Unveiling the protein landscape of the African trypanosome cell surface and chasing down potential targets for therapeutic intervention

Neglected tropical diseases are a diverse group of diseases that thrive mainly among the poorest of the world's populations. Three of the World Health Organisation's 10 most significant neglected tropical diseases - African trypanosomiasis (also known as sleeping sickness), leishmaniasis, and Chagas' disease - are caused by closely-related single-celled parasites. Human infection with African trypanosomes is brought about when an infected tsetse fly takes a blood meal. Without treatment the disease progresses through general ill health to coma and death. Current drugs in use against trypanosomes are old, toxic and failing due to emergence of resistance. Urgent new research is needed to identify potential new therapeutic options.

An unusual aspect of African trypanosomes is that they multiply in the human blood in full view of the body's defence systems. They do this by periodically changing their cell surface to escape recognition by the host. However, many molecules on the parasite surface perform essential functions and cannot be changed. Trypanosomes place these in a special, protected domain on the cell surface. Identifying surface-exposed invariant molecules and understanding how this protective segregation is maintained are of major scientific interest, as well as of practical utility in uncovering ways in which trypanosomes may be vulnerable to new therapies.

Using a combinatorial approach funded by the MRC, I have previously identified the composition of the African trypanosome cell surface. I now aim to exploit this knowledge to single out those invariant surface molecules that are essential to the survival of the parasite during infection. For this, I propose to harness some of the power of modern DNA sequencing technologies to test which cell surface genes, when silenced, cause the parasite to die inside the host. A similar method will be used to identify not only cell surface genes, but any genes that the parasite uses to maintain the cell surface organisation, which is so critical to escaping the host immune attack.

Finally, I propose to test those surface-exposed molecules for their potential as vaccines. My pilot experiments show that I can use genetically-modified parasite to screen for molecules on the parasite surface that are accessible to the immune system. I propose to develop this assay and test the most promising candidates in an animal model of human disease.
The proposed work will increase our understanding of the fundamental biology of a significant human parasite, and also, by exposing essential surface molecules, provide the first steps in developing new treatments.

The cell surface is the major point of interaction between an extracellular parasite like Trypanosoma brucei and its human host. This interface also represents the primary target for host immune attack. As a result, stable and accessible surface antigens that are unique to the parasite represent promising targets for both drug and vaccine development. With the availability of a validated, high-confidence cell surface proteome for the T. brucei bloodstream-form, testing for such surface antigens that are necessary for parasite survival becomes possible.

Here, I will use established methods for gene silencing by RNA interference coupled with target DNA sequencing to screen a surface protein-specific library for growth in vitro and in the animal model of disease. This will identify those surface-exposed molecules essential to parasite survival in the host and discriminate between those effecting uptake of nutrient, resistance to innate immunity and clearance of antibodies. I will use similar methods, plus tools for endogenous-locus tagging of membrane protein genes I have developed, to identify in a genome-wide RNAi-based screen the membrane barrier components that, when ablated, cause surface proteins to escape from specialised membrane domains. Finally, I will use a piloted in vitro assay to test if parasite surface proteins are accessible to antibodies and effectively killed by host serum components. This will inform an in vivo immunisation study to test for potential as vaccines.

The proposed work and its prospective applications will have impact both within and beyond academia, extending to the clinic and general public.

The proposed basic research on essential and exposed molecules of African trypanosomes will directly impact the field of molecular parasitology, but also offer advances in knowledge that can be exploited by researchers in cell signalling, infection & immunity, and translational medicine. To ensure immediate impact, our findings will be communicated through a project website, research talks, open-access publications, and transfer of research data to public repositories.

Many therapeutic strategies rely on surface-exposed molecules for drug delivery or immune attack. Identification of parasite-specific membrane proteins that are exposed to the host and key to survival will open the way to the design of therapeutic strategies to treat trypanosomiasis and related diseases. More widely, this project will develop methodologies for testing the potential of surface-exposed proteins as anti-trypanosome vaccines. This can have a clear impact in the study of other human infectious diseases whose parasite life cycle involves extracellular stages. We will actively promote the future use of our results and methods in this context, and engage with clinicians, vaccine researchers and drug-discovery pipelines so to maximise their impact.

The long-term impact is hoped to be improved treatment and control of African trypanosomiasis and related diseases in humans. This would have a significant impact on the quality of life for the millions of people currently affected by them. This proposal's discovery science and early stage development of novel strategies for medical intervention hold much promise, though it is expected that it will be a number of years before this impact is felt.

Engaging with and educating the public about our research is a short-term goal with long-term impacts. This also holds true for the impact stored in the training that will be received by the PDRA working on this project. The long-term impact will lie in the improved intellectual, economic or medical wellbeing of the general public through application of these skills in the future.