Biogenesis of mitochondrial ribosomes (mitoribosomes) in the unicellular parasite Trypanosoma brucei requires an exceptionally large toolkit of assembly factors, identified in stable precursors of large and small mitoribosomal subunits (mtLSU and mtSSU) by cryoEM. Here, using genetic modifications and proteomic characterization of the immunoprecipitated assemblosome, the earliest characterized mtSSU precursor, we determined that a cap of its distinctive protrusion of hitherto unknown composition consists of a p22 homotrimer. This protein was previously implicated in the uridine-insertion editing of the cytochrome c oxidase subunit II transcript. Our functional analysis confirmed this role but revealed that its ablation also causes a loss of mtSSU and a systemic reduction in mitochondrial translation, phenocopying the depletion of established mitoribosomal assembly factors. Consequently, the oxidative phosphorylation system and mitochondrial function are compromised. The p22 protein belongs to the p32 family. We showed that five of its six trypanosomal members are involved in mtSSU biogenesis. Notably, p32 proteins are associated with mitoribosomes in two other distant eukaryotic lineages. A eukaryote-wide mapping of p32 proteins documented that their presence correlates with the retention of mitochondrial genomes. Together, our findings redefine trypanosomal p22 as a dual-function coordinator of mitochondrial gene expression and reveal that the ancestral role of the p32 family is associated with mitochondrial translation.

ABSTRACT The insertase Oxa1 is required for protein insertion into the inner mitochondrial membrane and for the biogenesis of oxidative phosphorylation complexes. While most eukaryotes encode one or two Oxa1 proteins, we identified three paralogs in Trypanosoma brucei : TbOxa1-1, TbOxa1-2, and TbOxa1-3. Knock-out of individual paralogs followed by phenotypic analyses and proteomic characterization of submitochondrial fractions revealed distinct functions. Respiratory chain complexes I and IV are primarily affected by loss of TbOxa1-1, whereas complex III and ATP synthase depend on TbOxa1-2; the ablation of TbOxa1-3 results in minor phenotypes in culture. In TbOxa1-2-depleted cells, ATP synthase biogenesis is compromised by the defective import or processing of the nuclear-encoded subunit-c, which also requires a rhomboid peptidase-like protein. Further, the ablation of TbOxa1-2 triggers accumulation of membrane proteins in the matrix, supporting its role in conservative sorting. Together, our results demonstrate that the trypanosomal Oxa1 machinery evolved a paralog-specific division of labor to manage a highly divergent mitochondrial membrane proteome.

The mitochondrial FoF1-ATP synthase is a reversible nanomachine that normally produces ATP via oxidative phosphorylation but under stress conditions it can reverse to maintain the mitochondrial membrane potential at the expense of ATP, a process regulated by the conserved inhibitory factor 1 (IF1). We show that ATP synthase reversal also occurs during in vitro-induced differentiation of the unicellular parasite Trypanosoma brucei, partially mirroring events in the tsetse fly. Differentiation of insect forms is marked by increased expression of alternative oxidase and reduced levels of trypanosomal IF1 (TbIF1), changes that may promote ATP synthase reversal. Parasites lacking TbIF1 efficiently progressed to the mammalian-infective form, coinciding with increased ATP synthase reversal, a higher ADP/ATP ratio, elevated phosphorylation of AMP-activated protein kinase (AMPK), enhanced proline-supported respiration, and increased mitochondrial and cellular reactive oxygen species (ROS). In contrast, inducible TbIF1 overexpression diminished these hallmarks and locked parasites in the initial insect stage. Our findings reveal that TbIF1 downregulation enables life cycle progression and underscore a regulatory role for the ATP synthase-IF1 axis.

