Biology subjects

Tetaud, E.

Publications and source records attributed to Tetaud, E..

8 recordsLinked to original sources

bioRxiv2026

A lipid droplet triacylglycerol lipase governs neutral lipids mobilization and differentiation of Trypanosoma brucei

Lipid droplets (LD) are dynamic organelles largely distributed in all living cells that are essential notably for membrane biogenesis and cell proliferation. In Trypanosoma brucei, a protozoan parasite responsible for major lethal infectious diseases in humans and cattle, lipid acquisition and mobilization are essential for its survival and adaptation to its different hosts, but the roles of LD in these processes remain largely unknown. To address this gap, we identified and characterized a unique functional orthologue of the human adipose triacylglycerol lipase (ATGL), responsible for the initiation of neutral lipid mobilization, in T. brucei. In this study, we present TbPatatin-like (TbPat), the first ATGL ortholog in protozoan parasites. By combining reverse genetics, microscopy and -omics analysis, we deciphered the role of TbPat in lipolysis and neutral lipid maintenance in both insect and mammalian stages of African trypanosomes and imaged its distribution on the LD surface at an unprecedented resolution by using Cryo-Expansion microscopy. We found that inactivation of TbPat in mammalian form T. brucei led to a global upregulation of the mitochondrial metabolic proteome; furthermore, these cells appeared remarkably unable to differentiate into the procyclic insect stage. Through its expression and localization, TbPat appears to be at an essential checkpoint linking lipid environment and metabolism to more complex cell programs involving proteome, compartment remodeling and lifecycle completion.

microbiology↗

bioRxiv2025

Cellular adaptation of mitochondrial metabolism and biogenesis in response to partial ATP synthase uncoupling

Cellular energy metabolism relies on tight regulation of mitochondrial biogenesis and turnover to maintain bioenergetic homeostasis. Understanding the signaling pathways that modulate mitochondrial content in response to changing energetic demands is therefore essential. This work examines how partial mitochondrial uncoupling reshapes mitochondrial content and metabolism in yeast and how such adaptations mirror features of pathological or age-associated states. Using a Saccharomyces cerevisiae ATP synthase mutant lacking the epsilon subunit ({Delta}{varepsilon}) and carrying a compensatory mutation that allows survival under uncoupling ({Delta}{varepsilon}+c-L57F), mitochondrial abundance, organization, and function were compared with those of wild-type and rescue strains (mutant re-expressing the epsilon subunit of the ATP synthase). Analyses included respiration assays, mitochondrial DNA quantification, {beta}-galactosidase reporters, fluorescence microscopy of the mitochondrial network, and 2D-DiGE proteomics. Uncoupling in the mutant led to a strong increase in mitochondrial mass per cell, higher respiratory capacity, elevated cytochrome levels, and increased mtDNA copy number, together with augmented transcriptional activity consistent with activation of HAP-dependent mitochondrial biogenesis. Despite a fragmented network, mitochondria retained typical ultrastructural features, while proteomic profiling indicated a shift toward ATP production via substrate-level phosphorylation. Overall, partial uncoupling promotes reversible mitochondrial biogenesis and metabolic remodeling that depend on a preserved proton-motive force, highlighting mitochondrial signaling as a key regulator of cellular energy homeostasis and offer new perspectives for studying metabolic disorders and mitochondrial pathologies. GRAPHICAL ABSTRACTS O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/695256v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@19b1eceorg.highwire.dtl.DTLVardef@103a6ceorg.highwire.dtl.DTLVardef@bc48f6org.highwire.dtl.DTLVardef@15a191c_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIMitochondrial uncoupling triggers a marked increase in mitochondrial biogenesis in yeast. C_LIO_LIUncoupling leads to fragmentation of the mitochondrial network, while preserving mitochondrial ultrastructure. C_LIO_LIUncoupling induces metabolic remodeling that favors ATP production through substrate-level phosphorylation. C_LIO_LIThese cellular adaptations are reversible upon re-establishment of mitochondrial coupling. C_LIO_LIThe proton motive force serves as a key signal sensed by the cell. C_LI

molecular biology↗

bioRxiv2024

The glycosomal ATP-dependent phosphofructokinase of Trypanosoma brucei operates in the gluconeogenic direction in cellulo under in vivo-like conditions

