Assembly of the Plasmodium falciparum virulence complex

Abstract

After invading a human red blood cell (RBC), Plasmodium falciparum modifies the host RBC surface by exporting proteins that traffic to and interact with the RBC membrane skeleton. The physical properties of the RBC are altered and parasite derived structures called knobs arrive at the cell periphery. These knobs are comprised primarily of the knob-associated histidine rich protein (KAHRP), which acts as a scaffold for the presentation of the major virulence protein, P. falciparum erythrocyte membrane protein 1 (PfEMP1), at the membrane. Knobs are of particular interest as PfEMP1-mediated cytoadhesion is a key contributor to parasite virulence and to the complications that arise in cases of severe malaria. In this study I used a method for exposing the inner surface of the infected RBC membrane and visualised the physical organisation of the membrane proteins by scanning electron microscopy (SEM). The locations of particular proteins were also mapped using direct stochastic optical reconstruction microscopy (dSTORM). In a major advance, I combined these two imaging modalities in a correlative light and electron microscopy based approach (STORM-SEM) to investigate membrane remodelling and virulence complex assembly. I investigated how RBC membrane skeleton remodelling facilitates knob assembly upon the arrival of KAHRP at the RBC periphery and how PfEMP1 arrives at the membrane and assembles into knobs.

I showed that KAHRP is delivered to the RBC membrane as modular units. As the asexual parasite matures, the KAHRP modules re-arranged into five-membered ring-shaped oligomers. Correlative imaging revealed that the KAHRP modules are located at the perimeter of the physical knob. Parasites expressing C-terminally truncated KAHRP proteins were generated to analyse the regions of KAHRP required for oligomeric ring formation and membrane skeleton binding. I showed that the KAHRP spectrin-binding domain is needed for knob assembly and that the KAHRP 3’ repeat region is needed for formation of the canonical knob structure. Electron tomography of the mutant parasites revealed that correct formation of the ring-shaped KAHRP oligomer is required to maintain the correct arrangement of a spiral structure found at knob complexes.

Arrival of knobs at the RBC membrane has been linked to decreased RBC deformability. Interruption of remodelling of actin from the membrane skeleton using the mycotoxin cytochalasin D resulted in increased infected RBC deformability and a reduction in the number of knobs at the membrane. This indicates that remodelling of the host actin is required for the assembly of knobs. Recovery of cellular rigidity after removal of cytochalasin D highlighted the importance of membrane skeleton remodelling in the physical RBC changes that occur during infection.

The mechanism by which the major virulence protein, PfEMP1, is delivered to the virulence complex is not well understood. Using STORM-SEM, I provided evidence that PfEMP1 is delivered to the RBC membrane at regions away from the knobs. Temporal analysis suggests that PfEMP1 associates with the periphery of the physical knob structure before lateral insertion into the knob complex.

By combining STORM-SEM with cellular biology and biophysical measurements, this work has revealed new details of the remodelling events that underpin parasite virulence. Based on the data presented in this study I propose a new model for virulence complex assembly. In this model KAHRP is delivered to the RBC membrane skeleton as modular units. Actin mining and subsequent rearrangement of the spectrin meshwork facilitates self-association of KAHRP into a five-segment oligomer, enabling the assembly of an underlying spiral structure. Separately, PfEMP1 is inserted into the membrane, before lateral insertion into the virulence complex. It is hoped that the findings in this study will point to novel strategies to target parasite virulence machinery and to fight malaria infection.