Glucose 6-Phosphate Dehydrogenase from Trypanosomes: Selectivity for Steroids and Chemical Validation in Bloodstream Trypanosoma brucei.

Glucose 6-phosphate dehydrogenase (G6PDH) fulfills an essential role in cell physiology by catalyzing the production of NADPH+ and of a precursor for the de novo synthesis of ribose 5-phosphate. In trypanosomatids, G6PDH is essential for in vitro proliferation, antioxidant defense and, thereby, drug resistance mechanisms. So far, 16α-brominated epiandrosterone represents the most potent hit targeting trypanosomal G6PDH. Here, we extended the investigations on this important drug target and its inhibition by using a small subset of androstane derivatives. In Trypanosoma cruzi, immunofluorescence revealed a cytoplasmic distribution of G6PDH and the absence of signal in major organelles. Cytochemical assays confirmed parasitic G6PDH as the molecular target of epiandrosterone. Structure-activity analysis for a set of new (dehydro)epiandrosterone derivatives revealed that bromination at position 16α of the cyclopentane moiety yielded more potent T. cruzi G6PDH inhibitors than the corresponding ß-substituted analogues. For the 16α brominated compounds, the inclusion of an acetoxy group at position 3 either proved detrimental or enhanced the activity of the epiandrosterone or the dehydroepiandrosterone derivatives, respectively. Most derivatives presented single digit µM EC50 against infective T. brucei and the killing mechanism involved an early thiol-redox unbalance. This data suggests that infective African trypanosomes lack efficient NADPH+-synthesizing pathways, beyond the Pentose Phosphate, to maintain thiol-redox homeostasis.

Infection-driven activation of transglutaminase 2 boosts glucose uptake and hexosamine biosynthesis in epithelial cells.

Transglutaminase 2 (TG2) is a ubiquitously expressed enzyme with transamidating activity. We report here that both expression and activity of TG2 are enhanced in mammalian epithelial cells infected with the obligate intracellular bacteria Chlamydia trachomatis. Genetic or pharmacological inhibition of TG2 impairs bacterial development. We show that TG2 increases glucose import by up-regulating the transcription of the glucose transporter genes GLUT-1 and GLUT-3. Furthermore, TG2 activation drives one specific glucose-dependent pathway in the host, i.e., hexosamine biosynthesis. Mechanistically, we identify the glucosamine:fructose-6-phosphate amidotransferase (GFPT) among the substrates of TG2. GFPT modification by TG2 increases its enzymatic activity, resulting in higher levels of UDP-N-acetylglucosamine biosynthesis and protein O-GlcNAcylation. The correlation between TG2 transamidating activity and O-GlcNAcylation is disrupted in infected cells because host hexosamine biosynthesis is being exploited by the bacteria, in particular to assist their division. In conclusion, our work establishes TG2 as a key player in controlling glucose-derived metabolic pathways in mammalian cells, themselves hijacked by C. trachomatis to sustain their own metabolic needs.

Trypanosoma cruzi: death phenotypes induced by ortho-naphthoquinone substrates of the aldo-keto reductase (TcAKR). Role of this enzyme in the mechanism of action of ß-lapachone.

Several ortho-naphthoquinones (o-NQs) have trypanocidal activity against Trypanosoma cruzi, the aetiological agent of Chagas disease. Previously, we demonstrated that the aldo-keto reductase from this parasite (TcAKR) reduces o-NQs, such as ß-lapachone (ß-Lap) and 9,10-phenanthrenequinone (9,10-PQ), with concomitant reactive oxygen species (ROS) production. Recent characterization of TcAKR activity and expression in two T. cruzi strains, CL Brener and Nicaragua, showed that TcAKR expression is 2.2-fold higher in CL Brener than in Nicaragua. Here, we studied the trypanocidal effect and induction of several death phenotypes by ß-Lap and 9,10-PQ in epimastigotes of these two strains. The CL Brener strain was more resistant to both o-NQs than Nicaragua, indicating that greater TcAKR activity is unlikely to be a major influence on o-NQ toxicity. Evaluation of changes in ROS production, mitochondrial membrane potential, phosphatidylserine exposure and monodansylcadaverine labelling evidenced that ß-Lap and 9,10-PQ induce different death phenotypes depending on the combination of drug and T. cruzi strain analysed. To study whether TcAKR participates in o-NQ activation in intact parasites, ß-Lap and 9,10-PQ trypanocidal effect was next evaluated in TcAKR-overexpressing parasites. Only ß-Lap was more effective and induced greater ROS production in TcAKR-overexpressing epimastigotes than in controls, suggesting that TcAKR may participate in ß-Lap activation.

