apyrase and Chagas-Disease

Human populations are constantly plagued by hematophagous insects' bites, in particular the triatomine insects that are vectors of the Trypanosoma cruzi agent in Chagas disease. The pharmacologically-active molecules present in the salivary glands of hematophagous insects are injected into the human skin to initiate acquisition of blood meals. Sets of vasodilators, anti-platelet aggregators, anti-coagulants, immunogenic polypeptides, anesthetics, odorants, antibiotics, and detoxifying molecules have been disclosed with the aid of proteomics and recombinant cDNA techniques. These molecules can provide insights about the insect-pathogen-host interactions essential for understanding the physiopathology of the insect bite. The data and information presented in this review aim for the development of new drugs to prevent insect bites and the insect-transmitted endemic of Chagas disease.

Topics: Animals; Apyrase; Chagas Disease; Hemostasis; Host-Pathogen Interactions; Humans; Insect Bites and Stings; Salivary Glands; Salivary Proteins and Peptides; Triatoma; Vasodilator Agents

Distinct populations of Trypanosoma cruzi interact with mammalian cardiac muscle cells causing different inflammation patterns and low heart functionality. During T. cruzi infection, the extracellular ATP is hydrolyzed to tri- and/or diphosphate nucleotides, based on the infectivity, virulence, and regulation of the inflammatory response. T. cruzi carries out this hydrolysis through the T. cruzi ectonucleotidase, NTPDase-1 (TcNTPDase-1). This study aimed to evaluate the role of TcNTPDase-1 in culture rich in metacyclic trypomastigote forms (MT) and cell culture-derived trypomastigote forms (CT) from Colombiana (discrete typing unit - DTU I), VL-10 (DTU II), and CL (DTU VI) strains of T. cruzi. For this, we measured TcNTPDase-1 activity in suramin-treated and untreated parasites and infected J774 cells and C57BL/6 mice with suramin pre-treated parasites to assess parasitic and inflammatory cardiac profile in the acute phase of infection. Our data indicated a higher TcNTPDase-1 activity for ATP in culture rich in metacyclic trypomastigote forms from Colombiana strain in comparison to those from VL-10 and CL strains. The cell culture-derived trypomastigote forms from CL strain presented higher capacity to hydrolyze ATP than those from Colombiana and VL-10 strains. Suramin inhibited ATP hydrolysis in all studied parasite forms and strains. Suramin pre-treated parasites reduced J774 cell infection and increased nitrite production in vitro. In vivo studies showed a reduction of inflammatory infiltrate in the cardiac tissues of animals infected with cell culture-derived trypomastigote forms from suramin pre-treated Colombiana strain. In conclusion, TcNTPDase-1 activity in trypomastigotes forms drives part of the biological characteristics observed in distinct DTUs and may induce cardiac pathogenesis during T. cruzi infection.

Topics: Animals; Antigens, CD; Apyrase; Cell Line, Tumor; Chagas Disease; Mice; Mice, Inbred BALB C; Protozoan Proteins; Species Specificity; Trypanosoma cruzi; Virulence Factors

The aim of this study was to evaluate the activity of purinergic enzymes in lymphocytes and cardiac tissue of mice experimentally infected by Trypanosoma cruzi. Twelve female mice were used, divided into two groups (n = 6): uninfected and infected. On day 12 post-infection (PI), the animals were anesthetized and after euthanized, and samples were collected for analyses. Infected mice showed reduction in erythrocyte counts, hematocrit and hemoglobin concentration, as well as reduced number of total leukocytes in consequence of neutrophilia (P < 0.01). The number of monocytes increased in infected mice (P < 0.001), however the number of lymphocytes and eosinophils did not differ between groups (P > 0.05). The E-NTPDase (ATP and ADP substrate) and E-ADA activities in lymphocytes increased significantly in mice infected by T. cruzi (P < 0.01). In the heart, multiple pseudocysts containing amastigotes within cardiomyocytes were observed, as well as focally extensive severe necrosis associated with diffuse moderate to severe inflammatory infiltrate of lymphocytes. Although, the NTPDase activity (ATP and ADP substrate) in the cardiac homogenate did not differ between groups, a reduction on 5'-nucleotidase activity (P < 0.001) and an increase in the ADA activity in infected animals (P < 0.05) were observed. Thus, animals infected by T. cruzi experienced the disease, i.e., showed anemia, leucopenia, and heart lesions. Associated with this, purinergic enzymes showed altered activities, which might be related to the modulation of the inflammatory response.

