Protists of the order Trypanosomatida possess a single multifunctional flagellum, which powers cellular displacement and mediates attachment to tissues of the arthropod vector. The kinetoplastid flagellar cytoskeleton consists of a nine-microtubule doublet axoneme; further structural elaborations, which can vary between species and life cycle stages, include the assembly of axonemal dynein complexes, a pair of singlet microtubules and the extra-axonemal paraflagellar rod. The intracellular amastigote forms of Leishmania spp. build a short, non-motile cilium whose function has remained enigmatic. Here, we used a panel of 25 barcoded promastigote cell lines, including mutants lacking genes encoding flagellar assembly proteins, axonemal proteins required for normal motility, or flagellar membrane proteins to examine how these defects impact on their virulence in macrophages and mice. Mutants lacking the intraflagellar transport (IFT) protein 88 were avirulent indicating that assembly of a flagellum is necessary to allow for Leishmania survival in a mammalian host. A similarly severe loss of virulence was observed upon deletion of BBS2, a core component of the BBSome complex, which may act as a cargo adapter for IFT. By contrast, promastigotes that were unable to beat their flagella due to loss of core axonemal proteins could establish and sustain an infection and only showed a small reduction of parasite burden in vivo compared to the parental cell lines. These results confirm that flagellar motility is not necessary for mammalian infection, but flagellum assembly and the integrity of the BBSome are essential for pathogenicity.

• Keywords
• CRISPR screen, Leishmania, flagella, motility, virulence,
• MeSH
• Flagella * physiology   genetics   metabolism   MeSH
• Leishmania mexicana * pathogenicity   genetics   physiology   MeSH
• Macrophages parasitology   MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Protozoan Proteins genetics   metabolism   MeSH
• Virulence MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Protozoan Proteins MeSH

Unc-51-like kinase (ULK) family serine-threonine protein kinase homologues have been linked to the function of motile cilia in diverse species. Mutations in Fused/STK36 and ULK4 in mice resulted in hydrocephalus and other phenotypes consistent with ciliary defects. How either protein contributes to the assembly and function of motile cilia is not well understood. Here we studied the phenotypes of ULK4 and Fused gene knockout (KO) mutants in the flagellated protist Leishmania mexicana. Both KO mutants exhibited a variety of structural defects of the flagellum cytoskeleton. Biochemical approaches indicate spatial proximity of these proteins and indicate a direct interaction between the N-terminus of LmxULK4 and LmxFused. Both proteins display a dispersed localization throughout the cell body and flagellum, with enrichment near the flagellar base and tip. The stable expression of LmxULK4 was dependent on the presence of LmxFused. Fused/STK36 was previously shown to localize to mammalian motile cilia, and we demonstrate here that ULK4 also localizes to the motile cilia in mouse ependymal cells. Taken together these data suggest a model where the pseudokinase ULK4 is a positive regulator of the kinase Fused/ STK36 in a pathway required for stable assembly of motile cilia.

• MeSH
• Cilia metabolism   MeSH
• Flagella * metabolism   MeSH
• Microtubules metabolism   MeSH
• Mice MeSH
• Protein Serine-Threonine Kinases * metabolism   MeSH
• Mammals metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Protein Serine-Threonine Kinases * MeSH
• Stk36 protein, mouse MeSH Browser

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 α-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 thirteen 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 α-ketoglutarate consumption (15-times higher than glucose) is possible thanks to the balanced production and consumption of NADH at the substrate level and (iii) α-ketoglutarate is toxic for trypanosomes if not appropriately metabolized as observed for an α-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 α-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.

