Suramin exposure alters cellular metabolism and mitochondrial energy production in African trypanosomes.

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.

Glycosome heterogeneity in kinetoplastids.

Kinetoplastid parasites have essential organelles called glycosomes that are analogous to peroxisomes present in other eukaryotes. While many of the processes that regulate glycosomes are conserved, there are several unique aspects of their biology that are divergent from other systems and may be leveraged as therapeutic targets for the treatment of kinetoplastid diseases. Glycosomes are heterogeneous organelles that likely exist as sub-populations with different protein composition and function in a given cell, between individual cells, and between species. However, the limitations posed by the small size of these organelles makes the study of this heterogeneity difficult. Recent advances in the analysis of small vesicles by flow-cytometry provide an opportunity to overcome these limitations. In this review, we describe studies that document the diverse nature of glycosomes and propose an approach to using flow cytometry and organelle sorting to study the diverse composition and function of these organelles. Because the cellular machinery that regulates glycosome protein import and biogenesis is likely to contribute, at least in part, to glycosome heterogeneity we highlight some ways in which the glycosome protein import machinery differs from that of peroxisomes in other eukaryotes.

Short- and long-term effects of orally administered azithromycin on Trypanosoma brucei brucei-infected mice.

Human African trypanosomosis (HAT) and animal African trypanosomosis (AAT) are diseases of economic importance in humans and animals that affect more than 36 African countries. The currently available trypanocidal drugs are associated with side effects, and the parasites are continually developing resistance. Thus, effective and safe drugs are needed for the treatment of HAT and AAT. This study aimed to evaluate the effects of azithromycin (AZM) on Trypanosoma brucei brucei-infected mice. Mice were randomly divided into 7 groups consisting of a vehicle control group, 5 test groups and a diminazene aceturate (DA)-treated group. Mice were treated orally for 7 and 28 days, as short-term and long-term treatments, respectively. Short-term AZM treatment cured 23% (16 of 70) of the overall treated mice whereas long-term treatment resulted in the survival of 70% of the mice in the groups that received AZM at doses of 300 and 400 mg/kg. Trypanosomes treated in vitro with 25 µg/mL of AZM were subjected to transmission electron microscopy, which revealed the presence of increased numbers of glycosomes and acidocalcisomes in comparison to the vehicle group. The current study showed the trypanocidal effect of AZM on T. b. brucei in vivo. The demonstrated efficacy increased with an increase in treatment period and an increased concentration of AZM.

Biogenesis, maintenance and dynamics of glycosomes in trypanosomatid parasites.

Peroxisomes of organisms belonging to the protist group Kinetoplastea, which include trypanosomatid parasites of the genera Trypanosoma and Leishmania, are unique in playing a crucial role in glycolysis and other parts of intermediary metabolism. They sequester the majority of the glycolytic enzymes and hence are called glycosomes. Their glycosomal enzyme content can vary strongly, particularly quantitatively, between different trypanosomatid species, and within each species during its life cycle. Turnover of glycosomes by autophagy of redundant ones and biogenesis of a new population of organelles play a pivotal role in the efficient adaptation of the glycosomal metabolic repertoire to the sudden, major nutritional changes encountered during the transitions in their life cycle. The overall mechanism of glycosome biogenesis is similar to that of peroxisomes in other organisms, but the homologous peroxins involved display low sequence conservation as well as variations in motifs mediating crucial protein-protein interactions in the process. The correct compartmentalisation of enzymes is essential for the regulation of the trypanosomatids' metabolism and consequently for their viability. For Trypanosoma brucei it was shown that glycosomes also play a crucial role in its life-cycle regulation: a crucial developmental control switch involves the translocation of a protein phosphatase from the cytosol into the organelles. Many glycosomal proteins are differentially phosphorylated in different life-cycle stages, possibly indicative of regulation of enzyme activities as an additional means to adapt the metabolic network to the different environmental conditions encountered.

Localization of a Trypanosome Peroxin to the Endoplasmic Reticulum.

