The CRISPR-Cas9 system has become a valuable tool for genome editing in trypanosomatid parasites such as Trypanosoma and Leishmania species. Although these organisms have been genetically engineered for a long time using homologous recombination, CRISPR/Cas9 offers improved efficiency for genome editing. However, conventional strategies employing stable Cas9 expression require the persistent use of a specific genetic background (i.e., strains expressing Cas9), depend on selectable resistance markers, compromise genomic stability, and are not readily applicable to diverse strain backgrounds. Herein, we report an optimized marker-free CRISPR/Cas9 method based on transient ribonucleoprotein (RNP) delivery that overcomes these drawbacks. Our method eliminates the need for plasmid integration or antibiotic selection while maintaining high editing efficiency. The protocol comprises the following steps: (1) design of the guide RNA (gRNA), (2) design of the repair template (cassette), (3) assembly of the ribonucleoprotein (RNP) complex, (4) delivery by electroporation, and (5) clonal screening through PCR and sequencing. The procedure permits rapid (≤3 weeks) production of homozygous mutant lines in wild-type strains, including low-density culture strains. The reproducibility and ease of the technique render it particularly suited for multiplexed editing of polyploid genomes, multi-gene families, and several different genes at once, as well as validation of the essential nature of genes. Although designed for trypanosomatids, the workflow can be adapted to other kinetoplastids, offering a flexible platform for functional genomics.

Abstract In the mammalian host, Trypanosoma brucei proliferates as slender bloodstream forms before undergoing quorum-sensing (QS)-dependent differentiation into non-dividing transmissible stumpy-QS forms, a process that both controls parasitaemia and enables transmission to the tsetse fly. However, this model does not explain how transmission occurs in chronically infected patients and cattle, where parasite densities are often too low to trigger QS-mediated differentiation. We identify a relevant glycerol-dependent pathway driven by adipocyte-derived glycerol that offers an explanation for this transmission paradox. We show that, at low parasite density, and with physiological levels of glycerol (0.2 mM) and glucose (4 mM) mimicking adipose and hypodermal interstitial fluids, glycerol promotes the emergence of novel proliferative forms that we termed, slender-Glyc forms. These cells are transmissible, unlike the slender forms, since they ( i ) differentiate in vitro into procyclic forms, which appear in the midgut of the infected flies, and ( ii ) establish infections in the fly. In addition, at high parasite density, tissular amounts of glycerol extends the lifespan of stumpy-QS forms (named stumpy-QS/Glyc), increasing transmission chances for tissue parasites, especially in the skin. Altogether, our findings suggest that local metabolic conditions, either on their own or in combination with parasite density, can drive transmission capacity.

Trypanosomatids are protozoan parasites that remain a global health challenge due to the limited efficacy, safety, and durability of current treatments. Acetate: succinate CoA transferase (ASCT), together with succinyl-CoA synthase (SCS), forms the ASCT/SCS cycle that fuels ATP production and generates acetate, a central metabolic intermediate essential for mitochondrial pathways in these parasites. Although Trypanosoma brucei ASCT (TbASCT) shares 52% amino acid identity with mammalian succinyl-CoA:3-ketoacid CoA transferase (mSCOT), the latter catalyzes a rate-limiting step of ketone body catabolism. Because ASCT and SCOT perform distinct reactions, understanding their mechanistic divergence is crucial for identifying parasite-specific vulnerabilities and advancing selective drug discovery. Here, we report crystal structures of TbASCT bound to all substrates and products, revealing the molecular basis of substrate recognition and catalysis. In solution, TbASCT and mSCOT are homotetrameric and homodimeric, respectively. Despite similar monomer fold, the substrate-binding sites and catalytic mechanisms by which these two enzymes mediate different reactions remain unknown. Site-directed mutagenesis demonstrated that residues Arg162, Leu377, and Asp62 govern tetramer assembly, CoA binding, and acquisition of SCOT activity, respectively. Mutation of Leu377 abolished ASCT activity, while Arg162 mutant produced ASCT-active dimers with a 10-fold increase in SCOT/ASCT activity ratio. Notably, Asp62 mutants exhibited more than 4000-fold increase in this ratio, representing gain-of-function SCOT activity, a reaction absent in TbASCT. These mechanistic insights define the structural determinants that separate ASCT from SCOT function and illuminate opportunities to selectively inhibit ASCT without disrupting host SCOT, thereby informing the development of trypanosomatid-targeted therapeutics.

Abstract 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.

