ParaMet Research Projects
- 1. Identification of new metabolic pathways in trypanosomatids
Host Institutions: University Bordeaux Segalen, France, Dr Frederic Bringaud and Heidelberg University, Germany, Prof. Christine Clayton
http://www.rmsb.u-bordeaux2.fr/iMET/Bringaud_Lab/Bringaud_Lab.html
http://www.zmbh.uni-heidelberg.de/clayton/default.shtml
The function of approximately half of the genes encoded by the genome of trypanosomatids is unknown. Since most of them have no orthologs in the human genome, it is likely that potential drug targets belong to this large group of “hypothetical genes” possibly involved in as yet undiscovered metabolic pathways operating in the parasites. To identify new metabolic pathways, we have developed an approach based on the selection of viable null mutants by targeting parasite pathways to dissect their importance for parasite survival. We will identify redundant metabolic pathway(s) using reverse genetics and mass spectrometry based proteomics and transcriptomics with characterisation of impact on metabolism.
Keywords: Trypanosoma brucei, RNAi and KO mutants, metabolism, proteomics/transcriptomics
Qualifications and skills: Basic knowledge in metabolism is required. Experience in cell culture and molecular biology (DNA manipulation) is recommended.
- The metabolic consequences of gene knockout to pathway flux in trypanosomes
Host Institutions: INRA, Toulouse, France, Prof. Jean-Charles Portais and University Bordeaux Segalen, France, Dr Frederic Bringaud
http://www.lisbp.fr/en/index.html
http://www.rmsb.u-bordeaux2.fr/iMET/Bringaud_Lab/Bringaud_Lab.html
The objective of this IRP will be to establish a complete analytical methodology for detailed and comprehensive investigation of cellular metabolism in trypanosomes. Based on the complementary analytical expertise of Partners 1 (UoG), 4 (UB2) and 6 (INRA), different approaches will be used in combination to get a complete, functional, picture of the central carbon metabolism of the parasite. This includes both mass-spectrometry (MS)- based and Nuclear Magnetic Resonance (NMR)-based approaches aiming at the quantification of metabolites and their 13C-labelling patterns, as well as the establishment of 13C-labelling strategies aiming at the identification of metabolic pathway and quantification of their activities (metabolic fluxes). A specific objective will be the development and validation of novel LC-MS/MS approaches for better coverage of the metabolome and fluxome, including the analysis of Coenzyme A thioesters as well as the application of capillary ionic chromatography to the analysis of highly polar compounds. Because the metabolite content of trypanosomes is not currently established, and if and where appropriate, these approaches will be applied first to model organisms (e.g. E. coli) with known metabolite contents for validation purpose. These investigations will also include the analysis of the exometabolome (excreted compounds) of the parasites by a combination of NMR, MS and HPLC approaches. Because metabolic fluxes – i.e. the actual in vivo rates of biochemical reactions – represent the most accurate parameters for characterizing the actual operation of metabolic networks, the establishment of a complete strategy for fluxome analysis in trypanosomes will represent the central objective of this IPR. Taken together, these approaches will provide a highly valuable framework to analyse the operation of central metabolism in trypanosomes as well as the actual effects of the different mutations to be considered in the project.
Keywords: metabolomics, fluxomics, metabolic network analysis, mass spectrometry,
Qualifications and skills: background in biochemistry or chemistry. Skills in metabolomics, 13C-labelling studies of metabolism & fluxomics; analytical techniques (mass spectrometry, NMR), metabolic network analysis.