Putative Role of the Aldo-Keto Reductase from Trypanosoma cruzi in Benznidazole Metabolism.

Benznidazole (Bz), the drug used for treatment of Chagas' disease (caused by the protozoan Trypanosoma cruzi), is activated by a parasitic NADH-dependent type I nitroreductase (NTR I). However, several studies have shown that other enzymes are involved. The aim of this study was to evaluate whether the aldo-keto reductase from T. cruzi (TcAKR), a NADPH-dependent oxido-reductase previously described by our group, uses Bz as the substrate. We demonstrated that both recombinant and native TcAKR enzymes reduce Bz by using NADPH, but not NADH, as a cofactor. TcAKR-overexpressing epimastigotes showed higher NADPH-dependent Bz reductase activity and a 50% inhibitory concentration (IC50) value for Bz 1.8-fold higher than that of the controls, suggesting that TcAKR is involved in Bz detoxification instead of activation. To understand the role of TcAKR in Bz metabolism, we studied TcAKR expression and NADPH/NADH-dependent Bz reductase activities in two T. cruzi strains with differential susceptibility to Bz: CL Brener and Nicaragua. Taking into account the results obtained with TcAKR-overexpressing epimastigotes, we expected the more resistant strain, Nicaragua, to have higher TcAKR levels than CL Brener. However, the results were the opposite. CL Brener showed 2-fold higher TcAKR expression and 5.7-fold higher NADPH-Bz reduction than the Nicaragua strain. In addition, NADH-dependent Bz reductase activity, characteristic of NTR I, was also higher in CL Brener than in Nicaragua. We conclude that although TcAKR uses Bz as the substrate, TcAKR activity is not a determinant of Bz resistance in wild-type strains and may be overcome by other enzymes involved in Bz activation, such as NADPH- and NADH-dependent reductases.

Characterization of TcCYC6 from Trypanosoma cruzi, a gene with homology to mitotic cyclins.

Trypanosoma cruzi, the etiologic agent of Chagas disease, is a protozoan parasite with a life cycle that alternates between replicative and non-replicative forms, but the components and mechanisms that regulate its cell cycle are poorly described. In higher eukaryotes, cyclins are proteins that activate cyclin-dependent kinases (CDKs), by associating with them along the different stages of the cell cycle. These cyclin-CDK complexes exert their role as major modulators of the cell cycle by phosphorylating specific substrates. For the correct progression of the cell cycle, the mechanisms that regulate the activity of cyclins and their associated CDKs are diverse and must be controlled precisely. Different types of cyclins are involved in specific phases of the eukaryotic cell cycle, preferentially activating certain CDKs. In this work, we characterized TcCYC6, a putative coding sequence of T. cruzi which encodes a protein with homology to mitotic cyclins. The overexpression of this sequence, fused to a tag of nine amino acids from influenza virus hemagglutinin (TcCYC6-HA), showed to be detrimental for the proliferation of epimastigotes in axenic culture and affected the cell cycle progression. In silico analysis revealed an N-terminal segment similar to the consensus sequence of the destruction box, a hallmark for the degradation of several mitotic cyclins. We experimentally determined that the TcCYC6-HA turnover decreased in the presence of proteasome inhibitors, suggesting that TcCYC6 degradation occurs via ubiquitin-proteasome pathway. The results obtained in this study provide first evidence that TcCYC6 expression and degradation are finely regulated in T. cruzi.

Quantitative monitoring of the Chlamydia trachomatis developmental cycle using GFP-expressing bacteria, microscopy and flow cytometry.

