The Silicon Trypanosome

In this proposal we intend to set the foundation for a description of the cellular workings of parasitic protozoa called trypanosomes. Trypanosomes are responsible for the disease sleeping sickness in sub-Saharan Africa. The parasites are transmitted between people by biting tsetse flies. Once injected into the bloodstream they begin to proliferate and eventually invade the brain and central nervous system. Once inside the brain the presence of parasites leads to decreasing neurological function. Patients become depressed and cognitive function breaks down. They eventually become mad, fall into a coma and die. In recent years it has become possible to dissect trypanosomes at the molecular level. We have determined the sequence of their genetic code. We can measure the abundance of the individual proteins that are assembled within the trypanosome's structure. We can also measure the manner by which chemicals are transformed from one to another within the parasite. In short, we have at our disposal the parts list that comprises a trypanosome. We would like to exploit this information to assist in designing drugs that can perturb the parasite's inner workings. However, in order to achieve this, it is not enough to have a simple parts-list of the parasite. We need to understand how those parts assemble and how they interact with one another in order to create this living system, the trypanosome. Systems Biology is a recently emerged discipline that combines high throughput measurements of cellular parts, along with measurements of the dynamics of interactions between those parts and then employs high capacity computational modelling in efforts to describe how cellular constituents combine to create recognisable biological function. An ambition of systems biology is to reconstruct biological systems from descriptions of their component pieces with mathematical descriptions that describe how those pieces interact. Increasingly, models are emerging that describe biological function emerging from combined components of the cell. For several model organisms, including yeast and the bacterium Escherichia coli, models of cellular function are being combined into a project termed 'The silicon cell' which ultimately aims to include all component pieces of a cellular system and to describe the dynamics of the connectivity between them in order to predict how the system behaves as a whole. Profiting from the availability of the full genome sequence and methods to determine how genes are turned on to produce RNA transcripts that are then translated into proteins which ultimately control the flow of life through these cells we propose to generate a 'silicon trypanosome', i.e. we propose to build fully descriptive mathemical models of the flow of information that defines a trypanosome. We will take a bottom up approach, starting with a biochemical pathway, the so-called trypanothione pathway that dictates how well trypanosomes can deal when exposed to oxidative stresses. We have chosen this pathway because a great deal is already known about biochemical parameters of the component proteins, or enzymes, of this pathway. Furthermore the trypanothione pathway links directly through the NADPH generating pentose phosphate pathway to the glycolytic pathway, which consumes the parasite's major energy supply, glucose. A comprehensive mathematical model describing the glycolytic pathway in trypanosomes already exists, hence in a bottom up manner, extending into an adjacent pathway, offers a rational way towards a comprehensive model of the trypanosome. In addition to collecting data on the component pieces of the trypanosome we will alsoimplement a range of novel mathematical techniques to ensure the models we build are testable and robust. Ultimately we aim to use the models to predict the best ways to perturb the parasite's biological make up with the hope of generating new drugs.

The aim of this project is the creation of a comprehensive, multi-scale model of trypanosome physiology. Broadly the project can be divided into two phases: Step 1. Building a comprehensive, identifiable, predictive and testable model of trypanosome biology, starting with a detailed analysis of the trypanothione/redox network and the pentose phosphate pathway (including subcellular localization), transcriptomics, and protein abundance and flux. This expands on existing detailed models of various aspects of trypanosome biology, including detailed kinetic models of central metabolism previously published by our group. Step 2. Expansion of pathway knowledge by targeted molecular profiling and statistical ab initio network reconstruction. The following molecular profiling approaches will be employed: Various approaches will be taken to derive accurate quantitative measurements of metabolite levels in trypanosomes. LC-MS, GC-MS, HPLC and NMR, as well as specific bioassays, will all be used to assess metabolites of the polyamine, glutathione and trypanothione pathways. RNAseq will be employed to measure abundance and turnover of transcripts. Enzyme kinetic measurements will be used to acquire additional rate information on parts of the pathway for which information is not available. Mathematical modelling, metabolic control analysis, and statistical inference will be used in the construction of descriptive models across scales. Models will be tested and updated iteratively based on results feeding back through acquisition of new data sets following genetic and pharmacological perturbation of the pathways that assess model veracity.

WHO WILL BENEFIT FROM THIS RESEARCH? (a) Biomodellers: This project will involve the development of new statistical methodologies that will enable the Formal characterisation of model uncertainty. These methodologies will be of general utility to all scientists involved in modelling of high throughput datasets in which uncertainty is an inherent feature of the datasets. (b) Systems Biologists: Methods will be developed to integrate multiple 'omic' datasets, along with the dynamics of interactions between transcript, protein and metabolic flux. (c) Trypanosome biologists: Large data sets encompassing all transcripts, proteins and metabolites within a cell will be generated and made available to the trypanosome research community (beyond the pathway upon which we are focusing) (d) Neglected Diseases Drug development agencies: Organisations including the Drugs for Neglected Diseases initiative (DNDi), World Health Organisation (WHO), Consortium for Parasitic Drug Development (CPDD), and other major funding agencies (Bill and Melynda Gates Foundation, Wellcome Trust, European Commission) do not generally employ systems biology in their evaluation of drug targets. We believe that outputs from the project will include new ways of assessing suitability of particular proteins for targets and expect the approach to enter consideration in target evaluation. (e) Human African trypanosomiasis victims and farmers: Although this project will not produce new drugs for HAT directly we believe in the longer term our methodologies will expedite drug target evaluation and ultimately assist in drug development for this neglected tropical disease (and others too as our methods enter other drug development campaigns). Since animal trypanosomiasis is responsible for massive losses to livestock in Africa, the same developments in the drug discovery process will also be relevant to livestock farmers in Africa in the longer term. HOW WILL THEY BENEFIT AND WHAT WILL BE DONE TO ENSURE THAT THEY BENEFIT Biomodellers, Systems Biologists and Trypanosome Biologists will learn of the new methods through conference and publication in International journals, and through data becoming available at community websites e.g. the tritrypDB websites. Software will be available freely from the applicants' wedsites. We will actively demonstrate to the neglected diseases drug development agencies the potential of our methods and demonstrate how software developments can be integrated into existing software (e.g. TDR-targets at the WHO - http://tdrtargets.org/). As we mention above HAT victims will not benefit directly from this project, but from new drug developments that will stem from the methodologies we implement. The same applies to advances in treatment of animal trypanosomiasis.