Structural insight into activation mechanism of Toxoplasma gondii nucleoside triphosphate diphosphohydrolases by disulfide reduction

The intracellular parasite Toxoplasma gondii produces two nucleoside triphosphate diphosphohydrolases (NTPDase1 and -3). These tetrameric, cysteine-rich enzymes require activation by reductive cleavage of a hitherto unknown disulfide bond. Despite a 97% sequence identity, both isozymes differ largely in their ability to hydrolyze ATP and ADP. Here, we present crystal structures of inactive NTPDase3 as an apo form and in complex with the product AMP to resolutions of 2.0 and 2.2 Å, respectively. We find that the enzyme is present in an open conformation that precludes productive substrate binding and catalysis. The cysteine bridge 258–268 is identified to be responsible for locking of activity. Crystal structures of constitutively active variants of NTPDase1 and -3 generated by mutation of Cys²⁵⁸–Cys²⁶⁸ show that opening of the regulatory cysteine bridge induces a pronounced contraction of the whole tetramer. This is accompanied by a 12° domain closure motion resulting in the correct arrangement of all active site residues. A complex structure of activated NTPDase3 with a non-hydrolyzable ATP analog and the cofactor Mg²⁺ to a resolution of 2.85 Å indicates that catalytic differences between the NTPDases are primarily dictated by differences in positioning of the adenine base caused by substitution of Arg⁴⁹² and Glu⁴⁹³ in NTPDase1 by glycines in NTPDase3.     

ATP and ADP hydrolysis in fish, chicken and rat synaptosomes

Ecto-enzymes capable of hydrolyzing ATP and ADP (NTPDase) are present in the central nervous system of various species. In the present investigation we studied the synaptosomal NTPDase (ATP diphosphohydrolase, apyrase, E.C. 3.6.1.5) from fish, chicken and rats under different conditions and in the presence of several classical inhibitors. The cation concentration required for maximal activity was 0.5 mM for fish, 1.0 mM for chickens and 1.5 mM for rats with both substrates. The results showed that the pH optimum for all animal preparations was close to 8.0. The temperature used was 25–27°C for fish and 35–37°C for chicken and rat preparations. The inhibitors azide and fluoride only inhibited the preparation at high concentrations (10 mM). Lanthanum (0.1–0.4 mM), N-ethylmaleimide (0.4–3.0 mM) and ouabain (0.5–3.0 mM) had no effect on NTPDase activity from fish, chickens or rats. Orthovanadate (0.1–0.3 mM) only inhibited fish synaptosomal NTPDase. Trifluoperazine (0.05–0.2 mM) and suramin (0.03–0.3 mM) inhibited NTPDase at all concentrations tested. Suramin was the most potent compound in causing inhibition, presenting inhibition at 30 μM. Our results demonstrate that the synaptosomal NTPDase response to several factors is similar in fish, chickens and rats, and that the enzyme presents functional homology.     

Conformation of the high molecular weight form ofDunaliella salina phosphofructokinase in solution by means of small-angle X-ray diffraction and viscosity measurements

Summary The intrinsic viscosity of phosphofructokinase fromDunaliella salina in different states of aggregation was determined. The instrinsic viscosity [], of the biologically active tetramer, with a molecular weight of 320,000, was found to be 6.5 ml·g^–1 at 4°C. Moreover, for the inactive dimer, with a molecular weight of 160,000, a value of []=8.0 ml·g^–1 was determined. The high molecular weight aggregate of phosphofructokinase fromDunaliella salina, that shows little activity, has an intrinsic viscosity of 23.2 ml·g^–1, which is significantly higher than that found for the active tetramer and the inactive dimer.Small angle X-ray scattering experiments in solution of this high molecular from of phosphofructokinase fromDunaliella salina reveal a radius of gyration of the cross section ofR _c=49.0 Å at an ionic strength of 0.15 M and_pH 7.2. Furthermore, a comparison of the values obtained for the tetramer and the radius of gyration (R _g=52.9 Å) with those of typical spherical proteins (3–4 ml·g^–1) shows that the values of [] andR _g are significantly larger for the high molecular weight form of phosphofructokinase than for the spherical proteins. The high intrinsic viscosity of the polymeric form of phosphofructokinase suggests an end-to-end aggregation consisting of monomeric units with heights,h=80–90 Å, and a cylindrical diameter of approximately 140.0 Å, resulting in a long rod of a total length of 1,800 Å and a molecular weight of two million. On the basis of the experimentally observedR _c and [] values, using a prolate ellipsoid of revolution as a model, the hydrodynamic volume and the hydration, the axial ratio could be determined to be 12. The native tetrameric form contains 0.4 g H₂O/g protein, whereas the higher aggregate structure corresponds to a hydration of 0.60 g H₂O/g protein.     

