The knob protein KAHRP assembles into a ring-shaped structure that underpins virulence complex assembly

• Published in: PLoS pathogens 2019-05, Vol.15 (5), p.e1007761-e1007761
• Main Authors: Looker, Oliver, Blanch, Adam J, Liu, Boyin, Nunez-Iglesias, Juan, McMillan, Paul J, Tilley, Leann, Dixon, Matthew W A
• Format: Article
• Language: English
• Subjects: Actin; Amino acids; Antigen presentation; Antigens; Assembling; Assembly; Biochemistry; Biology and Life Sciences; Biotechnology; Blood; Blood cells; Blood vessels; Cytochalasin D; Dimpling; Disks; Disruption; Electron microscopy; Erythrocyte Membrane - metabolism; Erythrocyte Membrane - parasitology; Erythrocytes; Erythrocytes - metabolism; Erythrocytes - parasitology; Fluorescence; Histidine; Humans; Knobs; Malaria; Malaria, Falciparum - metabolism; Malaria, Falciparum - parasitology; Medicine and Health Sciences; Membrane proteins; Membranes; Microscopy; Microscopy, Electron, Scanning; Molecular biology; Muscle proteins; Parasites; Parasitology; Peptides - chemistry; Peptides - metabolism; Physical Sciences; Plasmodium falciparum; Plasmodium falciparum - pathogenicity; Proteins; Protozoan Proteins - chemistry; Protozoan Proteins - metabolism; Red blood cells; Research and Analysis Methods; Ring structures; Scaffolds; Scanning electron microscopy; Scanning microscopy; Stochasticity; Tomography; Virulence; Virulence (Microbiology); Visualization
• ISSN: 1553-7374; 1553-7366; 1553-7374
• DOI: 10.1371/journal.ppat.1007761

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.