Mitochondrial biogenesis and function in trypanosomes

Trypanosomatids are unicellular parasites that are as devastating in their consequences on health and economy as they are captivating as subjects of biological study. Our research is driven as much by the desire to alleviate the suffering caused by these parasites as by the fascination with the ways they have taken biological principles that underpin all life on earth, and twisted them into something bizarre and unique.

This project will study the mitochondrion in the sleeping sickness parasite Trypanosoma brucei. The mitochondrion is a cell organelle that is often described as the 'power plant' of eukaryotic cells (complex cells from multicellular organisms like humans, or unicellular organisms like yeast or trypanosomes that are not bacteria) but we now know that they provide the cell with much more than energy. The trypanosome mitochondrion hosts two particularly bizarre phenomena: a structure called 'kinetoplast' and a process called 'RNA editing'. Mitochondria have their own genome; usually a fairly short, circular piece of DNA. In trypanosomes, however, thousands of circular DNAs form a gigantic network that has been likened to medieval chainmail: the kinetoplast. When a trypanosome divides it has to accomplish faithful duplication of its kinetoplast so that each daughter cell inherits the complete set of genes that is encoded in this genome. Like other mitochondrial genomes, the kinetoplast encodes important proteins that the cell needs to survive. To make these proteins (this is called 'gene expression'), the DNA has first to be transcribed into a messenger molecule, the mRNA. Trypanosomes are unique in that they remodel many of their mitochondrial mRNAs in a very elaborate process called RNA editing. We know from past research in our lab and other labs around the world that maintaining the kinetoplast and expressing its genes is critical for parasite survival. If we interfere with one of these processes, the parasite dies.

A main goal of our research is to understand exactly how the cell makes and duplicates its kinetoplast, how it expresses the kinetoplast's genes to make proteins, and what these proteins actually do. This is complicated by the fact that trypanosomes have a complex life cycle. They are transmitted by tsetse flies, and the trypanosome that thrives in the mammalian bloodstream (and makes the mammal sick) changes the purpose of its mitochondrion once it has been taken up by the fly.

We have three goals in this project. Our first goal is to identify as many of the proteins that are involved in maintaining and duplicating the kinetoplast, and in expressing its genes, as possible. We and others have identified some of these proteins, but we believe there are many more. Because these processes are so unique there is a good chance that some of these proteins will be unique as well, which makes them potentially very suitable targets for new drugs. These are much needed since current treatment involves drugs that are toxic and drug resistance is emerging in various parts of the world.

Our second goal is to understand which of the kinetoplast genes are needed by the parasite when it changes from the mammalian bloodstream form to the one that can survive in the midgut of the tsetse fly. This transformation involves a particular intermediate form, called 'stumpy' because of its peculiar morphology, and the mitochondrial activities in the stumpy form are very poorly understood.

Our third goal is to understand how many genes exactly are encoded in the kinetoplast. We know of the ones that encode proteins, but there are many more that are not used to make proteins. Instead, they make small RNA molecules called 'guide RNAs' that are required for the RNA editing process. If we really want to understand how kinetoplast gene expression works we need to know how many guide RNAs there are. And because of advances in DNA sequencing technology we can now for the first time determine this.

Trypanosomatid parasites cause human and animal diseases with devastating health and economic consequences. Their survival crucially depends on mitochondrial (mt) function and expression of the organellar genome. Mitochondrial activity is highly regulated during the life cycle of the sleeping sickness parasite Trypanosoma brucei.

Aim 1 uses an RNAi screen to identify genes that are important for mtDNA maintenance and expression in T. brucei. Some of these genes will be trypanosomatid-specific genes and potential drug targets. Characterisation of the corresponding proteins will involve localisation by light and electron microscopy and functional categorisation using a panel of assays that interrogate the various steps along the maintenance/expression pathway. Some proteins will be characterised further by pull-down of tagged versions and identification of interaction partners by mass spectroscopy.