Salt-induced folding of a rabbit muscle pyruvate kinase intermediate at alkaline pH

The effect of alkaline denaturation on the structural and functional characteristics of rabbit muscle pyruvate kinase (PK) was investigated using enzymatic activity measurements and a combination of optical methods such as circular dichroism, fluorescence, and ANS binding. At a critical pH, 10.5, PK exists in an intermediate state (alkaline unfolded state) with predominant secondary structure along with some of the tertiary interactions and a strong binding to the hydrophobic dye ANS. This intermediate retains the enzymatic activity and corresponds to a dimeric state of the molecule. Above pH 10.5, a sudden fall in the spectral properties and enzymatic activity occurs suggesting the dissociation of the molecule followed by unfolding at very high pH. Addition of salts such as NaCl, KCl, and Na₂SO₄ to the alkali-induced state induces both secondary and tertiary structure to a level equivalent to that of native tetramer (salt-induced state). Chemical- and temperature-induced unfolding of the alkali-induced state as well as the salt-induced refolded state of PK reveal the presence of intermediate conformations in the unfolding pathway. The unfolding transition curves are noncoinciding and noncooperative along with ANS binding at intermediate concentrations of denaturants during unfolding. The observations presented in this paper suggest that the native pyruvate kinase tetramer dissociates to an active dimer around pH 10.5 and further to inactive monomer before attaining a completely unfolded monomeric conformation.     

Sulphate removal induces a major conformational change in Leishmania mexicana pyruvate kinase in the crystalline state

We report X-ray structures of pyruvate kinase from Leishmania mexicana (LmPYK) that are trapped in different conformations. These, together with the previously reported structure of LmPYK in its inactive (T-state) conformation, allow comparisons of three different conformers of the same species of pyruvate kinase (PYK). Four new site point mutants showing the effects of side-chain alteration at subunit interfaces are also enzymatically characterised. The LmPYK tetramer crystals grown with ammonium sulphate as precipitant adopt an active-like conformation, with sulphate ions at the active and effector sites. The sulphates occupy positions similar to those of the phosphates of ligands bound to active (R-state) and constitutively active (nonallosteric) PYKs from several species, and provide insight into the structural roles of the phosphates of the substrates and effectors. Crystal soaking in sulphate-free buffers was found to induce major conformational changes in the tetramer. In particular, the unwinding of the Aα6′ helix and the inward hinge movement of the B domain are coupled with a significant widening (4 Å) of the tetramer caused by lateral movement of the C domains. The two new LmPYK structures and the activity studies of site point mutations described in this article are consistent with a developing picture of allosteric activity in which localised changes in protein flexibility govern the distribution of conformer families adopted by the tetramer in its active and inactive states.     

ATP and ADP hydrolysis in the kidney and liver of fish, chickens and rats

We investigated NTPDase-like activity [ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases)] in liver and kidney membrane from silver catfish (Rhamdia quelen), chicken (Gallus gallus) and rat (Rattus norvegicus) under different conditions and in the presence of several inhibitors. The cation concentration required for maximal activity was 0.5, 1.5 and 2.0 mM for fish, chicken and rat liver, respectively (with ATP and ADP as substrates). The maximal activity in the kidney was observed at calcium concentrations of 0.5, 2.0, 1.5 mM (ATP) and 0.5, 1.5, 1.0 (ADP) for fish, chickens and rats, respectively. The results showed that the pH optimum for all animals and for the two tissues was close to 8.0. The temperature chosen was 25 °C for fish and 36 °C for chicken and rat preparations. Ouabain had no effect on the NTPDase-like activity of fish, chickens or rats. NTPDase activity was decreased in the presence of lanthanum in the chicken (ADP) and rat (ATP and ADP) liver. In the kidney, lanthanum inhibited fish ATP and rat ATP and ADP (0.2 mM) hydrolysis. N-ethylmaleimide (NEM) had an inhibitory effect on the kidney of all species at the concentration of 3.0 mM (ADP). Orthovanadate only inhibited fish membrane NTPDase; azide only inhibited the preparation at high concentrations (10 mM) and fluoride inhibited it at 10 mM (fish and chicken) and 5 mM (rat). Trifluoperazine (0.05–0.2 mM) and suramin (0.03–0.3 mM) inhibited NTPDase at all concentrations tested. These results suggest that NTPDase-like activity shows a different behavior among the vertebrate species and tissues studied. Additionally, we propose that NTPDase1 is the main enzyme present in this preparation.     

