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

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.

Clinical symptoms of malaria are associated with the cycles of parasite invasion of, and multiplication within, host red blood cells (RBCs). The ligands used by the parasite to target host cell receptors and invade RBCs are key determinants of virulence. Two parasite protein families are implicated in this process, the reticulocyte binding-like (RBL) and Duffy binding-like (DBL) proteins, but little is known about their role or their host cell receptors. I will define the role of these protein families in Plasmodium knowlesi, a parasite causing severe and fatal disease in South East Asia. Taking advantage of the high transfection efficiency and unique capability to grow this parasite in two different host cells, I will generate gene knock-out lines for each of the 5 P. knowlesi DBL/RBL genes - to define which proteins are essential for invasion of the two different host RBC. Transgenic parasite lines will be generated in which each of the RBL/DBL proteins has been modified by the addition of a fluorescent/epitope tag, which will be used to determine subcellular localisation, and through immunoprecipitation, identify host cell receptors and interacting parasite proteins. I will use electron tomography and super resolution microscopy for the phenotypic analysis of knock-out and tagged lines to determine the precise role of each protein in the invasion process. To identify proteins that are essential for invasion of both host RBC, I will adapt the Cas9 and DiCre conditional recombinase system for use in P. knowlesi. Finally, I will develop a transgenic P. knowlesi model to assess both naturally acquired and vaccine induced antibodies targeting P. vivax, an important malaria parasite that currently lacks an in vitro model. The unique advantages of P. knowlesi will provide an unprecedented insight into the mechanistic role of RBL/DBL proteins during invasion, and novel tools to support development of vaccines to target them.

Malaria is one of the most important infectious diseases of man, with 3.2 billion living at risk of the disease and between 350 and 500 million clinical cases a year. My research is driven by my desire to produce an impact on affected communities. My research proposal will help to unravel the process of red blood cell (RBC) invasion by malaria parasites, an important target for novel drugs and vaccines. The P. knowlesi malaria parasite that I will use to study this is recognised as an important emergent cause of severe zoonotic infections in South East Asia. Malaysia is currently working toward the elimination of malaria and whilst control measures are resulting in a decline in other human malaria parasites, P. knowlesi cases have risen >10-fold in areas of Malaysian Borneo between 2004-2011. Dealing with a malaria parasite with an animal reservoir will require the development of novel intervention strategies. In addressing parasite host cell tropism, this project will identify parasite factors which may be causing this rise in human infections. Through my Winston Churchill Memorial Trust Fellowship to Malaysia, I have developed strong links with both Malaysian Government Institutes (IMR), and field work teams, which I will use to convey my findings, inform policy and target field work. It was clear from my time in Malaysia that the UK retains a reputation for excellence in tropical disease through its historic links and continued commitment to global health research. I have already provided the P. knowlesi parasite line that I have developed to two Malaysian Institutions and provided training to a Malaysian PhD student seconded to my lab for 3 months to help establish its use in laboratories in Malaysia. Through the CDA I will continue this high level of support and interaction with Malaysian research groups, in alignment with the MRC's strategic aim 2 "Going Global".
By defining the ligands essential for human red blood invasion by P. knowlesi, I will identify novel vaccine candidates which in the long term could be used in humans or even the macaque reservoir. In addition to the potential validation of novel vaccine targets for P. knowlesi, I will collaborate with Simon Draper to develop a transgenic P. knowlesi model to test existing vaccine candidates for P. vivax. This means that my research can have an immediate impact on the development of malaria vaccines, since it establishes an important translational path to facilitate the development of novel vaccine targets as they emerge from my basic research in P. knowlesi.
My work will impact directly on healthcare in the UK. I have already provided parasite DNA and preparations from cultured parasites to the UK Malaria Reference laboratory to aid diagnosis of P. knowlesi infections in returning travellers. I regularly attend the All-party Parliamentary Committee for Malaria and Neglected Tropical Diseases at Westminster (MRC strategic Aim Two: Research to people). This allows me the opportunity to discuss my research with policy makers, including politicians, officials from the UK Department for International Development and key non-governmental organisations. In addition to the training and career development opportunities afforded to me (see Career Intentions), the project will provide a postdoctoral researcher with training in a superb collection of research skills. The combination of exposure to cutting edge imaging skills and training in a new malaria model with wide potential will be highly prized by the malaria research community, and the novelty of the system will provide plentiful opportunities to develop independence.
Finally, a significant hurdle to the delivery of impact for neglected tropical diseases is the relative lack of economic incentives for investment from Industry. Public awareness has a major role to play in promoting the required investment and I will use my experience of public engagement to highlight this (See communications plan for details).