- 3. The metabolic consequences of perturbation to the pentose phosphate pathway in trypanosomes and Leishmania
Host Institutions: University of Glasgow, UK, Prof Michael Barrett and University Bordeaux Segalen, France, Dr Frederic Bringaud
http://www.gla.ac.uk/researchinstitutes/iii/staff/Michaelbarrett/
http://www.rmsb.u-bordeaux2.fr/iMET/Bringaud_Lab/Bringaud_Lab.html
In addition to novel pathway identification as outlined in task 1.1 we will also study pathways known to have potential as therapeutic targets. The pentose phosphate pathway in African trypanosomes and Leishmania provides NADPH, a critical source of reducing power in cellular biosyntheses and also in protection against oxidative stress. Inhibition of various enzymes of the pathway in both parasites should have predictable impacts on the abundance of metabolites involved directly in the pathway. It will also have less predictable effects on other parts of metabolism. In this project the genes encoding enzymes of the pentose phosphate pathway will be systematically removed from African trypanosomes (UB2) and Leishmania (UoG). Effects on cellular growth and viability will be assessed and also responses to oxidative stress. Furthermore targeted metabolite analysis quantifying metabolites of the pentose phosphate pathway and untargeted analysis, identifying changes to the broader trypanosome metabolic network, will demonstrate precisely how changes to one pathway permeate through the metabolic network to affect other aspects of metabolism (UoG/INRA). The data will be used to build a metabolic model of this pathway allowing predictions of metabolic consequences of pathway perturbation to be tested against metabolomic observation.
Keywords: metabolomics, fluxomics, metabolic network analysis, mass spectrometry,
Qualifications and skills: background in biochemistry or chemistry. Skills in metabolomics, 13C-labelling studies of metabolism & fluxomics; analytical techniques (mass spectrometry, NMR), metabolic network analysis.
- Dissecting the link between cytosolic and mitochondrial glucose metabolism in Plasmodium falciparum
Host Institutions: University of Glasgow, UK, Prof. Sylke Müller and University of Leeds, UK, Prof. David Westhead & Dr Glenn McConkey
http://www.gla.ac.uk/researchinstitutes/iii/staff/sylkemuller/
http://bioinformatics.leeds.ac.uk/~david/
During intra-erythrocytic development Plasmodium falciparum rely primarily on glucose as their carbon source. They do not fully oxidise the sugar, but convert 85 % of glucose into lactate which is excreted from the parasitized red blood cell as a metabolic end product. The remaining 15 % of phosphoenolpyruvate (PEP) is utilised for a variety of essential metabolic processes such as fatty acid biosynthesis, nucleotide metabolism and redox metabolism. This project will use reverse genetics approaches to assess the importance of PEP-utilising pathways for P. falciparum survival in vitro. It will analyse the metabolic consequences of various gene deletions by focused metabolomics (UoG). The data will provide the basis for the generation of a mathematical model of carbohydrate metabolism in Plasmodium linking into WP 3 (UoL/INRA). The data will give insights into parasite carbon metabolism that may be exploitable for future drug development.
Keywords: malaria, carbon metabolism, metabolomics, metabolic labelling, genetic manipulation
Qualifications and skills: Applicants should have a good understanding of metabolism and biochemical and genetic manipulation to dissect the role of metabolic enzymes. Molecular and biochemical laboratory and excellent numerical skills are essential.
(A) The relationship between gene expression and metabolism.
Overall aim: To understand how gene expression responds to perturbations such as drug insult and how this relates to the metabolic networks.
- Transcriptomic and metabolomic responses to drug treatments and stress in trypanosomes
Host Institutions: Heidelberg University, Germany, Prof Christine Clayton and University of Glasgow, UK, Prof Michael Barrett
http://www.gla.ac.uk/researchinstitutes/iii/staff/Michaelbarrett/
http://www.zmbh.uni-heidelberg.de/clayton/default.shtml
Most eukaryotic and prokaryotic organisms respond to environmental stresses by compensatory alterations in the transcriptome and proteome. For example, inhibition of glucose uptake into cells might lead to compensatory up-regulation of transporter expression, in order to restore the flux of glucose into the cell. This means that the effects of drugs may be attenuated by the organism’s response. We do not know, however, to what extent this happens in Kinetoplastida. The sparse results so far suggest that this may depend upon the pathway involved. Some adaptive reactions have indeed been observed in response to inhibition of purine transport while other drug treatments such as with tunicamycin evince no obvious response at all. On the other hand regulatory responses that mimic a developmental switch are seen upon inhibition of glucose transport which renders the parasite hypersensitive to the treatment. The gene expression of trypanosomes either treated with inhibitors of known and unknown targets will be assessed (UHEI). Further, the metabolite profiles of these parasites will be analysed to determine whether and how changes in gene expression translate into metabolic adaptations when parasite metabolism is perturbed (UoG). The mechanisms by which the drugs change gene expression will also be investigated. Effects of parasites on host cell transcriptomes will be studied in parallel to host metabolic response to infection.
