DS0402 - Décryptage des fonctions biologiques élémentaires et de leur intégration

Non-glycolytic pathways in glycosomes: novel functions for trypanosome development and virulence – GLYCONOV

GLYCONOV - Non-glycolytic pathways in glycosomes: novel functions for trypanosome development and virulence

Trypanosomatids are parasites transmitted by insect vectors responsible for major human diseases, which represent ~24 million infected people worldwide. However, there is no vaccine and few drugs have been developed. The unconventional biological processes developed by these parasites, such as the glycosomal metabolic pathways studied here, are considered potential targets for the development of new drugs. It is in this process that the GLYCONOV project is positioned.

Towards the identification of new functions of unexplored or poorly explored glycosomal metabolic pathways in trypanosomes

Trypanosomes have a very atypical intermediate metabolism, including specific metabolic pathways located in the glycosomes, peroxisome-like organelles containing mainly glycolytic enzymes. GLYCONOV is an original project on unexplored or poorly explored non-glycolytic glycosomal metabolic pathways for which the GLYCONOV partners anticipate new roles in the development and interactions of the parasite with its hosts (reservoir mammals including humans and the tsetse fly , The insect vector). This project aiming at elucidating the role of three glycosomal metabolic pathways in the vector insect (PCF) and mammalian host (BSF) forms of Trypanosoma brucei is divided into 4 tasks. Task 1 involves analyzing the role(s) of gluconeogenesis and glycerol metabolism during the cyclic development of trypanosomes in the tsetse fly. In Task 2, we investigate the relationship between glycocomal IDHg and metabolism of ether lipid metabolism in PCFs. Indeed, we have shown that IDHg is essential for the development of the parasite in the tsetse fly and for the in vitro differentiation of PCF into metacyclic forms. Task 3 deals with the biosynthesis of sugar nucleotides, which are considered to be synthesized in trypanosome glycocomes, although our unpublished data suggests that the pathway leading to UGP-glucose, produced by UDP-glucose pyrophosphatase (UGP), is active in the cytosol instead of the glycosomes. In Task 4, we are also studying the role of these three metabolic pathways in bloodstream form trypanosomes (BSF).

The purpose of GLYCONOV is to get a comprehensive understanding of the role of 3 glycosomal metabolic pathways, in both T. brucei PCF and BSF, which have been shown by the GLYCONOV partners to be essential for viability of PCF trypanosomes and/or during development in the tsetse fly. This will be achieved by generating mutant cell lines (RNAi, knockout, expression of tagged- or point-mutated proteins, etc.) for key enzymatic steps of these pathways. In addition, a large panel of mutants are already available in our network (>100 have been produced so far) and some of them will also be analysed with the new tools developed by the GLYCONOV partners. These mutants will be analysed using a number of state-of-the-art qualitative and quantitative approaches including (i) metabolomic (NMR and IC-MS/MS), lipidomic and proteomic approaches, (ii) in vitro differentiation in the newly developed RBP6-driven differentiation model, and (iii) in vivo analyses of parasite development in the tsetse fly and mice models.

1- Task 1: We have characterized in detail the nature of the catabolic repression exerted by glycerol on the glucose metabolism. This repression results from competition for glycosomal ATP between HK (1st step of glycolysis) and GK (1st stage of glycerol degradation), which is in very large excess.
2. Task 1: We identified three different pathways (2 proven and one under analysis) for the synthesis of fructose 6-phosphate via gluconeogenesis, i.e. FBPase, reversion of PFK and probably SBPase (in progress).
3- Task 2: Against all odds, the glycosomal IDH (IDHG) accepts both NAD+ and NADP+ as a cofactor; The IDHg has been crystallized to understand its enzymatic activity and its role in glycosomes (article published in Protein Expr. Purif.).
4- Task 2: In the margin of this task, we have characterized the carbon sources used for lipid biosynthesis, the metabolic pathways involved, the regulation of metabolic fluxes between these different pathways and their role in vivo in the insect vector (article in review in PLoS Pathogens).
5- Task 3: When overexpressed in the glycosomes, the UGP is not active, whereas it is active in the cytosol, which is in favor of our hypothesis, i.e. the corresponding metabolic pathway is active only in the cytosol.
6- Task 4: When BSFs are grown in the presence of glycerol but without glucose, GK and especially FBPase are essential for growth, showing that bloodstream form trypanosomes carry out gluconeogenesis from glycerol.
7- In addition, two review articles dealing with the glycosomal metabolism of trypanosomes have been published in F1000 Res. and Int. J. Biochem. Cell. B, and Partner 1 is co-author of 2 articles published in PLoS Pathogens and Mol. Biochem. Parasitol. on the role of proline metabolism in vivo in the vector insect, and the role of NADH dehydrogenase in the metabolism of bloodstream trypanosomes, two aspects of the energy metabolism related to the GLYCONOV project.

This project will certainly contribute to increase knowledge about unusual metabolic pathways encountered in the glycocomes of trypanosomatids (gluconeogenesis and glycerol metabolism – Task 1, sugar nucleotide biosynthesis – Task 3), as well as pathways also present in the peroxisomes of other eukaryotes (IDHg and ether lipids biosynthesis – Task 2), which have only been poorly investigated so far. Indeed, a number of atypical processes have been first discovered in the trypanosome model before being found or understood in other eukaryotes, such as RNA editing, RNA interference (RNAi), etc. We anticipate that these discoveries will significantly contribute to understanding the non-glycolytic glycosomal metabolism in both the insect (PCF) and mammalian (BSF) stages of T. brucei, as well as the peroxisomal metabolism in other eukaryotes.