Aim 2 identifies mtDNA-encoded functions required for differentiation from mammalian bloodstream stage to tsetse midgut stage, and dissects the mechanism of the compensatory ATPase mutation that permits bloodstream stage survival in the absence of mtDNA. Pleomorphic T. brucei parasites that lack mtDNA or key nuclearly encoded complex I subunits will be tested for their ability to differentiate in vitro and in mice and flies. The cause of any differentiation defects will be investigated by analysis of mt and cellular metabolism. The composition and function of wild type ATPase will be compared with that of mutated enzyme in the presence and absence of mtDNA-encoded subunits.

Aim 3 identifies the complete T. brucei mtDNA minicircle and guide RNA repertoire by deep sequencing. Guide RNAs and their target mRNAs will be verified by in vivo cross-linking and deep sequencing. The fidelity of minicircle distribution to daughter cells will be determined by measuring changes in complexity over time and feeding the data into a mathematical model.

Academia.

In addition to direct benefits (see Academic Beneficiaries) the highly collaborative nature of this project will strengthen ties between labs and academic institutions on campus, within the UK (Edinburgh, Dundee, St Andrews, Cambridge, Glasgow), and between the UK and Europe, specifically France and the Czech Republic. This is important since in line with the MRC's aim to provide leadership in international partnerships. The MRC recognises that "international collaborations play an essential role in maintaining the high standards of research that the MRC funds". Our collaborations will be long-term and involve exchange of researchers between labs. We expect that they will be leveraged to win additional funding, for example through European funding schemes. Thus, we expect that this research will have a positive impact on science in the UK and Europe well beyond the immediate research outputs.

Commercial private sector / public sector.

We will teach skills that are highly relevant outside of academia. This project will train 2 PDRAs and 1 technician, and we are committed to the training of PhD students (currently three in the lab) and undergraduates. The technologies used in this project, such as genome wide RNAi screen, bespoke bioinformatics tools, deep sequencing analyses, are all at the cutting edge of science, not to mention transferable skills such as project management and communication. In addition, my laboratory is developing a good track record in translating basic science into applied research. We have a strong programme in drug discovery that involves a major industrial partner with Glaxo Smith Kline. All trainees coming through our lab will benefit from these activities since we operate in an open environment where ideas and data are constantly exchanged through lab meetings and informal discussions. Since many of our trainees will go on to have careers in the private and public sector we will contribute to the training of a highly skilled workforce.

Global Health.

This project does not directly aim to develop therapies for neglected diseases or commercially exploitable products. However, the trypanosome cell lines generated in Aim 2 will be very valuable tools in efforts to understand drug action in trypanosomes and to develop novel anti-trypanosomatid drugs. For example, our preliminary data using such cell lines confirms that interference with maintenance or expression of mitochondrial DNA is part of the mode of action of widely used veterinary drugs. We also anticipate benefits to Global Health in the long-term. The intended research aims to identify essential enzymes with highly specific functions in these parasites, which are potential new drug targets. We have a very good track record in feeding such discoveries into the translational pipeline: the drug discovery programme mentioned above is a direct consequence of identifying such a parasite-specific enzyme a few years ago. In addition, the project is aimed at increasing our fundamental understanding of mitochondrial biology, an area of very high medical relevance. It is increasingly recognised that mutations in the respiratory complexes that we study are linked to severe disorders in humans, and that many common human diseases are associated with a decline in mitochondrial activity. Respiratory switches like the one in trypanosomes that we propose to investigate, are also as a hallmark of many cancer cells. Although not expected in the time frame of this study, any exploitable results will be developed through Edinburgh Research and Innovation who have extensive expertise in this area. Data will be shared in accordance with MRC and University policy.

The wider public

As detailed elsewhere, we are committed to advancing public understanding in science and will communicate our field of research, including the broader question of inequalities in global health, to a wider audience in several ways (see Communications Plan).