Topics: 5'-Nucleotidase; Adenosine; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Antigens, CD; Apyrase; Chagas Disease; Disease Models, Animal; Female; Heart; Hematologic Tests; Hydrolysis; Lymphocytes; Mice; Myocardium; Myocytes, Cardiac; Parasitemia; Purines; Trypanosoma cruzi

Trypanosoma cruzi infection triggers a chronic inflammatory process in human host and purinergic system ecto-enzymes play an important role in modulating the inflammatory and immune responses. In this study, it was investigated ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase; EC 3.6.1.5; CD39) and ecto-adenosine deaminase (E-ADA; EC 3.5.4.4) activities in lymphocytes from patients with indeterminate form of Chagas' disease (IFCD). Twenty-five IFCD patients and 25 healthy subjects (control group) were selected. The peripheral lymphocytes were isolated and E-NTPDase and E-ADA activities were determined. Adenine nucleotides and adenosine levels were determined in serum by HPLC and the E-NTPDase1 expression in lymphocytes by Western blot analysis. E-NTPDase (ATP and ADP as substrates) and E-ADA (adenosine as substrate) activities were decreased in lymphocytes from IFCD patients (P<0.05 and P<0.01, respectively), while the E-NTPDase1 expression presented no changes in these patients. Serum ATP levels showed to be decreased (P<0.05) and both AMP (P<0.01) and adenosine (P<0.001) levels were increased in the IFCD group. The enzymatic alterations observed are in agreement with the immune response against T. cruzi infection in IFCD patients, since the decreased extracellular ATP and the increased adenosine levels trigger a Th2 anti-inflammatory response, which it is associated to adaptation of host to parasite, preventing clinical progress of disease.

Topics: Adenosine Deaminase; Animals; Antigens, CD; Apyrase; Case-Control Studies; Chagas Disease; Female; Gene Expression Regulation, Enzymologic; Humans; Lymphocytes; Male; Middle Aged

The protozoan Trypanosoma cruzi is the causative agent of Chagas disease. There are no vaccines or effective treatment, especially in the chronic phase when most patients are diagnosed. There is a clear necessity to develop new drugs and strategies for the control and treatment of Chagas disease. Recent papers have suggested the ecto-nucleotidases (from CD39 family) from pathogenic agents as important virulence factors. In this study we evaluated the influence of Ecto-Nucleoside-Triphosphate-Diphosphohydrolase (Ecto-NTPDase) activity on infectivity and virulence of T. cruzi using both in vivo and in vitro models.. We followed Ecto-NTPDase activities of Y strain infective forms (trypomastigotes) obtained during sequential sub-cultivation in mammalian cells. ATPase/ADPase activity ratios of cell-derived trypomastigotes decreased 3- to 6-fold and infectivity was substantially reduced during sequential sub-cultivation. Surprisingly, at third to fourth passages most of the cell-derived trypomastigotes could not penetrate mammalian cells and had differentiated into amastigote-like parasites that exhibited 3- to 4-fold lower levels of Ecto-NTPDase activities. To evidence the participation of T. cruzi Ecto-NTPDase1 in the infective process, we evaluated the effect of known Ecto-ATPDase inhibitors (ARL 67156, Gadolinium and Suramin), or anti-NTPDase-1 polyclonal antiserum on ATPase and ADPase hydrolytic activities in recombinant T. cruzi NTPDase-1 and in live trypomastigotes. All tests showed a partial inhibition of Ecto-ATPDase activities and a marked inhibition of trypomastigotes infectivity. Mice infections with Ecto-NTPDase-inhibited trypomastigotes produced lower levels of parasitemia and higher host survival than with non-inhibited control parasites.. Our results suggest that Ecto-ATPDases act as facilitators of infection and virulence in vitro and in vivo and emerge as target candidates in chemotherapy of Chagas disease.