Trypanosoma brucei is the causative agent of diseases that affect 30,000-50,000 people annually. Trypanosoma brucei harbors unique organelles named glycosomes that are essential to parasite survival, which requires growth under fluctuating environmental conditions. The mechanisms that govern the biogenesis of these organelles are poorly understood. Glycosomes are evolutionarily related to peroxisomes, which can proliferate de novo from the endoplasmic reticulum or through the growth and division of existing organelles depending on the organism and environmental conditions. The effect of environment on glycosome biogenesis is unknown. Here, we demonstrate that the glycosome membrane protein, TbPex13.1, is localized to glycosomes when cells are cultured under high glucose conditions and to the endoplasmic reticulum in low glucose conditions. This localization in low glucose was dependent on the presence of a C-terminal tripeptide sequence. Our findings suggest that glycosome biogenesis is influenced by extracellular glucose levels and adds to the growing body of evidence that de novo glycosome biogenesis occurs in trypanosomes. Because the movement of peroxisomal membrane proteins is a hallmark of ER-dependent peroxisome biogenesis, TbPex13.1 may be a useful marker for the study such processes in trypanosomes.

A novel stage-specific glycosomal nucleoside diphosphate kinase from Trypanosoma cruzi.

Nucleoside diphosphate kinases (NDPK) are key enzymes involved in the intracellular nucleotide maintenance in all living organisms, especially in trypanosomatids which are unable to synthesise purines de novo. Four putative NDPK isoforms were identified in the Trypanosoma cruzi Chagas, 1909 genome but only two of them were characterised so far. In this work, we studied a novel isoform from T. cruzi called TcNDPK3. This enzyme presents an atypical N-terminal extension similar to the DM10 domains. In T. cruzi, DM10 sequences targeted other NDPK isoform (TcNDPK2) to the cytoskeleton, but TcNDPK3 was localised in glycosomes despite lacking a typical peroxisomal targeting signal. In addition, TcNDPK3 was found only in the bloodstream trypomastigotes where glycolytic enzymes are very abundant. However, TcNDPK3 mRNA was also detected at lower levels in amastigotes suggesting regulation at protein and mRNA level. Finally, 33 TcNDPK3 gene orthologs were identified in the available kinetoplastid genomes. The characterisation of new glycosomal enzymes provides novel targets for drug development to use in therapies of trypanosomatid associated diseases.

Biochemical Fractionation of Trypanosomes for the Analysis of Glycosomal Protein Import Defects.

Insect-transmitted trypanosomatid parasite infections cause life-threatening neglected tropical diseases (NTDs), including African sleeping sickness, Chagas disease and leishmaniasis. In these parasites, glycosomes are unique organelles that are essential for the parasite survival. Proper biogenesis of glycosomes is crucial to ensure correct compartmentation of the glycosomal metabolism. Genetic or chemical disruption of the glycosome biogenesis leads to a mislocalization of the glycosomal enzymes into the cytosol, which results in toxicity to the parasites. Here, we describe a detailed protocol for biochemical fractionation of Trypanosoma brucei parasites to detect mislocalization of glycosomal proteins to the cytosol. This approach utilizes increasing concentrations of digitonin that first permeabilizes the plasma membrane, followed by permeabilization of other organelles, depending on their cholesterol content. Fractionated samples can be further analyzed using immunoblotting for specific marker proteins or quantified by the specific enzyme activities.

The Trypanosome UDP-Glucose Pyrophosphorylase Is Imported by Piggybacking into Glycosomes, Where Unconventional Sugar Nucleotide Synthesis Takes Place.

