The project of the iMET group meets the fundamental and applied research goal by pursuing our work on the intermediate and energy metabolism of trypanosomatids. Our project particularly concerns T. brucei, from which two parasitic forms have been adapted to in vitro culture, the bloodstream forms of the mammalian host and the procyclic form found in the midgut of the insect vector. Despite the tremendous progress made over the recent years (ref), the metabolism of the procyclic trypanosomes is far from being fully understood. Our aim is to get a comprehensive understanding of the metabolism in this parasite. This work may reveal essential and parasite specific steps, which could constitute potential therapeutic targets.
Our research program focuses on three different aspects of the intermediate and energy metabolism:
Metabolic Dialogue: Host/Parasite, Glycosome/Mitochondria
In the midgut of the insect, the procyclic stage of T. brucei is thought to encounter an amino acid-rich environment. However, in vitro this stage has been traditionally cultivated in glucose-rich media. Under these growth conditions, the procyclic trypanosomes convert glucose (the prefered carbon source), threonine and proline, into partially oxidized end products by aerobic fermentation. We are particularly interested in understanding the topology and regulation of the metabolic network involving degradation of these three carbon sources in the procyclic trypanosomes (ref). Interestingly, glucose and threonine catabolism leads to an important production of acetate into the mitochondrion of the parasite. We recently showed that acetate production is essential to feed lipid biosynthesis, which is initiated in the cytosol (ref). Our main projects concern the analysis of acetate metabolism in the procyclic trypanosomes, as well as in other stages (including the bloodstream form of T. brucei) or parasites.
Mitochondrial functions and dynamics in trypanosome life cycle
Mitochondria from trypanosomes are notorious for various peculiarities from the prototypical organelle. Unlike most other eukaryotes that contain numerous mitochondria, the pathogenic trypanosomes have a single, typically reticulated mitochondrion where mtDNA is restricted at the basis of the flagellum, only replicated once per cell cycle, which is synchronized with nuclear replication and division. During the trypanosome life cycle, the shape and functional plasticity of their single mitochondrion undergoes spectacular changes, reflecting adaptation to different environments. Indeed, the mitochondrion of this parasite exist in at least two major forms: (i) the fully active and developed one characteristic for the procyclic stage (a form transmitted by the tsetse fly Glossina spp.) that harbors the oxidative phosphorylation complexes (OXPHOS) for energy production (Figure A); (ii) the functionally and morphologically down-regulated form found in the bloodstream form (responsible for the actual disease in vertebrates) with energy produced through substrate level phosphorylation from glycolysis (Figure B). These alterations correlate with the adaptation of the parasite to its environments, alternating between the glucose-rich blood of a mammalian host and the proline-rich haemolymph and tissue fluids of the blood-feeding tsetse fly. The combination of these different states, among which the trypanosome mitochondrion oscillates, makes it an ideal model organelle for studying mitochondrial functional and dynamic transitions.
In most organisms’ mitochondria display a dynamic behaviour of constant fission and fusion and the size, appearance and organization of mitochondrial membranes (tubular-interconnected or small-fragmented) are determined by an equilibrium of antagonizing fusion and fission reactions that are regulated by several parameters, according to species, tissues and physiological conditions. The numerous mitochondrial fusion and fission factors appear conserved in all eukaryotes, with non-negligible differences between fungi and mammals.
Mitochondrial fusion has not been observed in trypanosomes. Evidence for mitochondrial fusion and genetic exchange of mtDNA in vivo was obtained from the study of kDNA inheritance patterns in genetic crosses conducted almost 20 years ago. Moreover, other lines of evidence strongly suggest that trypanosomatids undergo fusion and fission of mitochondria. Despite this functional conservation, none of the proteins that are part of the fusion machinery in yeast or mammals were found encoded in the parasite genome, with the exception of a single dynamin-like protein (Dlp) that has functions in both endocytosis and mitochondrial division. Mitochondrial dynamics components must thus have highly diverged, as recently reported for several other mitochondrial complexes. Our main goal is to identify and characterize the factors involved in mitochondrial dynamics and to investigate the physiological relevance of mitochondrial fusion/fission machineries to the parasite’s life cycle.
Virulence pathogenicity factors in African trypanosomes: from the parasite to the animal
Trypanosomosis ranks among the top ten global cattle diseases impacting on the poor 1. The Animal African Trypanosomosis (AAT) or “Nagana” is the livestock disease with the greatest impact on agricultural production and is a major obstacle to development, with estimated losses of several billion dollars per year 2. The species responsible for AAT are Trypanosoma congolense (Tco), Trypanosoma brucei brucei (Tbb) and Trypanosoma vivax (Tv). It is estimated that more than 100 million animals (cows, sheeps, goats) are raised in high-risk areas, with 3 million deaths each year. Current treatments are toxic, expensive and sometimes inefficient (parasitic resistance). Moreover, no vaccine is currently available because of the parasite’s major surface antigen, which varies all along the infection and fools the immune system of the mammalian host (“antigenic variation”). Also, the available diagnostic tools suffer from low sensitivity and low species-specificity. In this context, the search for new therapeutic and diagnostic targets is a priority. Unfortunately this parasitic disease remains neglected whereas in a “One health” context it affects the food safety and the development of the impacted populations 3. African trypanosomes are parasitic unicellular organisms, alternating between a mammalian host and an insect vector (the tsetse fly). In mammals, trypanosomes replicate extracellularly in the bloodstream and consequently, these parasites face mainly RBC and EC. However, the molecular interactions between trypanosomes and these cell types are poorly understood. i/ Interactions with RBC: Anemia is the most common and major symptom 4 and it is presently admitted that this clinical trait is multifactorial. Direct injury on RBC has been reported long time ago, however more investigations are needed to fully understand this phenomenon. Species-specific RBC features likely contribute to anemia since some cattle or wild species, called “trypanotolerant”, are “naturally” less sensitive to hemolysis by parasites and seem to better control the parasites and the anemia 5,6. ii/ Interactions with EC: Parasites can cross the endothelial barrier and invade tissues 7–9. Tbb is able to migrate between EC whereas Tco is strictly intravascular 10. These critical features and differences between species are not understood. Parasites may affect vascular permeability, a phenomenon still poorly understood but which may favor parasite diffusion through cell junctions 11. Several evidences suggest that lipases are important molecules involved in host-pathogen interactions (12,13, our data). These enzymes could participate in direct or indirect lysis of RBC by degrading or weakening membrane lipids, moreover they could have a role in the energetic metabolism of the parasites. Although lipases are poorly studied in trypanosomes, we have identified several lipase genes by in silico analysis and proteomics. Our goal is to characterize the role of these lipases in African trypanosomes by studying their expression, localization, activity and function.
The lipases project is linked to 2 other on-going projects :
– The development of a new diagnostic test for ATT in collaboration with the CIRAD (Montpellier, France) and the CIRDES (Bobo-Dioulasso, Burkina Faso).
Our experimental approach is based on the analysis of metabolic and behavior desorders generated by genetic modifications, such as gene knockout and/or inactivation of gene expression by RNAi (ref). The metabolic effects of the introduced alterations are investigated on a global basis by using a full range of methodologies, including qualitative and quantitative metabolomics, [13C]-labelling experiments coupled to NMR spectroscopy and mass spectrometry, proteomics, transcriptomics, metabolic modelling, and metabolic pathway analyses.