Abstract
Plasmodium falciparum mediates adhesion of infected red blood cells (RBCs) to blood vessel walls by assembling a multi-protein complex at the RBC surface. This virulence-mediating structure, called the knob, acts as a scaffold for the presentation of the major virulence antigen, P. falciparum Erythrocyte Membrane Protein-1 (PfEMP1). In this work we developed correlative STochastic Optical Reconstruction Microscopy–Scanning Electron Microscopy (STORM-SEM) to spatially and temporally map the delivery of the knob-associated histidine-rich protein (KAHRP) and PfEMP1 to the RBC membrane skeleton. We show that KAHRP is delivered as individual modules that assemble in situ, giving a ring-shaped fluorescence profile around a dimpled disk that can be visualized by SEM. Electron tomography of negatively-stained membranes reveals a previously observed spiral scaffold underpinning the assembled knobs. Truncation of the C-terminal region of KAHRP leads to loss of the ring structures, disruption of the raised disks and aberrant formation of the spiral scaffold, pointing to a critical role for KAHRP in assembling the physical knob structure. We show that host cell actin remodeling plays an important role in assembly of the virulence complex, with cytochalasin D blocking knob assembly. Additionally, PfEMP1 appears to be delivered to the RBC membrane, then inserted laterally into knob structures.
Highlights
Malaria is a devastating human disease that affects more than a quarter of the world’s population and kills more than 400,000 people annually [1]
The human malaria parasite Plasmodium falciparum causes severe disease, which is initiated by the adhesion of parasite-infected red blood cells (RBCs) to receptors on the walls of the host’s capillaries
Adhesion is mediated by a structure called the knob, which acts as a scaffold for the presentation of the virulence protein, P. falciparum erythrocyte membrane protein-1 (PfEMP1)
Summary
Malaria is a devastating human disease that affects more than a quarter of the world’s population and kills more than 400,000 people annually [1]. The majority of these deaths are caused by infection with Plasmodium falciparum, with children from sub-Saharan Africa most at risk. Adhesion of infected RBCs is mediated by a family of proteins called P. falciparum erythrocyte membrane protein-1 (PfEMP1). Once inserted into the RBC membrane, PfEMP1 can bind to endothelial cell receptors such as Endothelial Protein C Receptor (EPCR), Cluster Determinant 36 (CD36), Intercellular Adhesion Molecule-1 (ICAM), and glycosaminoglycans, driving parasite sequestration [6, 7]
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