Dissecting the Red Blood Cell Invasion Pathways of the Malaria Parasite Plasmodium knowlesi

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Malaria is one of the most important infectious diseases of man with more than half of the world's population living at risk of the disease, and resulting in more than half a million deaths per year. The disease is caused by Plasmodium species, single-celled parasites that can be transferred to humans by the bite of an infected mosquito. Symptoms of the disease result from the parasite destroying red blood cells by first entering them, growing and replicating inside them, before bursting out and invading other red blood cells in a continuous cycle. The parasites produce a range of adhesive proteins enabling them to bind to specific proteins on the surface of red blood cells and establish the process of red blood cell invasion. Because of their crucial role in the invasion process these parasite proteins are important vaccine targets. They also determine how effectively parasites can replicate and so can affect disease severity as well as determining which hosts are susceptible to malaria.In this project I will investigate the role of these proteins during the invasion process using a malaria parasite known as Plasmodium knowlesi. This parasite naturally infects macaque monkeys in South-East Asia, and was recently found to be a significant cause of severe and fatal human infections. In recent work I have developed methods to grow this parasite in culture with human red blood cells for the first time, and established highly efficient techniques to genetically modify the parasite. I will use these techniques to generate parasites in which I have deleted genes encoding the adhesive proteins. This will enable me to determine which are essential for invasion and which can be deleted without any effect on the invasion process. Using similar techniques I will also add fluorescent "tags" to each of the proteins coded by the target genes, so that I can determine where the adhesive proteins are in the cell and where they move during the invasion process. The "tags" will also allow me to identify parasite proteins that interact with the adhesive proteins as well as what they specifically bind to on the host red blood cell surface. I will analyse both the gene deletion and "tagged" parasite lines using cutting edge imaging technologies including electron tomography and super resolution microscopy, which have never before been used to visualise invasion of this parasite species. This will provide critical insight into the mechanism of invasion of all malaria parasites, as well as identifying precisely which parasite proteins and host proteins are required for P. knowlesi to invade human red blood cells. The latter is of particular importance as it may explain how a macaque malaria parasite is able to spread to infect humans and determine the potential for emergence of human-to-human transmission of the parasite.Whilst there is currently no vaccine for malaria, there is great interest and several vaccine candidates under development for the most common and serious cause of malaria P. falciparum. However, vaccine development for the second most common cause of malaria P. vivax, is hampered by the fact that it cannot be grown in the laboratory. This means that testing new vaccines would involve infecting people or non-human primates with P. vivax. The primary vaccine candidate for P. vivax is one of the parasite's adhesive proteins. P. knowlesi is closely related to P. vivax and also uses a similar version of this adhesive protein. By genetically modifying P. knowlesi to replace the gene encoding the adhesive protein with the version from P. vivax, it will be possible to determine whether a vaccine can induce antibodies that kill parasites in culture, before it is necessary to test it in people. Thus I will use the unique biology of P. knowlesi along with the technical advantages of the model to not only study the process of invasion but also generate important tools to expedite the development of vital malaria vaccines.