Expression of a Recombinant Toxoplasma gondii ROP2 Fragment as a Fusion Protein in Bacteria Circumvents Insolubility and Proteolytic Degradation

A 268-amino-acid-residue carboxy-terminal antigenic fragment of the Toxoplasma gondii rhoptry protein ROP2 (recROP2_t, residues 196–464) was expressed in Escherichia coli. This recombinant fragment was produced at low concentration and in a highly insoluble form. By contrast, the level of recROP2_t production was drastically greater when the same coding sequence was fused to the C-terminus of thioredoxin (TRX) or to the maltose-binding protein (MBP) gene. While both fusion proteins were found to be mainly insoluble, solubilization could be achieved without significant degradation. MBP was more efficient than TRX in increasing the recovery of soluble protein with more than 10% of total MBP–recROP2_t being readily expressed in a soluble form. Moreover, the insoluble form of MBP–recROP2_t could be correctly refolded with a recovery of more than 80%. Both forms of MBP–recROP2_t were purified to homogeneity by amylose chromatography. In contrast, the refolding of TRX–recROP2_t promoted aggregation of the protein, which was prevented by the use of zwitterionic detergent during the one-step purification by gel filtration. Subsequent proteolytic cleavages of purified TRX–recROP2_t and of MBP–recROP2_t led respectively to the complete degradation or to the truncation of the recROP2_t moiety. However, recROP2_t, despite the presence of the fusion partners, adopted a suitable conformation recognized by human serum-derived antibodies from T. gondii-seropositive individuals. Finally, both fusion proteins were able to induce specific humoral and cell-mediated immune response to the ROP2 fragment. Such fusions could represent an alternative to study the immunogenicity of T. gondii proteins which are difficult to produce because of insolubility and degradation.     

Trafficking and intracellular ATPase activity of human ecto-nucleotidase NTPDase3 and the effect of ER-targeted NTPDase3 on protein folding

Ecto-nucleoside triphosphate diphosphohydrolases, NTPDase1 (CD39) and NTPDase3, are integral plasma membrane proteins that hydrolyze extracellular nucleotides, thereby modulating the function of purinergic receptors. During processing in the secretory pathway, the active sites of ecto-nucleotidases are located in the lumen of vesicular compartments, thus raising the question whether the ecto-nucleotidases affect the ATP-dependent processes in these compartments, including protein folding in the endoplasmic reticulum (ER). It has been reported (J. Biol. Chem. (2001) 276, 41518-41525) that CD39 is not active until it reaches the plasma membrane, suggesting that terminal glycosylation in Golgi is critical for its activity. To investigate the subcellular location and the mechanism of ecto-nucleotidase activation, we expressed human NTPDase3 in COS-1 cells and blocked the secretory transport with monensin or brefeldin A, or by targeting to ER with a signal peptide. Cell surface biotinylation, sensitivity to glycosidases, and fluorescence microscopy analyses suggest that, in contrast to the previous report on CD39, NTPDase3 becomes catalytically active in the ER or in the ER-Golgi intermediate compartment, and that terminal glycosylation in Golgi is not essential for activity. Moreover, ER-targeted NTPDase3, but not wild-type NTPDase3 or ER-targeted inactive G221A mutant, significantly diminished the folding efficiency and the transport to the plasma membrane of coexpressed CD39 used as a reporter protein. These data suggest that ER-targeted NTPDase3 significantly depletes ATP in ER, whereas wild-type NTPDase3 is likely to acquire ATPase activity in a post-ER, but pre-Golgi, compartment, thus avoiding unproductive ATP hydrolysis and interference with protein folding in the ER. ER-targeted NTPDase3 may be a useful experimental tool to study the effects of ER ATP depletion on ER function under normal and stress conditions.     