Chlamydiae are obligate intracellular bacteria. These pathogens develop inside host cells through a biphasic cycle alternating between two morphologically distinct forms, the infectious elementary body and the replicative reticulate body. Recently, C. trachomatis strains stably expressing fluorescent proteins were obtained. The fluorochromes are expressed during the intracellular growth of the microbe, allowing bacterial visualization by fluorescence microscopy. Whether they are also present in the infectious form, the elementary body, to a detectable level has not been studied. Here, we show that a C. trachomatis strain transformed with a plasmid expressing the green fluorescent protein (GFP) accumulates sufficient quantities of the probe in elementary bodies for detection by microscopy and flow cytometry. Adhesion of single bacteria was detected. The precise kinetics of bacterial entry were determined by microscopy using automated procedures. We show that during the intracellular replication phase, GFP is a convenient read-out for bacterial growth with several advantages over current methods. In particular, infection rates within a non-homogenous cell population are easily quantified. Finally, in spite of their small size, individual elementary bodies are detected by flow cytometers, allowing for direct enumeration of a bacterial preparation. In conclusion, GFP-expressing chlamydiae are suitable to monitor, in a quantitative manner, progression throughout the developmental cycle. This will facilitate the identification of the developmental steps targeted by anti-chlamydial drugs or host factors.

Functional characterization of TcCYC2 cyclin from Trypanosoma cruzi.

In eukaryotes, an oscillating network of protein kinase activities drives the order and timing of the cell cycle progression. Complexes formed by cyclins associated to cyclin-dependent kinases (CDKs) are the central components of this network. Cyclins act as the activating subunits and their abundance is regulated by different mechanisms in order to promote or prevent kinase activity. Protein synthesis, proteasomal degradation and/or differential subcellular compartmentalization modulate cyclin expression levels along the cell cycle. We describe in this work the characterization of Trypanosoma cruzi Cyclin 2 (TcCYC2), which contributes to a better understanding of the cell cycle regulation in this protozoan parasite. We found TcCYC2 exhibited cyclin function in a yeast complementation assay and over-expression of hemagglutinin tagged TcCYC2-HA rendered shorter duplication times and smaller cell sizes in the epimastigote form of the parasite. Analysis of synchronized cultures showed that over-expression of TcCYC2-HA altered the timing epimastigotes pass through G2/M boundary or cytokinesis. Taken together, our results showed that TcCYC2 is a functional cyclin whose over-expression modifies the dynamics of the cell cycle as well as the morphology of epimastigote forms of T. cruzi, suggesting it plays an important role in the cell cycle regulation machinery.

SUMOylation pathway in Trypanosoma cruzi: functional characterization and proteomic analysis of target proteins.

SUMOylation is a relevant protein post-translational modification in eukaryotes. The C terminus of proteolytically activated small ubiquitin-like modifier (SUMO) is covalently linked to a lysine residue of the target protein by an isopeptide bond, through a mechanism that includes an E1-activating enzyme, an E2-conjugating enzyme, and transfer to the target, sometimes with the assistance of a ligase. The modification is reversed by a protease, also responsible for SUMO maturation. A number of proteins have been identified as SUMO targets, participating in the regulation of cell cycle progression, transcription, translation, ubiquitination, and DNA repair. In this study, we report that orthologous genes corresponding to the SUMOylation pathway are present in the etiological agent of Chagas disease, Trypanosoma cruzi. Furthermore, the SUMOylation system is functionally active in this protozoan parasite, having the requirements for SUMO maturation and conjugation. Immunofluorescence analysis showed that T. cruzi SUMO (TcSUMO) is predominantly found in the nucleus. To identify SUMOylation targets and get an insight into their physiological roles we generated transfectant T. cruzi epimastigote lines expressing a double-tagged T. cruzi SUMO, and SUMOylated proteins were enriched by tandem affinity chromatography. By two-dimensional liquid chromatography-mass spectrometry a total of 236 proteins with diverse biological functions were identified as potential T. cruzi SUMO targets. Of these, metacaspase-3 was biochemically validated as a bona fide SUMOylation substrate. Proteomic studies in other organisms have reported that orthologs of putative T. cruzi SUMOylated proteins are similarly modified, indicating conserved functions for protein SUMOylation in this early divergent eukaryote.