Published articles
1. Bringaud F., T.K. Smith, D.P. Nolan & L.M. Figueiredo (2017) Metabolic changes during the Trypanosoma brucei life cycle. F1000Res. In press
2. Wang X, Inaoka D.K., Shiba T., Balogun E.O., Allmann S., Watanabe Y.I., Boshart M., Kita K. & Harada S. (2017) Expression, purification, and crystallization of type 1 isocitrate dehydrogenase from Trypanosoma brucei. Protein Expr. Purif. 5928:30314-5.
3. Allmann S. & F. Bringaud (2017) Glycosomes: A comprehensive view of their metabolic roles in T. brucei. Int. J. Biochem. Cell. B. 85:85-90
4. Suárez Mantilla B., L. Marchese, N.A. Dyer, N. Ejeh, M. Biran, F. Bringaud, M.J. Lehane, A. Acosta-Serrano & A. Silber (2017) Trypanosoma brucei proline metabolism is essential for colonization of the tsetse. PLoS Pathog. 13:e1006158
5. Surve S.V., B.C. Jensen, M. Heestand, M. Mazet, T.K. Smith, F. Bringaud, M. Parsons & A. Schnaufer (2016) NADH dehydrogenase of Trypanosoma brucei is important for efficient acetate production in bloodstream forms. Mol. Biochem. Parasitol. 211:57-61

Trypanosomes and Leishmania spp. are vector-born parasitic protozoans responsible for major human diseases, which account for ~24 million persons infected worldwide, with over 600 million people at risk. Sleeping sickness, caused by Trypanosoma brucei, is a fatal disease if untreated. However, there is no vaccine, existing medications have severe side effects (including death) and drug resistance is increasing dramatically. Similar problems are encountered for Chagas disease (T. cruzi) and leishmaniases (Leishmania spp.). Trypanosomatids (including trypanosomes and Leishmania spp.) are also famous for a variety of very unusual biological, genetic and biochemical features that stimulate broad scientific and evolutionary interests. Among the numerous exotic mechanisms developed by these parasites, several atypical metabolic pathways have been discovered. In addition, these unconventional biological processes can also be considered as potential therapeutic targets. Since 2005, several trypanosomatid genome-sequencing projects (including T. brucei, T. cruzi and three Leishmania spp.) have been completed (with the involvement of Partner 1), providing a tool to determine their very atypical metabolic potentialities, such as metabolic capabilities of the enigmatic glycosomes only described in trypanosomatids and phylogenetically closely related protozoan groups. Glycosomes are peroxisome-like organelles primarily filled up with glycolytic enzymes (up to 90% of the glycosomal content). Although glycolysis has been extensively studied in both fly midgut PCF and mammalian BSF trypanosomes, very little is known about other predicted glycosomal pathways, and many questions regarding their (i) expression, (ii) topology, (iii) regulation, (iv) roles for development and virulence, and (iv) interaction with the other subcellular compartments remain largely unanswered.

Our project primarily concerns the analysis of unconventional metabolic pathways occurring in the glycosomes of trypanosomatids, using as model the African trypanosome T. brucei, which became the favourite model to study this parasitic group, since the development of the very powerful RNA interference (RNAi) reverse genetic tool. GLYCONOV is a basic research project with potential applications aiming to characterize new metabolic pathways recently highlighted by the GLYCONOV Partners in the insect stage (procyclic form – PCF) and the mammalian stages (bloodstream forms – BSF) of T. brucei.

Since 2002, GLYCONOV Partners have made an important contribution to the analysis of different pathways and enzymes of the intermediate and energy metabolism in T. brucei PCF, including the glycosomal pathway leading to succinate excretion from glucose degradation and pyruvate phosphate dikinase (PPDK), which participates in the maintenance of the glycosomal ATP/ADP balance. GLYCONOV Partners have also recently demonstrated that BSF metabolism is more elaborated than currently thought with unexpected essential glycosomal pathways. The aim of this project is to determine the role of three poorly investigated glycosomal metabolic pathways, recently highlighted by GLYCONOV partners, i.e. gluconeogenesis and glycerol metabolism (Task 1), a novel NADPH-generating pathway via glycosomal isocitrate dehydrogenase (IDHg) and its role for NADPH-consuming ether lipid biosynthesis (Task 2), and nucleotide sugar biosynthesis (Task 3), both in PCF (Tasks 1-3) and BSF trypanosomes (Task 4), in vitro and in vivo. Our network presents the advantage of combining state-of-the-art qualitative and quantitative global metabolomic approaches, as well as proteomics, to analyse a large collection of RNAi and knockout mutants (>100 available), affecting most of the known steps of the intermediary metabolism. GLYCONOV will contribute to understand the biological function of this enigmatic organelle and will highlight new essential enzymes that could ultimately be targeted to fight sleeping sickness.

Project coordination

Frédéric BRINGAUD (Laboratoire de Microbiologie Fondamentale et Pathogénicité)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

LMU Ludwig-Maximilians-Universität Munchen
LBM Laboratoire de Biogenèse Membranaire
IP INSTITUT PASTEUR - Unité de Biologie Cellulaire des Trypanosomes
MFP Laboratoire de Microbiologie Fondamentale et Pathogénicité

Help of the ANR 597,000 euros
Beginning and duration of the scientific project: December 2015 - 48 Months

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