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

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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.