Correlation Between Self-Association Modes and GTPase Activation of Dynamin

The GTPase activity of dynamin is obligatorily coupled, by a mechanism yet unknown, to the internalization of clathrin-coated endocytic vesicles. Dynamin oligomerizes in vitro and in vivo and both its mechanical and enzymatic activities appear to be mediated by this self-assembly. In this study we demonstrate that dynamin is characterized by a tetramer/monomer equilibrium with an equilibrium constant of 1.67 × 10¹⁷ M^–3. Stopped-flow fluorescence experiments show that the association rate constant for 2(3)-O-N-methylanthraniloyl (mant)GTP is 7.0 × 10^–5 M^–1 s^–1 and the dissociation rate constant is 2.1 s^–1, whereas the dissociation rate constant for mantdeoxyGDP is 93 s^–1. We also demonstrate the cooperativity of dynamin binding and GTPase activation on a microtubule lattice. Our results indicate that dynamin self-association is not a sufficient condition for the expression of maximal GTPase activity, which suggests that dynamin molecules must be in the proper conformation or orientation if they are to form an active oligomer.     

Variants of human thymidylate synthase with loop 181–197 stabilized in the inactive conformation

Loop 181–197 of human thymidylate synthase (hTS) populates two major conformations, essentially corresponding to the loop flipped by 180°. In one of the conformations, the catalytic Cys195 residue lies distant from the active site making the enzyme inactive. Ligands stabilizing this inactive conformation may function as allosteric inhibitors. To facilitate the search for such inhibitors, we have expressed and characterized several mutants designed to shift the equilibrium toward the inactive conformer. In most cases, the catalytic efficiency of the mutants was only somewhat impaired with values of k_cat/K_m reduced by factors in a 2–12 range. One of the mutants, M190K, is however unique in having the value of k_cat/K_m smaller by a factor of ～7500 than the wild type. The crystal structure of this mutant is similar to that of the wt hTS with loop 181–197 in the inactive conformation. However, the direct vicinity of the mutation, residues 188–194 of this loop, assumes a different conformation with the positions of C_α shifted up to 7.2 Å. This affects region 116–128, which became ordered in M190K while it is disordered in wt. The conformation of 116–128 is however different than that observed in hTS in the active conformation. The side chain of Lys190 does not form contacts and is in solvent region. The very low activity of M190K as compared to another mutant with a charged residue in this position, M190E, suggests that the protein is trapped in an inactive state that does not equilibrate easily with the active conformer.     

Structure of the RNA‐directed RNA Polymerase from the cystovirus ϕ12

We have determined the structure of P2, the self‐priming RdRp from cystovirus ?12 in two crystal forms (A, B) at resolutions of 1.7 Å and 2.1 Å. Form A contains Mg²⁺ bound at a site that deviates from the canonical noncatalytic position seen in form B. These structures provide insight into the temperature sensitivity of a ts‐mutant. However, the tunnel through which template ssRNA accesses the active site is partially occluded by a flexible loop; this feature, along with suboptimal positioning of other structural elements that prevent the formation of a stable initiation complex, indicate an inactive conformation in crystallo. Proteins 2013; 81:1479–1484. © 2013 Wiley Periodicals, Inc.     

The Dysfunction of CD4+CD25+ Regulatory T Cells Contributes to the Abortion of Mice Caused by Toxoplasma gondii Excreted-Secreted Antigens in Early Pregnancy

Toxoplasma gondii is an opportunistic intracellular parasite that is highly prevalent in human and warm-blooded animals throughout the world, leading to potentially severe congenital infections. Although the abortion caused by T. gondii is believed to be dependent on the timing of maternal infection during pregnancy, the mechanism remains unclear. This study was focused on the effects of T. gondii excreted-secreted antigens on pregnant outcomes and CD4⁺CD25⁺ Foxp3⁺ regulatory T cells at different stages of pregnancy. The results showed that in mice the frequency and suppressive function of CD4⁺CD25⁺ regulatory cells were diminished after injection of T. gondii excreted-secreted antigens at early and intermediate stages of pregnancy. The abortion caused by T. gondii excreted-secreted antigens at early pregnancy could be partly prevented by adoptively transferring of CD4⁺CD25⁺ cells from the mice injected with T. gondii excreted-secreted antigens at late pregnancy, but not from the mice with the same treatment at early pregnancy. Furthermore, T. gondii excreted-secreted antigens induced apoptosis of CD4⁺CD25⁺ regulatory cells of mice in early and intermediate stages of pregnancy by down-regulating their Bcl-2 expressions and Bcl-2/Bax ratio. This study provides new insights into the mechanism that T. gondii infection is the high risk factor for abortion in early pregnancy.     