The mitochondrial oxidative phosphorylation system powering most aerobic organisms is typically depicted as two spatially segregated machineries(1): electron transport chain complexes I-IV (ETC CI-CIV) that generates proton motive force and adenosine triphosphate (ATP) synthase (complex V, CV) that consumes it(2,3). Using the kinetoplastid parasite Trypanosoma brucei in its procyclic form, we characterize a bona fide ETC-ATP synthase supercomplex that directly incorporates CII, CIV dimer and ATP synthase (CIICIV2CV) alongside conventional assemblies. Cryo-EM structure shows a clear organizing principle in which ATP synthase engages CIV2 via a non-canonical hetero-dimerization module of two subunit g paralogs, g’ and g’’, replacing the canonical g/g dimerization interface required for ATP synthase dimerization(4). The resulting ~2 MDa assembly forms an arc-shaped membrane region, which stabilizes shallow positive membrane curvature as indicated by atomistic simulations. Genetic disruption of g’/g’’ selectively destabilizes the supercomplex and triggers pronounced cristae remodeling from discoidal to elongated tubular form, accompanied by a reduced maximal polarization capacity and comparatively modest effects on steady-state ATP output. These findings establish a structurally defined ETC-ATP synthase supercomplex and suggest its primary role in architectural quality control, constraining ATP synthase supramolecular organization to safeguard mitochondrial integrity rather than simply enhancing chemiosmotic coupling.

Mitochondrial membrane potential (ΔΨm) is a critical component of the protonmotive force that drives ATP synthesis and supports essential mitochondrial functions, including metabolite transport, ion homeostasis, and protein import. In the parasitic protist Trypanosoma brucei, ΔΨm regulation is uniquely adapted across life cycle stages to meet changing metabolic demands. In the insect-stage procyclic form (PF), ΔΨm is generated by a canonical electron transport system (ETS), while in the bloodstream form (BF), where complexes III and IV are absent, ΔΨm is maintained by the reverse operation of ATP synthase, consuming ATP to pump protons. In T. brucei evansi, which lacks mitochondrial DNA, the ATP synthase is unable to translocate protons, and ΔΨm is sustained solely by electrogenic ADP/ATP exchange through the mitochondrial carrier. This chapter presents three complementary fluorescence-based methods to evaluate ΔΨm in T. brucei and T. b. evansi cells, highlighting their applicability to both intact and permeabilized parasites. We detail the use of two ΔΨm-sensitive dyes-TMRE, a cell-permeable dye suited for live-cell assays, and Safranine O, used in permeabilized preparations-and describe protocols for flow cytometry and fluorescence spectroscopy, respectively. These approaches allow robust, qualitative and semi-quantitative analysis of ΔΨm under different metabolic and experimental conditions. We address specific challenges associated with using fluorescent dyes to measure ΔΨm including issues of dye concentration, cellular permeability and potential artifacts that can affect interpretation of ΔΨm measurements.

Neglected tropical diseases remain a major global health challenge, highlighting the need for new antiparasitic agents. In this study, a series of substituted 1-[5-(5-nitrofuran-2-yl)-1,3,4-thiadiazol-2-yl]piperidine-4-carboxamides was designed, synthesized, and evaluated for in vitro antileishmanial and antitrypanosomal activity. Compound 18 emerged as the most promising derivative, showing submicromolar activity against all tested parasites with acceptable selectivity toward THP-1 cells. Mechanistic studies in T. b. brucei bloodstream cells revealed a reversible cytostatic effect rather than apoptosis, and assessment of cellular and mitochondrial ROS levels indicated that oxidative stress was not a primary contributor to activity. In silico ADME analysis supported the drug-likeness of the synthesized compounds. Taken together, these findings identify 18 as a valuable lead for further antiparasitic drug development.

