The molecular mechanism for trypanosome cell death induced by ApoLI and its inactivation in human infective T. b. rhodesiense.

Trypanosomes are deadly, single celled parasites that are injected into the blood of humans, domestic livestock and wildlife when bitten by infected tsetse flies. They cause sleeping sickness in humans and wasting diseases in livestock, including nagana in cattle. These diseases have significant impact in sub-Saharan Africa. They lead to thousands of human deaths and cause significant human suffering by reducing the productivity of livestock.

Most species of African trypanosomes cannot infect humans, due to two molecular complexes found in human blood, known as trypanolytic factors. These contain a shared component, apolipoprotein LI (ApoLI) which kills the trypanosomes. ApoLI can punch holes in the membranes of the parasite, leading to death of the cell. The two subspecies of trypanosome that infect humans and cause sleeping sickness are able to inactivate ApoLI and can therefore survive in human blood and cause disease.

How ApoLI kills trypanosomes is not well understood. It is well established that ApoLI punches holes in membranes but it is not clear which membranes within the trypanosome cell are damaged. Some studies suggest that ApoLI bursts the lysosome, a compartment of the cell used to degrade cellular components. Others suggest that it punches holes in the outer cell membrane, while further studies suggest that it is the mitochondria, the energy 'power-houses' of the cell, that are damaged. We will use the latest microscopy methods to observe ApoLI as it moves around the cell, and to see what parts of the cell are affected, and how they are killed.

The mechanism used by ApoLI to punch holes is also not well understood. How does it interact with membranes and how does it form pores? How are these pores regulated? These questions are important for understanding how ApoLI kills trypanosomes. They are also important in understanding the natural function of ApoLI in the human body, as it is known that changes in ApoLI are associated with late-onset kidney disease. We will use structural biology methods to understand what ApoLI looks like, and to perform a variety of studies to see the mechanism by which it forms pores.

We will also study one of the two human infective species of trypanosome, Trypanosoma brucei rhodesiense. This parasite can resist the toxic effects of ApoLI because of the presence of a single molecule, the serum resistance associated protein, SRA. We have shown that SRA can interact directly with ApoLI under a variety of conditions and now aim to determine exactly how it inactivates ApoLI. Does it bind to ApoLI to prevent it from getting to the region of the cell in which it mediates its toxic effects? Or does it bind to ApoLI and prevent it from forming pores? Or perhaps it is both? We will again use structural biology methods to understand exactly how ApoLI interacts with SRA, and how it stops pore formation by ApoLI. We will also use the latest microscopy methods to investigate how SRA moves around within trypanosomes that are exposed to ApoLI.

These studies will provide molecule detail to allow us to understand how ApoLI kills trypanosomes and how human infective trypanosomes become resistant to this toxin. This has the potential to help us to design new therapeutics to prevent sleeping sickness. By understanding how SRA binds to and inhibits ApoLI, we can design new versions of ApoLI that are resistant to SRA-mediated inactivation. These can be supplied directly to sleeping sickness patients to kill trypanosomes. We can also develop small molecules that block the SRA-ApoLI interaction, allowing the bodies natural ApoLI-mediated defenses to kill the parasite. Finally, transgenic cattle are being developed which contain ApoLI to prevent trypanosome growth. Using versions of ApoLI which are resistant to SRA-mediated inactivation will decrease the chance of these cattle being infected and providing a reservoir for the growth and development of human infective trypanosomes.

This project combines the technical expertise of the Higgins, Carrington and Gilbert laboratories, allowing an integrated structural, biophysical and cellular approach.

We will combine structural techniques (x-ray crystallography, NMR and electron microscopy) with biophysical tools. Bacterial expression systems allow us to produce ApoLI and SRA, while hybridoma culture is used to generate monoclonal antibodies. We have obtained a crystal structure of SRA and will also use crystallography (and possibly NMR) to obtain structures of ApoLI and the SRA:ApoLI complex. Electron microscopy will be used to study ApoLI integrated into nanodiscs and liposomes. Liposome flotation and dye-release will be used to study membrane binding and pore formation. TIRF microscopy with photobleaching will reveal pore stoichiometries. Hydrogen-deuterium exchange mass spectrometry will reveal binding interfaces and biophysical tools, such as SPR, will characterize interactions. This combined set of structural and biophysical tools will reveal how ApoLI forms pores and how SRA inhibits these pores.

Cellular studies will allow us to integrate structural information into the context of trypanosome cell biology. We will use the latest microscopy techniques to study fluorescently labeled single molecules in live cells that have specifically labeled organelles. We will use these to study the dynamics of ApoLI and SRA movement and to correlate these changes with the effects of cell killing. We will use TIRF microscopy to study ApoLI at the cell surface and FRET-FLIM to determine if it forms clusters. We will design novel fluorescently labeled ApoLI variants that fluoresce when they integrate into membranes. We will use these tools, together with the integration of structural and biophysical information, to assess the molecular basis for ApoLI pore formation and its inactivation by SRA.

The research proposed here will have a rapid and direct academic impact in the UK and globally. In the long term, it may also have an economical and societal effect in Sub-Saharan Africa through design of more effective trypanolytic factors that will target human-infective trypanosomes, or the production of small molecules that will prevent trypanolytic factor inactivation by T. b. rhodesiense. It will impact 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 focuses on the important question of what makes a trypanosome human infective. It will provide direct molecular insight into the mode of action of the pore forming protein, ApoLI, showing how it kills most trypanosome species. It will also reveal how T. b. rhodesiense inactivates ApoLI, allowing it to survive in human sera and to cause sleeping sickness. Our findings will also impact more broadly. Polymorphisms in ApoLI are linked to late-onset kidney disease, and we will test the effect of these changes on the function of the protein. Furthermore, ApoLI is a member of the same class of pore forming protein as Bcl-2 proteins important in apoptosis (and cancer) and bacteria-killing colicin proteins and our findings will inform studies of these molecules. We will communicate our findings throughout the project, through local seminars, visits to institutes, presentations at conferences and open access publications.

Translational work:
This work will inform development of transgenic cattle that can resist trypanosome infection and therapeutics to treat sleeping sickness. Transgenic cattle that produce ApoLI are already under production to generate livestock resistant to trypanosome infection. The ApoLI-Hp fusion produced during this project will increase uptake of recombinant ApoLI into trypanosomes and will be ideal for a future generation of these cattle. Our knowledge of the molecular basis for ApoLI function and its interaction with SRA will also inform production of a new generation of modified ApoLI which can resist inactivation by SRA. These can be used to treat sleeping sickness patients infected with T. b. rhodesiense. 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 skills are highly transferable to other medically relevant biological studies, either in academia or industry. They will also receive training in transferable skills, including communication, project management, writing and IT. This will equip them to make major contributions to science and technology in the future. They will 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, equipping them to contribute to the scientific research base of the UK.

Outreach:
As described in the communications plan, both applicants arrange events to reach 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.