The F-type ATP synthase inhibitor bedaquiline (BDQ) is a potent inhibitor of mycobacterial growth and this inhibition cannot be rescued by fermentable carbon sources that would supply ATP by an alternative pathway (substrate level phosphorylation). To gain mechanistic insight into this phenomenon, we employed a metabolic engineering approach. We introduced into Mycobacterium smegmatis an alternative ATP production pathway by substrate-level phosphorylation, specifically through overexpression of trypanosomal acetate:succinate co-enzyme A (CoA) transferase (ASCT). Intriguingly, the overexpression of ASCT partially restored intracellular ATP levels and resulted in acquired tolerance to BDQ growth inhibition at low, but not high concentrations of BDQ. These results implicate intracellular ATP levels in modulating the growth inhibitory activity of BDQ at low concentrations. These findings shed light on the intricate interplay between BDQ and mycobacterial energy metabolism, while also providing a novel tool for the development of next-generation ATP synthase-specific inhibitors targeting mycobacteria.

l-Serine and l-threonine have versatile roles in metabolism. In addition to their use in protein synthesis, these amino acids participate in the biosynthesis pathways of other amino acids and even phospholipids. Furthermore, l-serine and l-threonine can be substrates for a serine/threonine dehydratase (Ser/ThrDH), resulting in pyruvate and 2-oxobutyrate, respectively, thus being amino acids with anaplerotic potential. Trypanosoma cruzi, the etiological agent of Chagas disease, uses amino acids in several biological processes: metacyclogenesis, infection, resistance to nutritional and oxidative stress, osmotic control, etc. This study investigated the import and metabolism of l-serine, l-threonine, and glycine in T. cruzi. Our results demonstrate that these amino acids are transported from the extracellular environment into T. cruzi cells through a saturable transport system that fits the Michaelis-Menten model. Our results show that l-serine and l-threonine can sustain epimastigote cell viability under nutritional stress conditions and stimulate oxygen consumption, maintaining intracellular ATP levels. Additionally, our findings indicate that serine plays a role in establishing the mitochondrial membrane potential in T. cruzi. Serine is also involved in energy metabolism via the serine-pyruvate pathway, which stimulates the production and subsequent excretion of acetate and alanine. Our results demonstrate the importance of l-serine and l-threonine in the energy metabolism of T. cruzi and provide new insights into the metabolic adaptations of this parasite during its life cycle.IMPORTANCETrypanosoma cruzi, the parasite responsible for Chagas disease, impacts 5-6 million individuals in the Americas and is rapidly spreading globally due to significant human migration. This parasitic organism undergoes a complex life cycle involving triatomine insects and mammalian hosts, thriving in diverse environments, such as various regions within the insect's digestive tract and mammalian cell cytoplasm. Crucially, its transmission hinges on its adaptive capabilities to varying environments. One of the most challenging environments is the insect's digestive tract, marked by nutrient scarcity between blood meals, redox imbalance, and osmotic stresses induced by the triatomine's metabolism. To endure these conditions, T. cruzi has developed a remarkably versatile metabolic network enabling it to metabolize sugars, lipids, and amino acids efficiently. However, the full extent of metabolites this parasite can thrive on remains incompletely understood. This study reveals that, beyond conventional carbon and energy sources (glucose, palmitic acids, proline, histidine, glutamine, and alanine), three additional metabolites (serine, threonine, and glycine) play vital roles in the parasite's survival during starvation. Remarkably, serine and threonine directly contribute to ATP production through a serine/threonine dehydratase enzyme not previously described in T. cruzi. The significance of this metabolic pathway for the parasite's survival sheds light on how metabolic networks aid in its endurance under extreme conditions and its ability to thrive in diverse metabolic settings.

In the mammalian bloodstream, Trypanosoma brucei , the parasite responsible for sleeping sickness, proliferates as slender forms before undergoing quorum-sensing (QS)-mediated differentiation into cell cycle-arrested stumpy forms (stumpy-QS), a transition that regulates parasitaemia and primes parasites for tsetse fly transmission. Beyond the bloodstream, T. brucei also occupies extravascular, adipose-rich tissues such as the skin, a potential reservoir for transmission. Here, we identify an alternative slender-to-stumpy differentiation pathway driven by glycerol, a host metabolite abundant in adipose-rich tissues, that could resolve the long-standing paradox of successful parasite transmission from human hosts during chronic infection despite low parasitaemia. We show that high (10 mM) and non-physiological glycerol concentrations under low glucose conditions (0.5 mM) induce differentiation, generating distinct stumpy-Glyc forms that resemble stumpy-QS parasites but have an extended lifespan. Under conditions mimicking dermal tissue interstitial fluids (4 mM glucose, 0.25 mM glycerol), we show that glycerol promotes the emergence of proliferative intermediate forms that retain transmission potential and can differentiate into fly host-specific procyclic forms in vitro and within tsetse flies. These findings open the door for reevaluation of the model of T. brucei transmission and supports a dominant role for adipocyte-derived glycerol in the skin in sustaining parasite transmission. ### Competing Interest Statement The authors have declared no competing interest. Centre national de la recherche scientifique Université de Bordeaux Agence nationale de la recherche (ANR), ANR-19-CE15-0004-01, ANR-18-CE15-0012, ANR-23-CE15-0040-01, ANR-11-LABX-0024, ANR-10-LABX-62-IBEID

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 new member of the dynamin superfamily protein (DSP) from T. brucei (TbMfnL), which shares 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 dynamics, such as optic atrophy 1 (Opa1) and mitofusin (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 inter-mitochondrial connections.