In the glucose-free environment of the midgut of the tsetse fly vector, the procyclic forms of Trypanosoma brucei primarily consume proline to feed its central carbon and energy metabolism. In this context, the parasite produces through gluconeogenesis glucose 6- phosphate (G6P), the precursor of essential metabolic pathways, from proline catabolism. We showed here that the parasite uses three different enzymes to perform the key gluconeogenic reaction producing fructose 6-phosphate (F6P) from fructose 1,6-bisphosphate, (i) fructose-1,6- bisphosphatase (FBPase), the canonical enzyme performing this reaction, (ii) sedoheptulose- 1,7-bisphosphatase (SBPase) and (iii) more surprisingly ATP-dependent phosphofructokinase (PFK), an enzyme considered to irreversibly catalyse the opposite reaction involved in glycolysis. These three enzymes, as well as six other glycolytic/gluconeogenic enzymes, are located in peroxisome-related organelles, named glycosomes. Incorporation of 13C-enriched glycerol (a more effective alternative to proline for monitoring gluconeogenic activity) into F6P and G6P was more affected in the PFK null mutant than in the FBPase null mutant, suggesting the PFK contributes at least as much as FBPase to gluconeogenesis. We also showed that glucose deprivation did not affect the quantities of PFK substrates and products, whereas a 500-fold increase in the substrate/product ratio was expected for PFK to carry out the gluconeogenic reaction. In conclusion, we showed for the first time that ATP-dependent PFK can function in vivo in the gluconeogenic direction, even in the presence of FBPase activity. This particular feature, which precludes loss of ATP through a futile cycle involving PFK and FBPase working simultaneously in the glycolytic and gluconeogenic directions, respectively, is probably due to the supramolecular organisation of the metabolic pathway within glycosomes to overcome thermodynamic barriers through metabolic channelling.

microbiology↗

bioRxiv2023

Streptococcus pyogenes Cas9 ribonucleoprotein delivery for efficient, rapid and marker-free gene editing in Trypanosoma and Leishmania

Kinetoplastids are unicellular eukaryotic flagellated parasites found in a wide range of hosts within the animal and plant kingdoms. They are known to be responsible in humans for African sleeping sickness (Trypanosoma brucei), Chagas disease (Trypanosoma cruzi), and various forms of leishmaniasis (Leishmania spp.), as well as several animal diseases with important economic impact (African trypanosomes, including T. congolense). Understanding the biology of these parasites necessarily implies the ability to manipulate their genomes. In this study, we demonstrate that transfection of a ribonucleoprotein complex, composed of recombinant Streptococcus pyogenes Cas9 (SpCas9) and an in vitro-synthesized guide RNA, results in rapid and efficient genetic modifications of trypanosomatids, in marker-free conditions. This approach was successfully developed to inactivate, delete and mutate candidate genes in various stages of the life cycle of T. brucei and T. congolense, and Leishmania promastigotes. The functionality of SpCas9 in these parasites now provides, to the research community working on these parasites, a rapid and efficient method of genome editing, without requiring plasmid construction and selection by antibiotics. Importantly, this approach is adaptable to any wild-type parasite, including field isolates.

microbiology↗

bioRxiv2023

Identification of a novel and ancestral machinery involved in mitochondrial membrane branching in Trypanosoma brucei

Unlike most other eukaryotes, where mitochondria continuously fuse and divide, the mitochondrion of trypanosome cells forms a single and continuously interconnected network that divides only during cytokinesis. However, the machinery governing mitochondrial remodeling and interconnection of trypanosome mitochondrion remain largely unknown. We functionally characterize a novel dynamin-superfamily protein (DSP) from T. brucei (TbMfnL) which shares close similarity with a family of homologs present in various eukaryotic and prokaryotic phyla, but not in opisthokonts like mammals and budding yeast. The sequence and domain organization of TbMfnL is distinct and it is phylogenetically very distant from the yeast and mammalian dynamin-related proteins involved in mitochondrial fusion/fission dynamincs, such as Opa1 and Mfn. TbMfnL localizes to the inner mitochondrial membrane facing the matrix, and upon overexpression, induces a strong increase in the interconnection and branching of mitochondrial filaments in a GTPase-dependent manner. TbMfnL is a component of a novel membrane remodeling machinery with an unprecedented matrix-side localization that is able to modulate the degree of intermitochondrial connections.

microbiology↗

bioRxiv2020

Fly stage trypanosomes recycle glucose catabolites and TCA cycle intermediates to stimulate growth in near physiological conditions