Glycosomes are peroxisome-related organelles of trypanosomatid parasites containing metabolic pathways, such as glycolysis and biosynthesis of sugar nucleotides, usually present in the cytosol of other eukaryotes. UDP-glucose pyrophosphorylase (UGP), the enzyme responsible for the synthesis of the sugar nucleotide UDP-glucose, is localized in the cytosol and glycosomes of the bloodstream and procyclic trypanosomes, despite the absence of any known peroxisome-targeting signal (PTS1 and PTS2). The questions that we address here are (i) is the unusual glycosomal biosynthetic pathway of sugar nucleotides functional and (ii) how is the PTS-free UGP imported into glycosomes? We showed that UGP is imported into glycosomes by piggybacking on the glycosomal PTS1-containing phosphoenolpyruvate carboxykinase (PEPCK) and identified the domains involved in the UGP/PEPCK interaction. Proximity ligation assays revealed that this interaction occurs in 3 to 10% of glycosomes, suggesting that these correspond to organelles competent for protein import. We also showed that UGP is essential for the growth of trypanosomes and that both the glycosomal and cytosolic metabolic pathways involving UGP are functional, since the lethality of the knockdown UGP mutant cell line (RNAiUGP, where RNAi indicates RNA interference) was rescued by expressing a recoded UGP (rUGP) in the organelle (RNAiUGP/EXPrUGP-GPDH, where GPDH is glycerol-3-phosphate dehydrogenase). Our conclusion was supported by targeted metabolomic analyses (ion chromatography-high-resolution mass spectrometry [IC-HRMS]) showing that UDP-glucose is no longer detectable in the RNAiUGP mutant, while it is still produced in cells expressing UGP exclusively in the cytosol (PEPCK null mutant) or glycosomes (RNAiUGP/EXPrUGP-GPDH). Trypanosomatids are the only known organisms to have selected functional peroxisomal (glycosomal) sugar nucleotide biosynthetic pathways in addition to the canonical cytosolic ones. IMPORTANCE Unusual compartmentalization of metabolic pathways within organelles is one of the most enigmatic features of trypanosomatids. These unicellular eukaryotes are the only organisms that sequestered glycolysis inside peroxisomes (glycosomes), although the selective advantage of this compartmentalization is still not clear. Trypanosomatids are also unique for the glycosomal localization of enzymes of the sugar nucleotide biosynthetic pathways, which are also present in the cytosol. Here, we showed that the cytosolic and glycosomal pathways are functional. As in all other eukaryotes, the cytosolic pathways feed glycosylation reactions; however, the role of the duplicated glycosomal pathways is currently unknown. We also showed that one of these enzymes (UGP) is imported into glycosomes by piggybacking on another glycosomal enzyme (PEPCK); they are not functionally related. The UGP/PEPCK association is unique since all piggybacking examples reported to date involve functionally related interacting partners, which broadens the possible combinations of carrier-cargo proteins being imported as hetero-oligomers.

The coiled-coil domain of glycosomal membrane-associated Leishmania donovani PEX14: cloning, overexpression, purification and preliminary crystallographic analysis.

The glycosomal membrane-associated Leishmania donovani protein PEX14, which plays a crucial role in protein import from the cytosol to the glycosomal matrix, consists of three domains: an N-terminal domain where the signalling molecule binds, a transmembrane domain and an 84-residue coiled-coil domain (CC) that is responsible for oligomerization. CCs are versatile domains that participate in a variety of functions including supramolecular assembly, cellular signalling and transport. Recombinant PEX14 CC was cloned, overexpressed, affinity-purified with in-column thrombin cleavage and further purified by size-exclusion chromatography. Crystals that diffracted to 1.98 Šresolution were obtained from a condition consisting of 1.4 M sodium citrate tribasic dihydrate, 0.1 M HEPES buffer pH 7.5. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 143.98, b = 32.62, c = 95.62 Å, ß = 94.68°. Structure determination and characterization are in progress.

Structure, Properties, and Function of Glycosomes in Trypanosoma cruzi.

Glycosomes are peroxisome-related organelles that have been identified in kinetoplastids and diplonemids. The hallmark of glycosomes is their harboring of the majority of the glycolytic enzymes. Our biochemical studies and proteome analysis of Trypanosoma cruzi glycosomes have located, in addition to enzymes of the glycolytic pathway, enzymes of several other metabolic processes in the organelles. These analyses revealed many aspects in common with glycosomes from other trypanosomatids as well as features that seem specific for T. cruzi. Their enzyme content indicates that T. cruzi glycosomes are multifunctional organelles, involved in both several catabolic processes such as glycolysis and anabolic ones. Specifically discussed in this minireview are the cross-talk between glycosomal metabolism and metabolic processes occurring in other cell compartments, and the importance of metabolite translocation systems in the glycosomal membrane to enable the coordination between the spatially separated processes. Possible mechanisms for metabolite translocation across the membrane are suggested by proteins identified in the organelle's membrane-homologs of the ABC and MCF transporter families-and the presence of channels as inferred previously from the detection of channel-forming proteins in glycosomal membrane preparations from the related parasite T. brucei. Together, these data provide insight in the way in which different parts of T. cruzi metabolism, although uniquely distributed over different compartments, are integrated and regulated. Moreover, this information reveals opportunities for the development of drugs against Chagas disease caused by these parasites and for which currently no adequate treatment is available.