Crystal structure of the nucleotide‐metabolizing enzyme NTPDase4

The ecto‐nucleoside triphosphate diphosphohydrolases (NTPDases) are a family of enzymes found on the cell surface and in the lumen of certain organelles, that are major regulators of purinergic signaling. Their intracellular roles, however, have not been clearly defined. NTPDase4 (UDPase, ENTPD4) is a Golgi protein potentially involved in nucleotide recycling as part of protein glycosylation, and is also found in lysosomes, where its purpose is unknown. To further our understanding of NTPDase4 function, we determined its crystal structure. The enzyme adopts a wide open, inactive conformation. Differences in the nucleotide‐binding site relative to its homologs could account for its substrate selectivity. The putative membrane‐interacting loop of cell‐surface NTPDases is drastically altered in NTPDase4, potentially affecting its interdomain dynamics at the Golgi membrane.     

Structural insight into mechanisms for dynamic regulation of PKM2

Pyruvate kinase isoform M2 (PKM2) converts phosphoenolpyruvate (PEP) to pyruvate and plays an important role in cancer metabolism. Here, we show that posttranslational modifications and a patient-derived mutation regulate pyruvate kinase activity of PKM2 through modulating the conformation of the PKM2 tetramer. We determined crystal structures of human PKM2 mutants and proposed a “seesaw” model to illustrate conformational changes between an inactive T-state and an active R-state tetramers of PKM2. Biochemical and structural analyses demonstrate that PKM2^Y105E (phosphorylation mimic of Y105) decreases pyruvate kinase activity by inhibiting FBP (fructose 1,6-bisphosphate)-induced R-state formation, and PKM2^K305Q (acetylation mimic of K305) abolishes the activity by hindering tetramer formation. K422R, a patient-derived mutation of PKM2, favors a stable, inactive T-state tetramer because of strong intermolecular interactions. Our study reveals the mechanism for dynamic regulation of PKM2 by posttranslational modifications and a patient-derived mutation and provides a structural basis for further investigation of other modifications and mutations of PKM2 yet to be discovered.     

Evaluation of the immune response elicited by multi-antigenic DNA vaccine expressing SAG1, ROP2 and GRA2 against Toxoplasma gondii

The parasite Toxoplasma gondii can infect most mammals and birds, sometimes causing severe pathology. Previous studies have reported that multi-antigenic vaccines were more effective than single-antigenic vaccine. It was also reported that the a single-gene vaccine with SAG1 or ROP2, GRA2 could only produce partial protection against T. gondii. In this study, we constructed a multi-antigenic DNA vaccine containing SAG1, ROP2 and GRA2, and evaluated its immune response. We used IL-12 as an adjuvant to enhance the immune response. We immunized BALB/c mice intramuscularly. After immunization, we evaluated the immune response using lymphocyte proliferation assay, cytokine and antibody measurements. The results showed that the group immunized with pcDNA3.1–SAG1–ROP2–GRA2 produced high Th1 immune response compared to other groups immunized with double-gene plasmid, empty plasmid or phosphate-buffered saline, respectively. Moreover, the co-immunization with IL-12 genes enhanced the immune response significantly and prolonged survival time. The current study showed that multi-antigenic DNA with IL-12 produced potent, effective and long-term protection against T. gondii challenge.     

Tobacco Etch Virus Protease: Crystal Structure of the Active Enzyme and Its Inactive Mutant

Tobacco Etch Virus Protease (TEV protease) is widely used as a tool for separation of recombinant target proteins from their fusion partners. The crystal structures of two mutants of TEV protease, the active autolysis-resistant mutant TEV-S219D in complex with the proteolysis product, and the inactive mutant TEV-C151A in complex with a substrate, have been determined at 1.8 and 2.2 Å resolution, respectively. The active sites of both mutants, including their oxyanion holes, have identical structures. The C-terminal residues 217–221 of the enzyme are involved in formation of the binding pockets S ₃–S ₆. This indicates that the autolysis of the peptide bond Met218–Ser219 exerts a strong effect on the fine-tuning of the substrate in the enzyme active site, which results in a considerable decrease in the enzymatic activity.     

