Dissecting the role of host receptor context and cytoskeletal disruption in malaria parasite invasion

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Every year, across the world more than 200 million people contract malaria, and more than half a million people die, the majority of them children under the age of five, as a result of this disease. The parasites that cause malaria survive by attaching to the surface of and then penetrating circulating red blood cells in which they then multiply. Red blood cells (RBCs) have a highly specialised membrane structure that results from complex interactions between proteins within the plasma membrane and a flexible underlying meshwork of protein filaments called the cytoskeleton that allow the cell to squeeze through capillaries. To penetrate the robust RBC membrane, the parasite attaches to proteins at the cell surface and induces a coordinated and localised disruption of this membrane-cytoskeletal architecture to facilitate invasion, a process that also shares some similarities with the transient disruptions required to enable RBC squeezing in the capillaries. Although several key proteins have been shown to be involved in or required for successful invasion, in the majority of cases insight into the role that these host cell proteins actually play in the invasion process is severely or completely lacking. One of the biggest obstacles to investigating the mechanism of parasite invasion from the perspective of the host RBC is the inability to directly manipulate protein expression in these cells. Unlike most cells, RBCs contain no DNA, preventing the application of genetic techniques commonly used to manipulate protein expression in other cell types. Recent developments made in the field of RBC development (erythropoiesis) have changed this. It is now possible to culture young RBCs (reticulocytes) that are susceptible to invasion by the parasite that causes severe malaria, from an immortal cell line that allows the precursors of RBCs (erythroblasts) to be grown indefinitely or safely stored.Excitingly, we have shown it is possible to manipulate protein expression in these nucleated cells using lentivirus and gene editing techniques to introduce changes which are maintained after the cells lose their nucleus to become RBCs. This technology can be used to prevent specific RBC proteins that are known to be involved in invasion from being expressed and also allows them to altered or replaced with mutated versions in which the localisation within the membrane, interactions with other proteins or properties of the protein itself have been changed. This technology has opened the door to the generation of RBCs with rare and even unique characteristics that can be used to explore which proteins are important for malaria parasite attachment or invasion, the importance of their membrane context and properties and how these host cell proteins participate in or are manipulated by the parasite during a successful invasion event.This project will use RBCs with novel characteristics generated using this approach together with normal donor RBCs to investigate the mechanism of malaria parasite attachment and invasion of RBCs from a unique host cell perspective. Using a combination of malaria parasite invasion assays, biochemical and imaging techniques it will uncover how RBC proteins with crucial but poorly understood roles in invasion participate in this process. Since attachment to or stimulus of RBC receptors also induces reconfiguration or disruption of membrane-cytoskeletal protein interactions we will also investigate the involvement and modification of key cytoskeletal adaptor proteins that mediate connections between both membrane and cytoskeletal proteins. By determining the nature of and degree to which mechanisms that facilitate RBC squeezing in the capillaries and successful invasion are shared (co-opted by the parasite) or unique we will strive to identify ways in which invasion may be targeted for inhibition without impairing the normal function of the RBC within the body.