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 show 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 catalyze 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 an approximately 500-fold increase in the substrate/product ratio was expected for PFK to carry out the gluconeogenic reaction. In conclusion, we show 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 possibly due to the supramolecular organization of the metabolic pathway within glycosomes to overcome thermodynamic barriers through metabolic channeling.

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 Trypanosoma 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 but requires only cloning and PCR screening of the clones. Importantly, this approach is adaptable to any wild-type parasite.

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. ### Competing Interest Statement The authors have declared no competing interest.

l-Serine (l-Ser) and l-Threonine (l-Thr) have versatile roles in metabolism. In addition to their use in protein synthesis, these amino acids participate in the biosynthesis pathways of other amino acids and even phospholipids. Furthermore, l-Ser and l-Thr can be substrates for a Ser/Thr dehydratase (Ser/ThrDH), resulting in pyruvate (Pyr) and 2-oxobutyrate, respectively, thus being amino acids with anaplerotic potential. Trypanosoma cruzi , the etiological agent of Chagas disease, uses amino acids in several biological processes: metacyclogenesis, infection, resistance to nutritional and oxidative stress, osmotic control, etc. In this study, we investigated the import and metabolism of l-Ser, l-Thr, and Gly in T. cruzi . Our results demonstrate that these amino acids are transported from the extracellular environment into T. cruzi cells through a saturable transport system that fits the Michaelis-Menten model. Our results show that l-Ser and l-Thr can sustain epimastigote (Epi) cell viability under nutritional stress (NS) conditions and can stimulate oxygen consumption to maintain intracellular ATP levels. Additionally, our findings indicate that l-Ser plays a role in establishing the mitochondrial membrane potential (ΔΨm) in T. cruzi . l-Ser is also involved in energy metabolism via the Ser-Pyr pathway, which stimulates the production and subsequent excretion of acetate and alanine. Our results demonstrate the importance of l-Ser and l-Thr in the energy metabolism of T. cruzi and provide new insights into the metabolic adaptations of this parasite during its life cycle. ### Competing Interest Statement The authors have declared no competing interest.

ABSTRACT African trypanosomes are eukaryotic parasites that exist in two main replicative forms; the procyclic form in the midgut of the insect vector, the tsetse fly Glossina spp. and the bloodstream form responsible for diseases in humans and cattle. Unlike most other eukaryotes, where mitochondria continuously fuse and divide, trypanosome mitochondria form a single and continuously interconnected network that only divides during cytokinesis. The machineries governing mitochondrial remodeling and interconnection, however, remain largely unknown. We characterize a dynamin-related protein (DRP) from T. brucei ( Tb DBF, previously called Tb MfnL) that depicts sequence similarities with Opa1 and Mfn, mammalian DRPs involved mitochondrial fusion. We showed that Tb DBF has closely related homologues in several organisms that are devoid of Mfn and Opa1, such as eukaryotes from different phyla, prokaryotes and archaea. Tb DBF is the first member of this new protein family to be functionally characterized. It localizes to the mitochondrial periphery and, upon overexpression, induces a strong increase in the interconnection and branching of mitochondrial filaments in a GTPase dependent manner. Its overexpression also promotes a major increase in cellular and mitochondrial volume and an increased consumption of the two major carbon sources used by the parasite (glucose and proline), as well as ethanolamine, a precursor of phosphatidyl-ethanolamine involved in membrane biogenesis and shaping. We propose that mitochondrial Tb DBF is a component of an ancestral membrane remodeling machinery that contributes to the formation of intermitochondrial connections.

The bloodstream form Trypanosoma brucei maintains essential mitochondrial membrane potential (ΔΨm) through the reverse activity of FoF1-ATP synthase. The ATP that drives this activity is thought to be generated by glycolysis and imported from the cytosol via an ATP/ADP carrier (AAC). We have shown that this carrier is the only carrier that can import ATP into the mitochondrial matrix to power the F o F 1 -ATPase. Contrary to expectations, its deletion has no effect on parasite growth, virulence and levels of ΔΨm, suggesting that ATP is produced intramitochondrially by substrate phosphorylation pathways. Therefore, we knocked out the succinyl-CoA synthetase (SCoAS) gene, a key enzyme that produces ATP through substrate phosphorylation. Its absence resulted in changes in the metabolic landscape of the parasite, lower virulence, and reduced mitochondrial ATP content. This minimal mitochondrial ATP pool was maintained by AAC activity as evidenced by the 25- fold increase in sensitivity of the mutant parasites to AAC inhibitor carboxyatractyloside. Under nutrient-limited conditions, suppression of SCoAS expression by RNA interference negatively affected cell growth and levels of ΔΨm. We concluded that the bloodstream mitochondrion is capable of generating ATP via substrate phosphorylation pathways, the importance of which depends on environmental conditions.

