Structural studies of the clustering of PfEMP1 proteins on the surface of Plasmodium falciparum-infected erythrocytes

Malaria is one of the most devastating diseases that affect humanity. It kills about 2 million people each year and causes about 500 million serious cases. The disease is caused by tiny parasites, known as Plasmodium. The deadly symptoms of the disease, including fever, anaemia and even coma and death, occur during the blood phase of the parasite life cycle. Here, the parasites invade red blood cells and live and divide within them, using the red cells for protection from the immune system and as a source of nutrients to fuel parasite replication.

After invasion the parasites remodel red cells, causing dramatic changes that make them a suitable home. One of these changes is the formation of structures called knobs on the red cell surface. Sticky proteins, known as PfEMP1 proteins, become clustered at these knobs. These sticky proteins interact with different molecules on the blood vessel surfaces or with human tissues such as brain or placenta. They also cause red blood cells to stick together to form tiny clumps known as rosettes. By sticking throughout the body, the infected red cells hide from detection, allowing the parasite to grow and divide in peace and prolonging the infection. But this stickiness also causes some of the most severe symptoms of the disease. When infected red cells and rosettes become clustered in the brain, blood flow is disrupted, leading to cerebral malaria and causing coma and death. The accumulation of infected red cells on the placenta is also deadly, causing the severe symptoms of malaria during pregnancy.

We are studying the molecules that the parasite uses to cause the formation of knobs and to cause adhesive proteins to cluster at these knobs. We will focus on proteins called KAHRP, spectrin and PfEMP1. The parasite protein, KAHRP, interacts with the red cell protein, spectrin, and acts as the major scaffold for knob formation. PfEMP1 proteins can then interact with KAHRP, causing them to become clustered at the knobs. We will use a variety of techniques to study the precise structural details of how these three proteins interact with one another. By understanding how knobs are formed, and how PfEMP1 proteins are clustered, we aim to provide information that will guide the development of medicines to prevent knob formation or the development of stickiness. These treatments will be useful to prevent many of the most deadly symptoms of malaria.

Malaria is one of the most deadly diseases affecting humanity, causing around 2 million deaths, and 500 million serious cases each year. The symptoms of the disease occur during the erythrocytic phase of the Plasmodium life cycle, when the parasite divides within infected erythrocytes. During this phase, the parasite causes extensive remodelling of the erythrocyte. Structures called knobs form on the erythrocyte surface and adhesive PfEMP1 proteins are clustered at these knobs. This causes infected erythrocyte to adhere to human tissues or to the microvasculature, protecting the parasite from detection by the spleen and prolonging the infection. This adhesion also causes many of the most severe symptoms of malaria, with adhesive erythrocyte blocking tiny capillaries in the brain during cerebral malaria and binding to the placenta during pregnancy-associated malaria.

The parasite protein KAHRP appears to act as the hub around which the knobs are formed. Important in the correct localisation of KAHRP, and the subsequent formation of knobs, is the interaction between KAHRP and the erythrocyte cytoskeletal protein, spectrin. We have determined the spectrin-binding region of KAHRP and have crystals of the KAHRP binding region of spectrin. We will use structural and biophysical methods to characterise this interaction in stereochemical detail.

The intracellular domains of PfEMP1 proteins (the VARC domains) interact with KAHRP and this is thought to be important in clustering PfEMP1 proteins at knobs. We will use structural and biophysical methods, including protein crystallography and nuclear magnetic resonance (NMR) spectroscopy to investigate this interaction in stereochemical detail. We have shown that different peptides from KAHRP interact with VARC and that in the absence of binding partner, VARC is partially disordered. We will use NMR to study VARC-KAHRP complexes and will determine the structures of ordered complexes between KAHRP binding peptides and VARC subdomains.

Finally we will initiate parasite mutagenesis studies in which we replace the KAHRP gene by genes containing mutant KAHRP lacking regions needed to bind to spectrin or VARC. We will study erythrocytes infected with these parasite strains and use confocal microscopy, electron microscopy and adhesion studies to determine the roles of different interactions in knob formation and function.

These studies will provide detailed structural insight into the formation of knobs and will guide future attempts to develop inhibitors to block knob formation - molecules that will be useful as therapeutics for treatment of the symptoms of severe malaria.