How does the trypanosome haptoglobin-hemoglobin receptor interact with nutrients and immunity molecules? Receptor structure to cellular mechanism.

Trypanosomes are single celled parasites that cause significant disease in Sub Saharan Africa. They infect humans, causing African sleeping sickness. They also infect a variety of domestic animals, including cattle, leading to disease that reduces food production capability.

Trypanosomes live and divide in the blood of their human or animal host. To survive in this environment they have a highly adapted cell surface. This allows them to take up nutrient molecules from the blood, which they can use as sources of energy or building materials as they grow and divide. However, it also puts them in peril. If they are recognised by antibodies, they can be detected and destroyed by the body's immune defences.

The human body also contains several protein complexes called trypanolytic factors. If taken up into the parasite, these cause trypanosomes to burst and die. Species of trypanosome that can infect humans have developed ways to avoid trypanolytic factor uptake or to block the process that leads to lysis.

How do the trypanosomes both take up nutrient molecules from the blood and to avoid recognition by antibodies? They are covered by a coat of a protein called VSG. This coat is dynamic with VSG molecules being taken up into the cell together with attached antibodies. The antibodies are degraded and the VSG returned to the surface. The parasite has the ability to express a large number of different VSG molecules, and they can switch which VSG they use to avoid detection by the immune system. The receptor proteins that are used to take up nutrient molecules must bind their cargos in the context of this VSG layer and yet avoid recognition by the immune system.

In this proposal we will use a variety of different experimental tools to study one of these receptors - the haptoglobin-haemoglobin receptor (HpHbR). This receptor lies at the heart of a conflict for the parasite. It is involved in the uptake of HpHb into the trypanosome cell, from which the parasite obtains haem molecules to allow it to build its own cellular structures. But it is also a major route for the uptake of trypanolytic factors, leading to parasite death.

We will use structural biology methods to understand, in molecular detail, how the receptor recognises its different cargos, including HpHb and trypanolytic factors. We will combine this with cell biology, allowing us to understand how ligand uptake occurs in the context of the parasite surface. This will be the first study that combines structural and cellular experiments to gain insight into a trypanosome receptor, revealing how the trypanosome surface is adapted to allow nutrient uptake while avoiding destruction by the body. It will also show how an uptake route for a toxin molecules functions. This is a route currently used by natural trypanolytic factors and has the potential to be subverted to allow drug and toxin molecules to be specifically targeted to this deadly parasite.

This project combines the technical expertise of the Carrington and Higgins laboratories, allowing an integrated structural and cellular approach to understand the biology of the trypanosome haptoglobin-haemoglobin receptor (HpHbR).

We will combine structural techniques (x-ray crystallography, NMR and electron microscopy) with biophysical tools and molecular modeling, to understand how HpHbR interacts with ligands. Bacterial and baculovirus expression systems allow us to produce HpHbRs and haptoglobin variants. We have successfully solved crystal structures of T congolense HpHbR, alone and in complex with haemoglobin, and determined the structure of human HpHb complex. We have crystals (diffracting to 1.9Å resolution) and NMR data showing that we are close to determining structures of HpHbR from T. brucei variants. We will continue with these studies to determine the structure of HpHbR from a human infective trypanosome species in complex with human HpHb and HprHb and to understand the structural effect of polymorphisms that disrupt ligand binding in HpHbR from T. b. gambiense. We will use electron microscopy and image processing to study the architecture of the trypanolytic complexes and their interactions with HpHbR.

Cellular studies will allow us to integrate structural information into the context of the dynamic endocytic system and VSG surface coat. Here we follow up the finding that HpHbR can protrude above the VSG layer, making it available to ligands but also accessible to IgG binding. We will use uptake assays and high-resolution microscopy techniques to follow ligand and IgG uptake. We have prepared an HpHbR null mutant of T. brucei and will introduce variant receptors into this context, allowing us to examine the importance of the position of the HpHb binding site relative to the VSG layer. We will use flagella mutants of trypanosomes to study the role of hydrodynamic flow in HpHbR function, ligand uptake and immunoglobulin clearance.

We realistically expect the research proposed here to have a rapid and direct academic impact, both in the UK and globally. In the long term, it may also have an economical and societal effect in Sub-Saharan Africa through the better exploitation of the trypanolytic factors and through improved uptake routes for therapeutics to target trypanosomes. We expect impact within the academic community, through translation to guide the development of therapeutics, through training of postdoctoral workers, through research placements for school and undergraduates and through outreach activities.

The academic community:
This work will provide direct insight into the trypanolytic factor uptake system, revealing why some trypanosome species are human infective while others are not. It will also give broader insight into the trypanosome surface, revealing how receptor proteins within this layer recognize and internalize their ligands, while avoiding immune clearance. This will be of great interest to the broad academic community interested in the parasite surface. We will communicate these findings throughout the project, through local seminars, visits to institutes, presentations at conferences and open access publications.

Translational work:
This work has the potential to interact with therapeutic development that is currently ongoing through collaborations between the applicants and monoclonal antibody companies. Novel methods are being sought for routes by which cytotoxic agents can be specifically target for uptake into trypanosomes. A better understanding of uptake mechanisms will be important to guide these developments. In particular, through collaboration with Medimmune, we are starting to develop monoclonal antibody therapies in which toxin molecules are coupled to antibodies that can be taken up into trypanosome cells through the haptoglobin-haemoglobin receptor. The insights developed from this study, in which we will test which regions of the receptor are surface exposed in the context of the VSG coat, and how attached antibodies are internalized, will be essential to this project. We therefore expect the results from this project to feed directly into the development of therapeutics to treat African Trypanosomiasis.

Training:
The postdoctoral research fellows employed through this funding will be trained in either parasite biology or structural biology. These are research skills that are highly transferable to the study of other medically relevant biological systems, either in academia or industry. They will also receive training in transferable skills, including communication, project management, writing and IT. This will provide them with skills that will equip them to make major contributions to science and technology in the future. Importantly, they will also be mentored in career progression throughout the three years and beyond to help them to exploit this training.

Education:
Both applicants provide research opportunities for school and undergraduate research placements in their laboratories. These projects involve engagement in an original research project, allowing the students to develop laboratory experience. In many cases, this has resulted in students deciding to study biochemistry or to take a graduate research degree, thereby moving in a direction in which they can contribute to scientific research base of the UK.

Outreach:
As described in the communications plan, both applicants arrange events to reach out to different groups of people who are not actively engaged in research. These groups are fascinated to hear about the interactions of pathogens with their human hosts and the work proposed here will directly feed into our outreach presentations. In many cases these presentations are to school groups and are also opportunities to encourage these students to study medically relevant subjects at University.