactions between erythrocyte multi-pass receptors and P. falciparum merozoite surface ligands that are involved in invasion

Invasion of erythrocytes by P. falciparum merozoites is a complex multistep process involving a series of molecular recognition events between parasite ligands and their receptors on the erythrocyte surface. Although many potential P. falciparum invasion ligands are known, the erythrocyte receptors they interact with have been identified only for a handful of cases. Studying extracellular binding events between membrane-embedded proteins in vitro is challenging and beyond the reach of standard approaches such as co-immunoprecipitation and yeast two-hybrid screens. These interactions are generally of very low affinity and often require specific motifs generated via post-translational processing; furthermore, amphipathic membrane-spanning proteins are difficult to manipulate biochemically. The Duffy Antigen Receptor for Chemokines (DARC), a multi-pass G-protein coupled erythrocyte receptor is known to be important for invasion by P. vivax but no multi-pass receptor has been established to play a role in P. falciparum invasion. With the aim of identifying such a receptor, we have developed a flow cytometry based, high-throughput strategy that can detect transient, extracellular protein: protein interactions. We are in the process of screening the soluble, recombinant ectodomains of 39 P. falciparum merozoite surface proteins against an expression library of 45 erythrocyte multi-pass surface proteins. We will present our screening strategy and preliminary results.