Trypanosoma brucei, a protist responsible for human African trypanosomiasis (sleeping sickness), is transmitted by the tsetse fly, where the procyclic forms of the parasite develop in the proline-rich (1-2 mM) and glucose-depleted digestive tract. Proline is essential for the midgut colonization of the parasite in the insect vector, however other carbon sources could be available and used to feed its central metabolism. Here we show that procyclic trypanosomes can consume and metabolize metabolic intermediates, including those excreted from glucose catabolism (succinate, alanine and pyruvate), with the exception of acetate, which is the ultimate end-product excreted by the parasite. Among the tested metabolites, tricarboxylic acid (TCA) cycle intermediates (succinate, malate and -ketoglutarate) stimulated growth of the parasite in the presence of 2 mM proline. The pathways used for their metabolism were mapped by proton-NMR metabolic profiling and phenotypic analyses of a dozen RNAi and/or null mutants affecting central carbon metabolism. We showed that (i) malate is converted to succinate by both the reducing and oxidative branches of the TCA cycle, which demonstrates that procyclic trypanosomes can use the full TCA cycle, (ii) the enormous rate of -ketoglutarate consumption (15-times higher than glucose) is possible thanks to the balanced production and consumption of NADH at the substrate level and (iii) -ketoglutarate is toxic for trypanosomes if not appropriately metabolized as observed for an -ketoglutarate dehydrogenase null mutant. In addition, epimastigotes produced from procyclics upon overexpression of RBP6, showed a growth defect in the presence of 2 mM proline, which is rescued by -ketoglutarate, suggesting that physiological amounts of proline are not sufficient per se for the development of trypanosomes in the fly. In conclusion, these data show that trypanosomes can metabolize multiple metabolites, in addition to proline, which allows them to confront challenging environments in the fly. Author SummaryIn the midgut of its insect vector, trypanosomes rely on proline to feed their energy metabolism. However, the availability of other potential carbon sources that can be used by the parasite is currently unknown. Here we show that tricarboxylic acid (TCA) cycle intermediates, i.e. succinate, malate and -ketoglutarate, stimulate growth of procyclic trypanosomes incubated in medium containing 2 mM proline, which is in the range of the amounts measured in the midgut of the fly. Some of these additional carbon sources are needed for the development of epimastigotes, which differentiate from procyclics in the midgut of the fly, since their growth defect observed in the presence of 2 mM proline is rescued by addition of -ketoglutarate. In addition, we have implemented new approaches to study a poorly explored branch of the TCA cycle converting malate to -ketoglutarate, which was previously described as non-functional in the parasite, regardless of the glucose levels available. The discovery of this branch reveals that a full TCA cycle can operate in procyclic trypanosomes. Our data broaden the metabolic potential of trypanosomes and pave the way for a better understanding of the parasites metabolism in various organ systems of the tsetse fly, where it evolves.

microbiology↗

bioRxiv2020

TMEM70 promotes ATP synthase assembly within cristae via transient interactions with subunit c

Mitochondrial ATP-synthesis is catalyzed by a F1Fo-ATP synthase, an enzyme of dual genetic origin enriched at the edge of cristae where it plays a key role in their structure/stability. The enzymes biogenesis remains poorly understood, both from a mechanistic and a compartmentalization point of view. The present study provides novel molecular insights into this process through investigations on a human protein called TMEM70 with an unclear role in the assembly of ATP synthase. A recent study has revealed the existence of physical interactions between TMEM70 and the subunit c (Su.c), a protein present in 8 identical copies forming a transmembrane oligomeric ring (c-ring) within the ATP synthase proton translocating domain (FO). Herein we analyzed the ATP-synthase assembly in cells lacking TMEM70, mitochondrial DNA or F1 subunits and observe a reciprocal dependence of TMEM70 and Su.c levels, regardless of the status of other ATP synthase subunits or of mitochondrial bioenergetics. Immunoprecipitation and two-dimensional blue-native/SDS-PAGE reveal that TMEM70 forms large oligomers composed of 8 TMEM70 dimers and that TMEM70 oligomers interact with Su.c not yet incorporated into ATP synthase complexes. Moreover, discrete TMEM70-Su.c complexes with increasing Su.c contents can be detected, suggesting a role for TMEM70 oligomers in the gradual assembly of the c-ring. Furthermore, we demonstrate using expansion super-resolution microscopy the specific localization of TMEM70 at the inner cristae membrane, distinct from the MICOS component MIC60. Taken together, our results show that TMEM70 oligomers provide a scaffold for c-ring assembly and that mammalian ATP synthase is assembled within inner cristae membranes.

cell biology↗

bioRxiv2019

\"Metabolic contest\", a new way to control carbon source preference

Microorganisms must make the right choice for nutrient consumption to adapt to their changing environment. As a consequence, bacteria and yeasts have developed regulatory mechanisms involving nutrient sensing and signaling, allowing to redirect cell metabolism to maximize the consumption of an energy-efficient carbon source. Here, we report a new mechanism, named \"metabolic contest\", for regulating the use of carbon sources without nutrient sensing and signaling. In contrast to most microorganisms, trypanosomes show a glycerol-to-glucose preference that depends on the combination of three conditions: (i) the sequestration of both metabolic pathways in the same subcellular compartment, here in the peroxisomal-like organelles named glycosomes; (ii) the competition for the same substrate, here ATP, with the first enzymatic step of the glycerol and glucose metabolic pathways being both ATP-dependent (glycerol kinase and hexokinase, respectively) and (iii) an unbalanced activity between the competing enzymes, here the glycerol kinase activity being ~80-fold higher than the hexokinase activity.

microbiology↗