- Discovering the epigenetic-metabolic interface in Plasmodium
Host Institutions: Radboud University of Nijmegen, NL, Prof Henk Stunnenberg and University of Glasgow, UK, Prof Andy Waters
http://www.ncmls.eu/NCMLS/MenuStructures/PI/theme3/HenkStunnenberg.asp
http://www.gla.ac.uk/researchinstitutes/iii/staff/andywaters/
Efficient utilization of nutrient sources requires constant adaptation and changes in the expression of genes, including those encoding metabolic enzymes. In recent years it has become clear that “epigenetic” events, whereby information is passed on through biochemical changes not directly encoded within DNA, play an important role in the metabolic diversity of organisms, including parasites. Such essential regulatory mechanism(s) potentially serve as promising targets to control parasitic organisms, like the human malaria parasite P. falciparum. However the link between gene expression regulation and metabolism is poorly understood. This project aims to decipher key epigenetic regulatory mechanisms and analyze the effect of metabolic perturbations on both gene expression and epigenetic marking. The approach takes advantage of the latest genomic technologies (next generation sequencing) as well as chemical or genetic means to disrupt the function of epigenetic enzymes (e.g. sirtuins). Genome-wide RNA-seq and ChIP-seq data will be generated from parasites grown on different nutrient sources or in which specific epigenetic/metabolic processes are perturbed and compared to parasites grown under “standard” conditions. These approaches will provide information on the link between metabolism and epigenetic regulation and yield knowledge that can be used in our battle against malaria.
Keywords: malaria, Plasmodium, genomics, transcriptomics, gene-regulation
Qualifications and skills: Master degree (or equivalent). Enthusiasm/Interest for epigenetics and malaria research is a prerequisite. Experience in molecular biology techniques (e.g. cloning, PCR, RNA/DNA isolation) or cell culturing is an advantage. Working with animal models (i.e. mice) might become necessary during the course of the project.
(B) Mathematical modeling of parasite metabolism.
Overall Aim: Systems biology has heralded the advent of genome scale reconstructions of metabolic networks. These provide an environment for contextualisation of metabolomic and transcriptomic/genomic data and also predictions on where metabolism may be vulnerable to inhibition. Here we will produce models of apicomplexan and kinetoplastid metabolism to interpret data from WPs 1, 2 and 4 and to guide experiments in other WPs. Comparative analyses of host and parasite metabolic models will highlight essential metabolites acquired from the host and produced de novo.
- Modelling apicomplexan metabolic and transcriptional regulatory networks
Host Institutions: University of Leeds, UK, Prof David Westhead & Dr Glenn McConkey and INRA, France, Dr Fabien Jourdan
http://bioinformatics.leeds.ac.uk/~david/
http://www.international.inra.fr/
In order to provide context to help interpret data from the above projects Constraint based modelling methods, like flux balance analysis (FBA), provide a route whereby genome scale models of metabolic networks can be constructed without detailed knowledge of enzyme kinetic parameters. They have been popularised in recent years through significant predictive success in many studies in yeast and bacteria. Westhead and McConkey are presently building such models of the malaria parasite, parameterising them with experimental data (e.g. biomass composition) and validating growth rate predictions for growth in vitro in red blood cells. Other similar models have been constructed for malaria and Leishmania. This project will continue this work by compiling genome scale models of the different parasites studied over the ITN, using and developing existing models where possible or building de novo where information is not presently available. Models will incorporate metabolic process and transcriptional regulation based on approaches that have been effective in other species.
