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

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.

Genomic and transcriptomic evidence for descent from Plasmodium and loss of blood schizogony in Hepatocystis parasites from naturally infected red colobus monkeys

“…However, they contain a repeat region with striking similarity to that in Plasmodium kahrp, a gene involved in presenting proteins on the red blood cell surface (15). Three members were highly expressed in in vivo blood stages, with one correlating well with the presence of early stages (S8A Fig; HEP_00211100, Pearson's r=0.80 with rings).…”

“…Additionally, Merozoite TRAP-like protein (MTRAP), essential for Plasmodium gamete egress from erythrocytes (26) and two genes (HEP_00254800 and HEP_00195400) with orthologues involved in osmiophilic body formation (27,28) had high dN values. Overall, clusters involved in ookinete (15) and general mosquito stages ). This is also reflected in the Hepatocystis genes with the highest dN values, which include oocyst rupture protein 2 (orp2; dN = 1.08), ap2-o, ap2-sp2, secreted ookinete protein (psop7) and osmiophilic body protein (g377).…”

The Plasmodium falciparum protein VCAP1 controls Maurer’s cleft morphology, knob architecture and PfEMP1 trafficking

“…To investigate the architecture of the knobs in more detail we made use of a recently developed method for imaging knobs at the cytoplasmic surface of infected RBCs (2). Infected RBCs were tightly linked to lectin-coated slides and subjected to shearing.…”

“…We examined the ability of CS2 and ΔVCAP1 infected RBCs to filter through a bed of beads designed to mimic splenic fenestrations. RBCs infected with ΔVCAP1 parasites synchronised to 20-24 h post invasion (the window during which knob assembly and host cell remodelling occurs (2)) exhibited a 23% increase in the filterability of the parasites compared to CS2 controls (Fig 6E). We next examined the sensitivity of infected RBCs to osmotic lysis.…”

“…During this cycle the parasite induces marked changes to the host RBC, including the elaboration of new organelles in the RBC cytoplasm, known as the Maurer’s clefts and the establishment of protrusions at the RBC membrane, known as knobs. The knobs comprise a spiral protein structure supported by the knob-associated histidine-rich protein, (KAHRP) (2). The knob acts as a scaffold for the presentation of the major virulence antigen P. falciparum erythrocyte membrane protein 1 ( Pf EMP1).…”