Inhibitors of glycosomal protein import provide new leads against trypanosomiasis.

Vector-borne trypanosomatid parasite infections in tropical and sub-tropical countries constitute a major threat to humans and livestock. Trypanosoma brucei parasites are transmitted by tsetse fly and lead to African sleeping sickness in humans and Nagana in cattle. In Latin American countries, Trypanosoma cruzi infections spread by triatomine kissing bugs lead to Chagas disease. Various species of Leishmania transmitted to humans by phlebotomine sandflies manifest in a spectrum of diseases termed Leishmaniasis. 20 million people are currently infected with trypanosomatid parasites, leading to over 30,000 deaths annually and half billion people at risk of the infection. It is estimated that 300,000 Chagas infected people reside in the United States and 100,000 in Europe. Glycosomes are peroxisome-like organelles found only in trypanosomatids. Glycolysis occurs in the cytosol in all other organisms, but glycolytic enzymes and other metabolic pathways are compartmentalized inside glycosomes in trypanosomatids. Glycosomes are essential for the parasite survival and hence thought to be an attractive drug target. Our recent study [Dawidowski et al. Science (2017)] is the first to report small molecule inhibitors of glycosomal protein import. Using structure-based drug design, we developed small molecule inhibitors of the Trypanosoma PEX5-PEX14 protein-protein interaction that disrupt glycosomal protein import and kill the parasites. Oral treatment of T. brucei infected mice with PEX14 inhibitor significantly reduced the parasite levels with no adverse effect on mice. The study provides the grounds for further development of the glycosome inhibitors into clinical candidates and validates the parasite protein-protein interactions as drug targets.

Clear Shot at Primary Aim: Susceptibility of Trypanosoma cruzi Organelles, Structures and Molecular Targets to Drug Treatment.

Chagas disease, caused by Trypanosoma cruzi, stands out due to its socio-economic effects on low-income tropical populations. This disease affects millions of people worldwide. The current chemotherapy for it is based on benznidazole (Bz) and nifurtimox (Nif) and is unsatisfactory. In this review, we will focus on the search for potential target organelles and molecules for the chemotherapy of Chagas disease. We consider as potential target organelles those that are absent or significantly different in host cells and present in the clinically relevant forms of the parasite (trypomastigotes and amastigotes), which are the mitochondrion, cytoskeletal-related structures, the acidocalcisomes/ contractile vacuole complex and glycosomes. Most molecular targets are key enzymes involved in processes that are essential to parasite survival, such as sterol biosynthesis, antioxidant defences and bioenergetic pathways. Among the molecular targets, enzymes of the sterol pathway, particularly C14α-sterol demethylase, are still the most promising target, even if clinical trials with posaconazole and E1224 have failed to sustain efficacy. We believe that in the near future, the Chagas community will have a "clear shot" at new drug candidates for Chagas disease based on the accumulated knowledge about trypanosomatid biochemistry, preclinical studies, advances in screening technologies, the efforts of medicinal chemists in the synthesis of both azolic and non-azolic inhibitors, and the interest of pharmaceutical companies in the development of new antifungal agents, which form a critical mass of information.

In or out? On the tightness of glycosomal compartmentalization of metabolites and enzymes in Trypanosoma brucei.

Trypanosomatids sequester large parts of glucose metabolism inside specialised peroxisomes, called glycosomes. Many studies have shown that correct glycosomal compartmentalization of glycolytic enzymes is essential for bloodstream-form Trypanosoma brucei. The recent finding of pore-forming activities in glycosomal membrane preparations and extensions of the trypanosome glycolysis computer model with size-selective pores sparked again an old debate on the extent of (im)permeability of the glycosomal membrane and whether glycosomally located glycolytic enzymes could and should also be present with some activity in the cytosol. This review presents a critical discussion of the experimental and theoretical evidence for and against the different hypotheses.