FORTH, T., MCCONKEY, G.A., & WESTHEAD, D.R. (2010) MetNetMaker: a free and open-source tool for the creation of novel metabolic networks in SBML format. Bioinformatics. 26(18):2352-3.
WHITAKER, J. W., WESTHEAD, D. R. & MCCONKEY, G. A. (2009) Alio intuitu: the automated reconstruction of the metabolic networks of parasites. Trends in Parasitology, 25, 396-7.
Keywords: Computational biology, network analysis, drug target identification, genomics
Qualifications and skills: Competent knowledge in fields of biology and computer science, particularly single-celled eukaryotes; understand genomic data analysis; strong analytical, problem solving, data evaluation and statistical skills; proficient in SQL, PERL, Oracle DB, UNIX, C++ and the common MS office applications; laboratory experience helpful.
- Modelling kinetoplastid metabolic and transcriptional regulatory networks
Host Institutions: INRA, France, Dr Fabien Jourdan and University of Leeds, UK, Prof David Westhead
http://bioinformatics.leeds.ac.uk/~david/
Genome reconstructions of trypanosome metabolism will be carefully annotated in view of expert opinion and literature citations. These will then be used to produce a constraints based model of kinetoplastid metabolism to provide both a contextual basis for interpretation of data in other work packages and also predictive power to generate testable hypotheses on potential drug targets with similar techniques as outlined in project 7. Metabolic regulation will be investigated by analysing the structure of the flux coupling graph.
Model reconstruction will be based on the Trypanocyc database and will take into account manual curation already provided by several experts of Trypansoma brucei. The bioinformatics work will be achieved within the MetExplore web server (http://www.metexplore.fr/).
WHITAKER, J. W., MCCONKEY, G. A. & WESTHEAD, D. R. (2009) The transferome of metabolic genes explored: analysis of the horizontal transfer of enzyme encoding genes in unicellular eukaryotes. Genome Biology, 10, 36-42.
WHITAKER, J. W., LETUNIC, I., MCCONKEY, G. A. & WESTHEAD, D. R. (2009) metaTIGER: a metabolic evolution resource. Nucleic Acids Research, 37, D531-8.
Keywords: Metabolic network, Constraint Based Models, Flux Coupling Graph
Qualifications and skills: Bioinformatics, Metabolic network modelling, programming skills.
(C) Characterise new drug targets.
Overall Aim: To identify and characterise new drug targets in protozoa. Crucially, host cell metabolism will be studied in parallel to minimise the risks of side effects.
- Analysis of the role of protein kinases in trypanosome metabolic regulation
Host Institutions: University of Glasgow, UK, Prof. J.C. Mottram and Intervet,Germany, Prof. P. Selzer
http://www.gla.ac.uk/researchinstitutes/iii/staff/jeremymottram/
Recent evidence suggests that proteins critical to central metabolism in trypanosomes are phosphorylated, indicating a role for protein kinases in regulating metabolic processes. We have identified 183 protein kinases (ePKs) in the Trypanosoma brucei genome and have created a protein kinase focus RNAi library, comprising 183 RNAi plasmids and RNAi cell lines. This powerful resource will be used to investigate the role of protein phosphorylation in regulation of metabolic function. In this project we shall (i) Create a reporter line in which the trypanosome specific organelles called glycosomes are tagged with GFP and carry out an RNAi screen for kinases involved in glycosome function (; (ii) develop procedures for phosphoproteomic and/or metabolomic profiling of protein kinase RNAi induced cell lines ; (iii) carry out phosphoproteomic and/or metabolic profiling on selected RNAi lines identified to alter glycosome function . This project will result in the identification of protein kinases essential for metabolic regulation. Those kinases that are of particular interest will be analysed by x-ray crystallography to determine their 3 dimensional structure and their potential as drug targets will be assessed in silico and in the laboratory.