• MeSH
• Citric Acid Cycle drug effects   MeSH
• Glucose metabolism   MeSH
• Insect Vectors parasitology   MeSH
• Tsetse Flies drug effects   parasitology   MeSH
• Oxidation-Reduction drug effects   MeSH
• Proline metabolism   pharmacology   MeSH
• RNA Interference physiology   MeSH
• Trypanosoma brucei brucei drug effects   metabolism   MeSH
• Trypanosoma drug effects   metabolism   MeSH
• Trypanosomiasis, African drug therapy   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Glucose MeSH
• Proline MeSH

Livestock diseases caused by Trypanosoma congolense, T. vivax and T. brucei, collectively known as nagana, are responsible for billions of dollars in lost food production annually. There is an urgent need for novel therapeutics. Encouragingly, promising antitrypanosomal benzoxaboroles are under veterinary development. Here, we show that the most efficacious subclass of these compounds are prodrugs activated by trypanosome serine carboxypeptidases (CBPs). Drug-resistance to a development candidate, AN11736, emerged readily in T. brucei, due to partial deletion within the locus containing three tandem copies of the CBP genes. T. congolense parasites, which possess a larger array of related CBPs, also developed resistance to AN11736 through deletion within the locus. A genome-scale screen in T. brucei confirmed CBP loss-of-function as the primary mechanism of resistance and CRISPR-Cas9 editing proved that partial deletion within the locus was sufficient to confer resistance. CBP re-expression in either T. brucei or T. congolense AN11736-resistant lines restored drug-susceptibility. CBPs act by cleaving the benzoxaborole AN11736 to a carboxylic acid derivative, revealing a prodrug activation mechanism. Loss of CBP activity results in massive reduction in net uptake of AN11736, indicating that entry is facilitated by the concentration gradient created by prodrug metabolism.

• MeSH
• Livestock MeSH
• Carboxypeptidases metabolism   MeSH
• Carboxylic Acids metabolism   MeSH
• Drug Resistance MeSH
• Mice MeSH
• Parasitemia veterinary   MeSH
• Prodrugs metabolism   MeSH
• Protozoan Proteins metabolism   MeSH
• Boron Compounds metabolism   MeSH
• Trypanocidal Agents metabolism   MeSH
• Trypanosoma brucei brucei drug effects   enzymology   MeSH
• Trypanosoma congolense drug effects   enzymology   MeSH
• Trypanosoma vivax drug effects   enzymology   MeSH
• Trypanosomiasis, African drug therapy   parasitology   veterinary   MeSH
• Valine analogs & derivatives   metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• AN11736 MeSH Browser
• Carboxypeptidases MeSH
• Carboxylic Acids MeSH
• Prodrugs MeSH
• Protozoan Proteins MeSH
• serine carboxypeptidase MeSH Browser
• Boron Compounds MeSH
• Trypanocidal Agents MeSH
• Valine MeSH

Introduced about a century ago, suramin remains a frontline drug for the management of early-stage East African trypanosomiasis (sleeping sickness). Cellular entry into the causative agent, the protozoan parasite Trypanosoma brucei, occurs through receptor-mediated endocytosis involving the parasite's invariant surface glycoprotein 75 (ISG75), followed by transport into the cytosol via a lysosomal transporter. The molecular basis of the trypanocidal activity of suramin remains unclear, but some evidence suggests broad, but specific, impacts on trypanosome metabolism (i.e. polypharmacology). Here we observed that suramin is rapidly accumulated in trypanosome cells proportionally to ISG75 abundance. Although we found little evidence that suramin disrupts glycolytic or glycosomal pathways, we noted increased mitochondrial ATP production, but a net decrease in cellular ATP levels. Metabolomics highlighted additional impacts on mitochondrial metabolism, including partial Krebs' cycle activation and significant accumulation of pyruvate, corroborated by increased expression of mitochondrial enzymes and transporters. Significantly, the vast majority of suramin-induced proteins were normally more abundant in the insect forms compared with the blood stage of the parasite, including several proteins associated with differentiation. We conclude that suramin has multiple and complex effects on trypanosomes, but unexpectedly partially activates mitochondrial ATP-generating activity. We propose that despite apparent compensatory mechanisms in drug-challenged cells, the suramin-induced collapse of cellular ATP ultimately leads to trypanosome cell death.

