Optical tweezers reveal that PfEBA and PfRH ligands, not PfMSP1, play a central role in Plasmodium-falciparum merozoite-erythrocyte attachment

Malaria pathogenesis and parasite multiplication both depend on the ability of Plasmodium falciparum merozoites to invade human erythrocytes. Invasion is a complex multi-step process that is known to involve multiple P. falciparum proteins but dissecting the precise role of individual proteins has to date been limited by the availability of quantifiable phenotypic assays. In this study, we apply a new approach to assigning function to invasion proteins by using optical tweezers to directly manipulate recently egressed merozoites and erythrocytes and quantify the strength of attachment between them, as well as the frequency with which such attachments occur. Using a range of inhibitors, antibodies, and genetically modified P. falciparum strains, we quantitated the contribution of individual P. falciparum proteins to these merozoite-erythrocyte attachment phenotypes for the first time. Most of the interactions investigated did not affect the force needed to pull merozoites and erythrocytes apart, including loss of the major P. falciparum merozoite surface protein PfMSP1 and PfGAP45, part of the glideosome actinomyosin motor complex. The only factors that significantly reduced the strength of merozoite-erythrocyte attachment were ones that disrupted the function of members of the EBA-175 like Antigen (PfEBA) family and Reticulocyte Binding Protein Homologue (PfRH) invasion ligand families. While these assays also reinforced the known redundancy within these families, with the deletion of some ligands not impacting detachment force, it appears that the PfEBA/PfRH families play a central role in merozoite attachment, not the major merozoite surface protein PfMSP1. Author summaryMalaria is a devastating disease caused by a parasitic infection. The deadliest species is Plasmodium falciparum, which causes more than 600,000 deaths annually. The parasites life cycle is complex, but all the symptoms of malaria are caused when the parasites replicate in human red blood cells. Replication depends on the invasion of the red blood cells by the parasites which is a complex process involving multiple molecular interactions and multiple steps. Invasion begins with the attachment of the parasite to the red blood cell, making this step of particular interest in the development of new therapeutics. We assessed which interactions are key to the strength of attachment using an optical tweezer assay, which allowed us to directly measure the binding force between individual parasites and red blood cells whilst using a range of molecular and genetic tools that target specific interactions known to have a role in invasion. This showed that loss of a protein commonly thought to be critical to the early stages of binding (PfMSP1) had no effect on attachment strength, whereas disruptions of several members from two families of proteins (the Erythrocyte Binding Like protein family and the reticulocyte binding-like protein family) affect attachment strength.