The catalytic F1 domain of the mitochondrial ATP synthase is conserved across eukaryotes, with the only known exceptions found within euglenozoans. One distinctive trait identified in Trypanosoma brucei is the proteolysis of subunit α that excises an internal octapeptide, resulting in functional N- and C-terminal polypeptides. The significance of this cleavage remained unclear. Here, we determined that the yeast subunit α expressed in T. brucei was not proteolytically processed, despite significant structural similarities. The proteolytic recognition sequence was identified to be largely contained within the octapeptide and replacing it with a flexible linker rendered the protein resistant to cleavage. This uncleaved subunit α restored growth after RNAi depletion of endogenous subunit α by incorporating into F1Fo-ATP synthase complexes capable of both ATP synthesis and hydrolysis. A FRET-based assay revealed that peptides consisting of either the first or second octapeptide cleavage site experienced significantly more proteolysis when incubated with cytosolic lysates compared to mitochondrial extracts. Furthermore, expressing just the mature C-terminal polypeptide resulted in mitochondrial localization, suggesting it contains an internal targeting signal. Together, these results indicate that proteolysis of subunit α occurs in the cytosol prior to mitochondrial import, highlighting a unique processing step in T. brucei ATP synthase assembly.

Metamonada is a eukaryotic supergroup of free-living and parasitic anaerobic protists. Their characteristic feature is the presence of highly reduced mitochondria that have lost the ability to produce ATP by oxidative phosphorylation and in some cases even by substrate phosphorylation, with all ATP being imported from the cytosol. Given this striking difference in cellular ATP metabolism when compared to aerobic mitochondria, we studied the presence of mitochondrial carrier proteins (MCPs) mediating the transport of ATP across the inner mitochondrial membrane. Our bioinformatic analyses revealed remarkable reduction of MCP repertoire in Metamonada with striking loss of the major ADP/ATP carrier (AAC). Instead, nearly all species retained carriers orthologous to human SLC25A43 protein, a little-characterized MCP. Heterologous expression of metamonad SLC25A43 carriers confirmed their mitochondrial localization, and functional analysis revealed that SLC25A43 orthologues represent a distinct group of ATP transporters, which we designate as ATP-importing carriers (AIC). Together, our findings suggest that AIC facilitate the ATP import into highly reduced anaerobic mitochondria, compensating for their diminished or absent energy metabolism.

Protein import across the mitochondrial inner membrane typically depends on two protein translocases of the inner membrane (TIM) complexes and the membrane potential. The protozoan parasite Trypanosoma brucei, however, has a single, divergent TIM complex. Unlike other trypanosomal TIM subunits, TbTim20 is not essential for the normal growth of the insect or bloodstream forms of T. brucei, leaving its role uncertain. Specific mutations in the γ-subunit of the F1FO-ATPase, such as γL262P, permit bloodstream form trypanosomes to grow without mitochondrial DNA (kinetoplast or kDNA). Here, we show that RNAi-mediated depletion of TbTim20 inhibits growth of this cell line, but only if it lacks the kDNA. Titration of mitochondrial uncouplers and direct membrane potential measurements reveal that TbTim20 becomes more critical as the membrane potential decreases across all tested cell lines. Proteomic analysis of the uninduced and induced γL262P TbTim20-RNAi cell line, which lacks kDNA and exhibits the lowest membrane potential, shows depletion of a subset of imported proteins. This subset includes ATPase subunits, suggesting a mechanism by which TbTim20-silenced cell lines become more sensitive to uncouplers. Thus, we propose that TbTim20 supports the import of a subset of proteins whose import is hypersensitive to a low membrane potential.

Six new pyranonaphthoquinone derivatives, gunacin A-E (2-7), along with the known compounds gunacin (1) and the isocoumarin derivative (+) orthosporin (8), were isolated from the fungus Exobasidium sp. Their chemical structures were elucidated by X-ray crystallography, extensive spectroscopic analysis supported by ROESY experiments, and mass spectrometry. Two tested compounds (1, 5) demonstrated high activity against Leishmania mexicana and four salivarian Trypanosoma species, with the lowest detected EC50 value of 0.02-0.24 μM, a value that is comparable to those of currently used drugs. In addition, compounds 1, 3, 5, 6, and 7 demonstrated antibacterial properties at micromolar concentrations, while 1, 5, 6, and 7 exhibited moderate antifungal activity (MIC 33.3-66.7 μM). In cytotoxicity assays, the compounds exhibited a range of toxicity against mammalian Jurkat, RAT2, MDCK cell lines, HeLa cells, and fibroblasts, with inhibition levels varying from strong to minimal inhibition (EC50 = 0.03-125 μM). This study is among the first to explore Exobasidium, a genus of phytopathogenic fungi and highlights the untapped potential of smut fungi (Basidiomycota: Ustilaginomycetes). The discovery of gunacins, which exhibit potent antiprotozoal activity at submicromolar concentrations, suggests a promising avenue for the development of antiprotozoal agents.

