Journal Article

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

McMillan, Paul J., Tilley, Leann, Looker, Oliver, Liu, Boyin, Blanch, Adam J., Nunez-Iglesias, Juan, Dixon, Matthew W. A.

2019

Overview

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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: 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