Molecular analyses of Toxoplasma gondii calmodulin-like domain protein kinase isoform 3

Ca²⁺ signaling is thought to play an important role in Toxoplasma gondii motility, including invasion of and egress from host cells. Recently, it has been reported that phosphorylation of the glideosome apparatus components of T. gondii occurs during invasion. To elucidate the role of T. gondii calmodulin-like domain protein kinase in the signaling pathway that bridges Ca²⁺ stimulation and motility, we characterized T. gondii calmodulin-like domain protein kinase isoform 3 (TgCDPKif3). TgCDPKif3 is homologous to Plasmodium falciparum calcium-dependent protein kinase 1, which has been reported to phosphorylate P. falciparum glideosome components. TgCDPKif3 was purified as a fusion protein that was labeled with [γ-³²P]ATP, and the label was subsequently removed by phosphatase treatment. Phosphorylation was eliminated when the putative catalytic lysine residue of TgCDPKif3 was replaced with alanine. TgCDPKif3 phosphorylated Histone II_AS as a representative substrate in a Ca²⁺-dependent manner at a high Ca²⁺ concentration. TgCDPKif3 was localized to the apical ends of tachyzoites. TgCDPKif3 showed the translocation between intra- and extracellular tachyzoites. TgCDPKif3 could phosphorylate T. gondii aldolase 1 (TgALD1) in vitro. The interaction between TgCDPKif3 and TgALD1 was confirmed by the co-immunoprecipitation assay in mammal cells. We suggested that TgCDPKif3 could participate in the motility of T. gondii through the phosphorylation of glideosome complex member.     

Parasiticidal activity of human α-defensin-5 against <Emphasis Type="Italic">Toxoplasma gondii</Emphasis>

Human defensins play a fundamental role in the initiation of innate immune responses to some microbial pathogens. In this paper, we show that human α-defensin-5 displays a parasiticidal role against Toxoplasma gondii, the causative agent of toxoplasmosis. Exposure of the tachyzoite form of T. gondii to defensin induced aggregation and significantly reduced parasite viability in a concentration-dependent peptide. Pre-incubation of tachyzoites with human α-defensin-5 followed by exposure to a mouse embryonal cell line (NIH/3T3) significantly reduced T. gondii infection in these cells. Thus, human α-defensin-5 is an innate immune molecule that causes severe toxocity to T. gondii and plays an important role in reducing cellular infection. This is the first report showing that human α-defensin-5 causes aggregation, leading to Toxoplasma destruction.     

Diphenyl diselenide modulates nucleotidases,reducing inflammatory responses in the liver of <Emphasis Type="Italic">Toxoplasma gondii</Emphasis>-infected mice

The aim of this study was to verify the effect of diphenyl diselenide (PhSe)₂ on hepatic nucleotidases and on the concentration of purines in mice infected by Toxoplasma gondii. The animals were divided into four groups: Group A (uninfected), Group B (uninfected and treated with (PhSe)₂), Group C (infected), and Group D (infected and treated with (PhSe)₂). The inoculation (groups C and D) was performed with 50 cysts of T. gondii (ME-49 strain). Mice from groups B and D were treated with 5 μmol kg^?1 of (PhSe)₂. Liver tissue from infected mice showed less severe inflammation, elevated ATP/ADO ratio, elevated NTPDase, 5′nucleotidase, and ADA activities compared to the uninfected group (Group A; P < 0.05). However, infected and treated mice showed decreased ATP levels and elevated ADO levels, as well as higher NTPDase and 5′nucleotidase activities and decreased ADA activity in the hepatic tissue compared to the infected group (P < 0.05). Moreover, the (PhSe)₂ treatment of infected mice reduced the hepatic inflammation and showed an immunomodulatory effect on ectonucleotidases of hepatic lymphocytes, which it returned to basal levels. Therefore, chronic infection by T. gondii induces hepatic inflammation in mice, and it is possible that purine levels and nucleotidase activities in hepatic tissue are related to the pathogenesis of the infection in this tissue. The treatment with (PhSe)₂ was able to reverse the hepatic inflammation in mice chronically infected, possibly due to the modulation of purinergic enzymes that produce an anti-inflammatory profile through the purinergic system in the liver tissue.