Keywords: Trypanosoma brucei, protein kinase, metabolism, genetics, parasite
Qualifications and skills: molecular biology, biochemistry, genetics
- 10. Identification of natural products with antimalarial activity by targeting pyrimidine metabolism.
Host Institutions: Instituto de Parasitología y Biomedicina "López-Neyra", Spain, Prof. Dolores Gonzalez Pacanowska and MEDINA, Spain, Dr Olga Genilloud
Microbial extracts have provided an excellent number of drugs over the past 50 years and constitute a source of unique chemical diversity synthesized in relatively simple metabolic pathways. On the other hand, pyrimidine biosynthesis constitutes a major metabolic pathway for therapeutic intervention in malaria parasites since important differences exist with regard to the host metabolism regarding requirements for synthesis de novo. The activities in this project will involve high throughput screening of new chemicals, parasite biology, enzyme kinetics and natural products. Firstly we aim at the identification of compounds with antimalarial activity by phenotypic screening of microbial extract collections available at the Medina Foundation (CSIC-MEDINA). Secondly we will take a target-based approach that will focus on screening of microbial extracts (CSIC-MEDINA) or of chemical libraries (UoL) with selected enzymes involved in pyrimidine metabolism validated as targets by chemical and genetic means. The partnership will provide expertise in the areas of antimalarial drug discovery, compound profiling and preclinical studies and a unique perspective in translational research. The activity may lead to the discovery of innovative therapies and will provide training in the area of drug discovery and development from both an industrial and academic perspective.
11. Identification on new targets in Plasmodium by unravelling the mode of action of phenotypic hits
Host Institutions: MEDINA, Spain, Dr Olga Genilloud and Instituto de Parasitología y Biomedicina "López-Neyra", Spain, Prof. Dolores Gonzalez Pacanowska
Here we will explore the mode of action of hits/leads arising in phenotypic screening activities. To this aim we shall evaluate the inhibition of a set of enzymes available in the CSIC lab in order to establish potential targets and compare the action on the corresponding human target enzymes. Likewise specific target metabolic pathways will be explored by (i) profiling of the metabolome of Plasmodium parasites treated with hit compounds and (ii) investigating selected pathways by performing precursor incorporation studies.
12. Characterization of unexplored key steps of phospholipid metabolism in Plasmodium and their potential use as pharmacological targets.
Host Institutions: University of Montpellier 2, France, Prof Henri Vial/Dr Rachel Cerdan and University of Glasgow, UK, Prof Sylke Müller
http://www.umr5235.univ-montp2.fr/spip.php?rubrique10
http://www.gla.ac.uk/researchinstitutes/iii/staff/sylkemuller/
In intraerythrocytic malaria parasites, parasite-derived glycerophospholipids are the main membrane constituents. Phospholipid synthesis has been validated as a drug target and a number of toxic choline analogues have moved progressively towards development of a clinical candidate in humans (phase 2 trials are ongoing). We now wish to capitalise on diverse and complementary strengths to characterize other antimalarial targets. The most abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine , are synthesized by a variety of metabolic pathways, whose combined presence is not usually found in other organisms. In addition, base-exchange mechanism and phosphatidylglycerol pathways remain unexplored. Our interests in these metabolisms are based on their essentiality, on the identification of regulatory steps and on host specificity. We also propose biochemical, cellular and structural characterization of key proteins. For example two phospholipid-related proteins (one transporter and one enzyme) both druggable and suitable for pharmacological targeting are of higher interest for our current investigation. The goal is to fully characterize these potential targets including the 3D structure determinations using both X-ray crystallography and nuclear magnetic resonance spectroscopy (UM2). The description at the atomic level of the target-substrate/drug interactions will provide support for the subsequent identification of new inhibitor scaffolds by in silico rational drug-design studies. Genetic studies for functional characterization will be performed at UoG.
Keywords: phospholipid metabolism, functional and structural characterizations, potential targets
Qualifications and skills: biochemistry, structural biology, microbiology