• Keywords
• Trypanosoma brucei, differentiation, drug action, drug mechanisms, energy homeostasis, glycosomes, metabolomics, parasite metabolism, polypharmacology, proteomics, sleeping sickness, suramin, trypanosome,
• MeSH
• Adenosine Triphosphate metabolism   MeSH
• Energy Metabolism drug effects   MeSH
• Flagella drug effects   metabolism   ultrastructure   MeSH
• Glycolysis drug effects   MeSH
• Pyruvic Acid metabolism   MeSH
• Membrane Potential, Mitochondrial drug effects   MeSH
• Metabolome drug effects   MeSH
• Microbodies drug effects   metabolism   ultrastructure   MeSH
• Mitochondria drug effects   metabolism   ultrastructure   MeSH
• Models, Molecular MeSH
• Proline metabolism   MeSH
• Proteome metabolism   MeSH
• Proton-Translocating ATPases metabolism   MeSH
• Protozoan Proteins metabolism   MeSH
• Suramin pharmacology   MeSH
• Trypanosoma brucei brucei metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• Pyruvic Acid MeSH
• Proline MeSH
• Proteome MeSH
• Proton-Translocating ATPases MeSH
• Protozoan Proteins MeSH
• Suramin MeSH

Mitochondrial metabolic remodeling is a hallmark of the Trypanosoma brucei digenetic life cycle because the insect stage utilizes a cost-effective oxidative phosphorylation (OxPhos) to generate ATP, while bloodstream cells switch to aerobic glycolysis. Due to difficulties in acquiring enough parasites from the tsetse fly vector, the dynamics of the parasite's metabolic rewiring in the vector have remained obscure. Here, we took advantage of in vitro-induced differentiation to follow changes at the RNA, protein, and metabolite levels. This multi-omics and cell-based profiling showed an immediate redirection of electron flow from the cytochrome-mediated pathway to an alternative oxidase (AOX), an increase in proline consumption, elevated activity of complex II, and certain tricarboxylic acid (TCA) cycle enzymes, which led to mitochondrial membrane hyperpolarization and increased reactive oxygen species (ROS) levels. Interestingly, these ROS molecules appear to act as signaling molecules driving developmental progression because ectopic expression of catalase, a ROS scavenger, halted the in vitro-induced differentiation. Our results provide insights into the mechanisms of the parasite's mitochondrial rewiring and reinforce the emerging concept that mitochondria act as signaling organelles through release of ROS to drive cellular differentiation.

• MeSH
• Adenosine Triphosphate biosynthesis   MeSH
• Cell Differentiation drug effects   MeSH
• Cell Respiration drug effects   MeSH
• Cell Line MeSH
• Electrons MeSH
• Glucose pharmacology   MeSH
• Membrane Potential, Mitochondrial drug effects   MeSH
• Metabolic Networks and Pathways drug effects   MeSH
• Metabolomics * MeSH
• Mitochondrial Proteins metabolism   MeSH
• Mitochondria drug effects   metabolism   MeSH
• Oxidation-Reduction MeSH
• Oxidoreductases metabolism   MeSH
• Proline metabolism   MeSH
• Proteome metabolism   MeSH
• Protozoan Proteins metabolism   MeSH
• Reactive Oxygen Species metabolism   MeSH
• Plant Proteins metabolism   MeSH
• Signal Transduction MeSH
• Transcriptome genetics   MeSH
• Electron Transport drug effects   MeSH
• Trypanosoma brucei brucei drug effects   genetics   growth & development   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• alternative oxidase MeSH Browser
• Glucose MeSH
• Mitochondrial Proteins MeSH
• Oxidoreductases MeSH
• Proline MeSH
• Proteome MeSH
• Protozoan Proteins MeSH
• Reactive Oxygen Species MeSH
• Plant Proteins MeSH