The biogenesis of the mitoribosomal small subunit involves a dynamic network of assembly factors. Conserved methyltransferases Mettl15 and Mettl17 act on the solvent-exposed surface of rRNA. Binding of Mettl17 is associated with the early assembly stage, whereas Mettl15 is involved in the late stage. Here, we integrate structural data from Trypanosoma brucei with mammalian homologs and molecular dynamics simulations. We reveal how the interplay of Mettl15 and Mettl17 in intermediate steps links the distinct stages of small subunit assembly. The analysis suggests a model wherein Mettl17 acts as a platform for Mettl15 recruitment. Subsequent release of Mettl17 allows a conformational change of Mettl15 for substrate recognition. Upon methylation, Mettl15 adopts a loosely bound state which leads to its replacement by initiation factors, concluding the assembly. Together, our results indicate that assembly factors Mettl15 and Mettl17 cooperate to regulate the biogenesis process.

Inhibition of hypoxanthine–guanine–xanthine phosphoribosyltransferase activity decreases the pool of 6-oxo and 6-amino purine nucleoside monophosphates required for DNA and RNA synthesis, resulting in a reduction in cell growth. Therefore, inhibitors of this enzyme have potential to control infections, caused by Plasmodium falciparum and Plasmodium vivax, Trypanosoma brucei, Mycobacterium tuberculosis, and Helicobacter pylori. Five compounds synthesized here that contain a purine base covalently linked by a prolinol group to one or two phosphonate groups have Ki values ranging from 3 nM to >10 μM, depending on the structure of the inhibitor and the biological origin of the enzyme. X-ray crystal structures show that, on binding, these prolinol-containing inhibitors stimulated the movement of active site loops in the enzyme. Against TBr in cell culture, a prodrug exhibited an EC50 of 10 μM. Thus, these compounds are excellent candidates for further development as drug leads against infectious diseases as well as being potential anticancer agents.

Trypanosoma brucei is a causative agent of the Human and Animal African Trypanosomiases. The mammalian stage parasites infect various tissues and organs including the bloodstream, central nervous system, skin, adipose tissue and lungs. They rely on ATP produced in glycolysis, consuming large amounts of glucose, which is readily available in the mammalian host. In addition to glucose, glycerol can also be used as a source of carbon and ATP and as a substrate for gluconeogenesis. However, the physiological relevance of glycerol-fed gluconeogenesis for the mammalian-infective life cycle forms remains elusive. To demonstrate its (in)dispensability, first we must identify the enzyme(s) of the pathway. Loss of the canonical gluconeogenic enzyme, fructose-1,6-bisphosphatase, does not abolish the process hence at least one other enzyme must participate in gluconeogenesis in trypanosomes. Using a combination of CRISPR/Cas9 gene editing and RNA interference, we generated mutants for four enzymes potentially capable of contributing to gluconeogenesis: fructose-1,6-bisphoshatase, sedoheptulose-1,7-bisphosphatase, phosphofructokinase and transaldolase, alone or in various combinations. Metabolomic analyses revealed that flux through gluconeogenesis was maintained irrespective of which of these genes were lost. Our data render unlikely a previously hypothesised role of a reverse phosphofructokinase reaction in gluconeogenesis and preclude the participation of a novel biochemical pathway involving transaldolase in the process. The sustained metabolic flux in gluconeogenesis in our mutants, including a triple-null strain, indicates the presence of a unique enzyme participating in gluconeogenesis. Additionally, the data provide new insights into gluconeogenesis and the pentose phosphate pathway, and improve the current understanding of carbon metabolism of the mammalian-infective stages of T. brucei.