The long slender bloodstream form Trypanosoma brucei maintains its essential mitochondrial membrane potential (ΔΨm) through the proton-pumping activity of the FoF1-ATP synthase operating in the reverse mode. The ATP that drives this hydrolytic reaction has long been thought to be generated by glycolysis and imported from the cytosol via an ATP/ADP carrier (AAC). Indeed, we demonstrate that AAC is the only carrier that can import ATP into the mitochondrial matrix to power the hydrolytic activity of the FoF1-ATP synthase. However, contrary to expectations, the deletion of AAC has no effect on parasite growth, virulence or levels of ΔΨm. This suggests that ATP is produced by substrate-level phosphorylation pathways in the mitochondrion. Therefore, we knocked out the succinyl-CoA synthetase (SCS) gene, a key mitochondrial enzyme that produces ATP through substrate-level phosphorylation in this parasite. Its absence resulted in changes to the metabolic landscape of the parasite, lowered virulence, and reduced mitochondrial ATP content. Strikingly, these SCS mutant parasites become more dependent on AAC as demonstrated by a 25-fold increase in their sensitivity to the AAC inhibitor, carboxyatractyloside. Since the parasites were able to adapt to the loss of SCS in culture, we also analyzed the more immediate phenotypes that manifest when SCS expression is rapidly suppressed by RNAi. Importantly, when performed under nutrient-limited conditions mimicking various host environments, SCS depletion strongly affected parasite growth and levels of ΔΨm. In totality, the data establish that the long slender bloodstream form mitochondrion is capable of generating ATP via substrate-level phosphorylation pathways.

Phospholipases (PLs) and Lysophospholipases (LysoPLs) are a diverse group of esterases responsible for phospholipid or lysophospholipid hydrolysis. They are involved in several biological processes, including lipid catabolism, modulation of the immune response and membrane maintenance. PLs are classified depending on their site of hydrolysis as PLA1, PLA2, PLC and PLD. In many pathogenic microorganisms, from bacteria to fungi, PLAs and LysoPLs have been described as critical virulence and/or pathogenicity factors. In protozoan parasites, a group containing major human and animal pathogens, growing literature show that PLAs and LysoPLs are also involved in the host infection. Their ubiquitous presence and role in host-pathogen interactions make them particularly interesting to study. In this review, we summarize the literature on PLAs and LysoPLs in several protozoan parasites of medical relevance, and discuss the growing interest for them as potential drug and vaccine targets.

When Trypanosoma brucei parasites, the causative agent of sleeping sickness, colonize the adipose tissue, they rewire gene expression. Whether this adaptation affects population behavior and disease treatment remained unknown. By using a mathematical model, we estimate that the population of adipose tissue forms (ATFs) proliferates slower than blood parasites. Analysis of the ATFs proteome, measurement of protein synthesis and proliferation rates confirm that the ATFs divide on average every 12 h, instead of 6 h in the blood. Importantly, the population of ATFs is heterogeneous with parasites doubling times ranging between 5 h and 35 h. Slow-proliferating parasites remain capable of reverting to the fast proliferation profile in blood conditions. Intravital imaging shows that ATFs are refractory to drug treatment. We propose that in adipose tissue, a subpopulation of T. brucei parasites acquire a slow growing behavior, which contributes to disease chronicity and treatment failure.

Phospholipases are esterases involved in lipid catabolism. In pathogenic micro-organisms (bacteria, fungi, parasites) they often play a critical role in virulence and pathogenicity. A few phospholipases (PL) have been characterised so far at the gene and protein level in unicellular parasites including African trypanosomes (AT). They could play a role in different processes such as host–pathogen interaction, antigenic variation, intermediary metabolism. By mining the genome database of AT we found putative new phospholipase candidate genes and here we provided biochemical evidence that one of these has lipolytic activity. This protein has a unique non-canonical glycosome targeting signal responsible for its dual localisation in the cytosol and the peroxisomes-related organelles named glycosomes. We also show that this new phospholipase is excreted by these pathogens and that antibodies directed against this protein are generated during an experimental infection with T. brucei gambiense, a subspecies responsible for infection in humans. This feature makes this protein a possible tool for diagnosis.