Consumption of Galactose by Trypanosoma cruzi Epimastigotes Generates Resistance against Oxidative Stress.

In this study, we demonstrate that Trypanosoma cruzi epimastigotes previously grown in LIT medium supplemented with 20 mM galactose and exposed to sub-lethal concentrations of hydrogen peroxide (100 µM) showed two-fold and five-fold viability when compared to epimastigotes grown in LIT medium supplemented with two different glucose concentrations (20 mM and 1.5 mM), respectively. Similar results were obtained when exposing epimastigotes from all treatments to methylene blue 30 µM. Additionally, through differential centrifugation and the selective permeabilization of cellular membranes with digitonin, we found that phosphoglucomutase activity (a key enzyme in galactose metabolism) occurs predominantly within the cytosolic compartment. Furthermore, after partially permeabilizing epimastigotes with digitonin (0.025 mg × mg-1 of protein), intact glycosomes treated with 20 mM galactose released a higher hexose phosphate concentration to the cytosol in the form of glucose-1-phosphate, when compared to intact glycosomes treated with 20 mM glucose, which predominantly released glucose-6-phosphate. These results shine a light on T. cruzi's galactose metabolism and its interplay with mechanisms that enable resistance to oxidative stress.

IgE antibodies against Trypanosoma cruzi arginine kinase in patients with chronic Chagas disease.

Arginine kinase (AK) is an enzyme present in various invertebrates, as well as in some trypanosomatids such as T. cruzi, the etiological agent that causes Chagas disease. In invertebrates, this protein acts as an allergen inducing an IgE-type humoral immune response. Since AK is a highly conserved protein, we decided to study whether patients with chronic Chagas disease (CCD) produce specific antibodies against T. cruzi AK (TcAK). Plasma from patients with CCD, with and without cardiac alterations and non-infected individuals were evaluated for the presence of anti-TcAK IgG and IgE antibodies by ELISA, including detection of specific IgG subclasses. Our results showed that the levels of specific anti-TcAK IgG and IgE were different between infected and non-infected individuals, but comparable between those with different clinical manifestations. Interestingly, anti-TcAK IgG4 antibodies associated with IgE-mediated allergenic processes were also increased in CCD patients. Finally, we found that several of the predicted B cell epitopes in TcAK matched allergenic peptides previously described for its homologues in other organisms. Our results revealed for the first time a parasite's specific IgE antibody target and suggest that TcAK could contribute to delineate an inefficient B cell response by prompting a bias towards a Th2 profile. These findings also shed light on a potential allergenic response in the context of T. cruzi infection.

Genetic validation of aldolase and glyceraldehyde-3-phosphate dehydrogenase as drug targets in Trypanosoma brucei.

Aldolase (ALD) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Trypanosoma brucei are considered to be promising targets for chemotherapeutic treatment of African sleeping sickness, because glycolysis is the single source of ATP for the parasite when living in the human bloodstream. Moreover, these enzymes appeared to possess distinct kinetic and structural properties that have already been exploited for the discovery of effective and selective inhibitors with trypanocidal activity. Here we present an experimental, quantitative assessment of the importance of these enzymes for the glycolytic pathway. This was achieved by decreasing the concentrations of ALD and GAPDH by RNA interference. The effects of these knockdowns on parasite growth, levels of various enzymes and transcripts, enzyme activities and glucose consumption were studied. A partial depletion of ALD and GAPDH was already sufficient to rapidly kill the trypanosomes. An effect was also observed on the activity of some other glycolytic enzymes.

Molecular and biochemical characterization of novel glucokinases from Trypanosoma cruzi and Leishmania spp.

Glucokinase genes, found in the genome databases of Trypanosoma cruzi and Leishmania major, were cloned and sequenced. Their expression in Escherichia coli resulted in the synthesis of soluble and active enzymes, TcGlcK and LmjGlcK, with a molecular mass of 43 kDa and 46 kDa, respectively. The enzymes were purified, and values of their kinetic parameters determined. The K(m) values for glucose were 1.0 mM for TcGlcK and 3.3 mM for LmjGlcK. For ATP, the K(m) values were 0.36 mM (TcGlcK) and 0.35 mM (LmjGlcK). A lower K(m) value for glucose (2.55 mM) was found when the (His)(6)-tag was removed from the recombinant LmjGlcK, whereas the TcGlcK retained the same value. The V(max)'s of the T. cruzi and L. major GlcKs were 36.3 and 30.9 U/mg of protein, respectively. No inhibition was exerted by glucose-6-phosphate. Similarly, no inhibition by inorganic pyrophosphate was found in contrast to previous observations made for the T. cruzi and L. mexicana hexokinases. Both trypanosomatid enzymes were only able to phosphorylate glucose indicating that they are true glucokinases. Gel-filtration chromatography showed that the GlcK of both trypanosomatids may occur as a monomer or dimer, dependent on the protein concentration. Both GlcK sequences have a type-1 peroxisome-targeting signal. Indeed, they were shown to be present inside glycosomes using three different methods. These glucokinases present highest, albeit still a moderate 24% sequence identity with their counterpart from Trichomonas vaginalis, which has been classified into group A of the hexokinase family. This group comprises mainly eubacterial and cyanobacterial glucokinases. Indeed, multiple sequence comparisons, as well as kinetic properties, strongly support the notion that these trypanosomatid enzymes belong to group A of the hexokinases, in which they, according to a phylogenetic analysis, form a separate cluster.

Molecular and biochemical characterization of hexokinase from Trypanosoma cruzi.

The Trypanosoma cruzi hexokinase gene has been cloned, sequenced, and expressed as an active enzyme in Escherichia coli. Sequence analysis revealed 67% identity with its counterpart in Trypanosoma brucei but low similarity with all other available hexokinase sequences including those of human. It contains an N-terminal peroxisome-targeting signal (PTS-2) and has a calculated basic isoelectric point (pI = 9.67), a feature often associated with glycosomal proteins. The polypeptide has a predicted mass of approximately 50 kDa similar to that of many non-vertebrate hexokinases and the vertebrate hexokinase isoenzyme IV. The natural enzyme was purified to homogeneity from T. cruzi epimastigotes and appeared to exist in several aggregation states, an apparent tetramer being the predominant form. Its kinetic properties were compared with those of the purified recombinant protein. Higher K(m) values for glucose and ATP were found for the (His)(6)-tag-containing recombinant hexokinase. However, removal of the tag produced an enzyme displaying similar values as the natural enzyme (K(m) for glucose = 43 and 60 microM for the natural and the recombinant protein, respectively). None of these enzymes presented activity with fructose. As reported previously for hexokinases from several trypanosomatids, no inhibition was exerted by glucose 6-phosphate (G6-P). In contrast, a mixed-type inhibition was observed with inorganic pyrophosphate (PPi, K(i) = 0.5mM).