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.

The passage of protons across membranes through F 1 F o -ATP synthases spins their rotors and drives synthesis of ATP. While the principle of torque generation by proton transfer is known, the mechanisms and routes of proton access and release and their evolution are not fully understood. Here, we show that the entry site and path of protons in the lumenal half-channel of mitochondrial ATP synthases are largely defined by a short N-terminal α-helix of subunit-a. In Trypanosoma brucei and other Euglenozoa, the α-helix is part of another polypeptide chain that is a product of subunit-a gene fragmentation. This α-helix and other elements forming the proton pathway are widely conserved across eukaryotes and in Alphaproteobacteria, the closest extant relatives of mitochondria, but not in other bacteria. The α-helix blocks one of two proton routes found in Escherichia coli , resulting in the single proton entry site in mitochondrial and alphaproteobacterial ATP synthases. Thus, the shape of the access half-channel predates eukaryotes and originated in the lineage from which mitochondria evolved by endosymbiosis.

AbstractTrypanosoma bruceiis a causative agent of the Human and Animal African Trypanosomiases. The mammalian stage parasites infect various tissues and organs including the bloodstream, central nervous system, skin, adipose tissue and lungs. They rely on ATP produced in glycolysis, consuming large amounts of glucose, which is readily available in the mammalian host. In addition to glucose, glycerol can also be used as a source of carbon and ATP and as a substrate for gluconeogenesis. However, the physiological relevance of glycerol-fed gluconeogenesis for the mammalian-infective life cycle forms remains elusive. To demonstrate its (in)dispensability, first we must identify the enzyme(s) of the pathway. Loss of the canonical gluconeogenic enzyme, fructose-1,6-bisphosphatase, does not abolish the process hence at least one other enzyme must participate in gluconeogenesis in trypanosomes. Using a combination of CRISPR/Cas9 gene editing and RNA interference, we generated mutants for four enzymes potentially capable of contributing to gluconeogenesis: fructose-1,6-bisphoshatase, sedoheptulose-1,7-bisphosphatase, phosphofructokinase and transaldolase, alone or in various combinations. Metabolomic analyses revealed that flux through gluconeogenesis was maintained irrespective of which of these genes were lost. Our data render unlikely a previously hypothesised role of a reverse phosphofructokinase reaction in gluconeogenesis and preclude the participation of a novel biochemical pathway involving transaldolase in the process. The sustained metabolic flux in gluconeogenesis in our mutants, including a triple-null strain, indicates the presence of a unique enzyme participating in gluconeogenesis. Additionally, the data provide new insights into gluconeogenesis and the pentose phosphate pathway, and improve the current understanding of carbon metabolism of the mammalian-infective stages ofT. brucei.Author SummaryTrypanosoma bruceiis a unicellular parasite causing sleeping sickness in humans and nagana disease in cattle. The parasite invades the bloodstream and cerebrospinal fluid and only recently, it has been shown to infect additional tissues such as skin, adipose tissue, or lungs. While the glucose-based metabolism of the bloodstream form is well understood, the parasite’s metabolism in these secondary tissues has not been sufficiently explored, despite its importance for drug development. One possibility is the use of gluconeogenesis since the mammalian-infective stages can use glycerol as a carbon and ATP source. First, enzymes involved in gluconeogenesis have to be identified, then it can be tested if the pathway is advantageous for the survival of the parasite. We generated mutants in four different enzymes potentially involved in this metabolic pathway. Surprisingly, the flux in gluconeogenesis was maintained in all cell lines tested, implying that another non-canonical enzyme participates in the production of glucose from glycerol in these parasites.