Topics: Animals; Antigens, CD; Apyrase; Chagas Disease; Chlorocebus aethiops; Mice; Trypanosoma cruzi; Vero Cells; Virulence; Virulence Factors

Evolutionary and closer structural relationships are demonstrated by phylogenetic analysis, peptide prediction and molecular modelling between Solanum tuberosum apyrase, Schistosoma mansoni SmATPase 2 and Leishmania braziliensis NDPase. Specific protein domains are suggested to be potentially involved in the immune response, and also seem to be conserved during host and parasite co-evolution. Significant IgG antibody reactivity was observed in sera from patients with American cutaneous leishmaniasis (ACL) and schistosomiasis using potato apyrase as antigen in ELISA. S. mansoni adult worm or egg, L. braziliensis promastigote (Lb) and Trypanosoma cruzi epimastigote (EPI) have ATP diphosphohydrolases, and antigenic preparations of them were evaluated. In ACL patients, IgG seropositivity was about 43% and 90% for Lb and potato apyrase, respectively, while IgM was lower (40%) or IgG (100%) seropositivity for both soluble egg (SEA) and adult worm (SWAP) antigens was higher than that found for potato apyrase (IgM=10%; IgG=39%). In Chagas disease, IgG seropositivity for EPI and potato apyrase was 97% and 17%, respectively, while the IgM was low (3%) for both antigens. The study of the conserved domains from both parasite proteins and potato apyrase could lead to the development of new drug targets or molecular markers.

Topics: Amino Acid Sequence; Animals; Antibodies, Protozoan; Apyrase; Chagas Disease; Conserved Sequence; Epitope Mapping; Humans; Leishmania braziliensis; Leishmaniasis, Cutaneous; Molecular Sequence Data; Parasites; Phylogeny; Protein Structure, Tertiary; Schistosoma mansoni; Schistosomiasis; Sequence Alignment; Solanum tuberosum

Triatoma infestans is a hemiptera, vector of Chagas' disease that feeds exclusively on vertebrate blood in all life stages. Hematophagous insects' salivary glands (SG) produce potent pharmacological compounds that counteract host hemostasis, including anticlotting, antiplatelet, and vasodilatory molecules. To obtain a further insight into the salivary biochemical and pharmacological complexity of this insect, a cDNA library from its SG was randomly sequenced. Also, salivary proteins were submitted to two-dimensional gel (2D-gel) electrophoresis followed by MS analysis. We present the analysis of a set of 1534 (SG) cDNA sequences, 645 of which coded for proteins of a putative secretory nature. Most salivary proteins described as lipocalins matched peptide sequences obtained from proteomic results.

Topics: Amino Acid Sequence; Animals; Apyrase; Capsid Proteins; Chagas Disease; Defensins; DNA Transposable Elements; Gene Expression Profiling; Gene Library; Host-Parasite Interactions; Inositol Phosphates; Lipocalins; Molecular Sequence Data; Nymph; Proteomics; Receptors, Odorant; Saliva; Salivary Glands; Salivary Proteins and Peptides; Sequence Analysis, DNA; Serpins; Triatoma

Apyrases are nucleoside triphosphate-diphosphohydrolases that remove Pi from ATP and ADP. The blood feeding reduviid Triatoma infestans, which transmits the Trypanosoma cruzi agent of Chagas disease to animals and man, presents in its salivary glands five apyrases with molecular masses of 88, 82, 79, 68 and 67 kDa. These triatomine apyrases have been associated with the prevention of ADP induced platelet aggregation in the host. Here we provide biochemical data showing that these apyrases are stored in the lumen of the salivary gland D1 pairs, and that about one half of the pool of the enzyme is consumed during feeding. After the feeding recovery of apyrases to maximal activity level takes days, thus suggesting de novo protein synthesis. This hypothesis is supported by quantitative RT-PCR analysis which shows an upregulation of the 79 kDa apyrase mRNA level after feeding.

Topics: Animals; Apyrase; Base Sequence; Blood; Chagas Disease; DNA; Eating; Gene Expression; Humans; Insect Vectors; Kinetics; Molecular Weight; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Salivary Glands; Triatoma; Trypanosoma cruzi