Leishmania infantum amastigote nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1): Its inhibition as a new insight into mode of action of pentamidine.

Nucleoside triphosphate diphosphohydrolase (NTPDase) 1 from intracellular amastigotes of Leishmania infantum-infected macrophage was identified by immunocytochemistry and confocal laser scanning microscopy using antibodies that specifically recognize its B-domain. This enzyme was previously characterized in Leishmania promastigote form, and here it is shown to be susceptible to pentamidine isethionate (PEN). In initial assays, this antileishmanial compound (100 µM) reduced 60% phosphohydrolytic activity of promastigotes preparation. An active NTPDase 1 was then isolated by non-denaturing gel electrophoresis, and PEN (10 µM) inhibited 74% and 35% of the ATPase and ADPase activities, respectively, of this pure protein. In addition, PEN 0.1-1 µM inhibited 56% potato apyrase activity, a plant protein that shares high identity with Leishmania NTPDase 1. In contrast, amphotericin B, fluconazole, ketoconazole or allopurinol did not significantly affect phosphohydrolytic activity of either promastigotes preparation or potato apyrase. This work suggests amastigote NTPDase 1 as a new molecular target, and inhibition of its catalytic activity by pentamidine can be part of the mode of action of this drug contributing with the knowledge of its antileishmanial effect.

Aminoquinoline compounds: Effect of 7-chloro-4-quinolinylhydrazone derivatives against Leishmania amazonensis.

In this study, we have investigated the antileishmanial activity of ten 7-chloro-4-quinolinylhydrazone derivatives. Among the compounds tested, compounds 2a and 2j presented activity against promastigotes (IC50 values of 52.5 and 21.1 µM, respectively) and compounds 2a and 2c were active against intracellular amastigotes (IC50 of 8.1 and 15.6 µM, respectively) of Leishmania amazonensis. The majority of compounds did not show toxicity against murine macrophages. Compound 2a exhibited low cytotoxicity to human erythrocytes and induced an oxidative imbalance in promastigote forms, reflected by an increase in the formation of reactive oxygen species (ROS) and a reduction of mitochondrial membrane potential. No alteration in the plasma membrane integrity of parasites was observed. Taken together, these results suggest that compound 2a is a selective antileishmanial agent, and preliminary observations suggest that its effects appear to be mediated by mitochondrial dysfunction.

Leishmania (Viannia) braziliensis nucleoside triphosphate diphosphohydrolase (NTPDase 1): localization and in vitro inhibition of promastigotes growth by polyclonal antibodies.

Nucleoside triphosphate diphosphohydrolase (NTPDase) activity was recently characterized in Leishmania (Viannia) braziliensis promastigotes (Lb), and an antigenic conserved domain (r82-121) from the specific NTPDase 1 isoform was identified. In this work, mouse polyclonal antibodies produced against two synthetic peptides derived from this domain (LbB1LJ, r82-103; LbB2LJ, r102-121) were used. The anti-LbB1LJ or anti-LbB2LJ antibodies were immobilized on protein A-sepharose and immunoprecipitated the NTPDase 1 of 48 kDa and depleted approximately 40% of the phosphohydrolytic activity from detergent-homogenized Lb preparation. Ultrastructural immunocytochemical microscopy identified the NTPDase 1 on the parasite surface and in its subcellular cytoplasmic vesicles, mitochondria, kinetoplast and nucleus. The ATPase and ADPase activities of detergent-homogenized Lb preparation were partially inhibited by anti-LbB1LJ antibody (43-79%), which was more effective than that inhibition (18-47%) by anti-LbB2LJ antibody. In addition, the immune serum anti-LbB1LJ (67%) or anti-LbB2LJ (33%) was cytotoxic, significantly reducing the promastigotes growth in vitro. The results appoint the conserved domain from the L. braziliensis NTPDase as an important target for inhibitor design and the potential application of these biomolecules in experimental protocols of disease control.

Immunostimulatory property of a synthetic peptide belonging to the soluble ATP diphosphohydrolase isoform (SmATPDase 2) and immunolocalisation of this protein in the Schistosoma mansoni egg.

A peptide (SmB2LJ; r175-194) that belongs to a conserved domain from Schistosoma mansoni SmATPDase 2 and is shared with potato apyrase, as predicted by in silico analysis as antigenic, was synthesised and its immunostimulatory property was analysed. When inoculated in BALB/c mice, this peptide induced high levels of SmB2LJ-specific IgG1 and IgG2a subtypes, as detected by enzyme linked immunosorbent assay. In addition, dot blots were found to be positive for immune sera against potato apyrase and SmB2LJ. These results suggest that the conserved domain r175-194 from the S. mansoni SmATPDase 2 is antigenic. Western blots were performed and the anti-SmB2LJ antibody recognised in adult worm (soluble worm antigen preparation) or soluble egg antigen antigenic preparations two bands of approximately 63 and 55 kDa, molecular masses similar to those predicted for adult worm SmATPDase 2. This finding strongly suggests the expression of this same isoform in S. mansoni eggs. To assess localisation of SmATPDase 2, confocal fluorescence microscopy was performed using cryostat sections of infected mouse liver and polyclonal antiserum against SmB2LJ. Positive reactions were identified on the external surface from the miracidium in von Lichtenberg's envelope and, in the outer side of the egg-shell, showing that this soluble isoform is secreted from the S. mansoni eggs.

Detection of IgG1 and IgG4 subtypes reactive against potato apyrase in schistosomiasis patients.

In this paper, we showed for the first time that the conserved domains within Schistosoma mansoni ATP diphosphohydrolase isoforms, shared with potato apyrase, possess epitopes for the IgG1 and IgG4 subtypes, as 24 (80%) of the 30 schistosomiasis patients were seropositive for this vegetable protein. The analyses for each patient cured (n = 14) after treatment (AT) with praziquantel revealed variable IgG1 and IgG4 reactivity against potato apyrase. Different antigenic epitopes shared between the vegetable and parasite proteins could be involved in susceptibility or resistance to S. mansoni AT with praziquantel and these possibilities should be explored.

Screening of plant derived chalcones on the inhibition of potato apyrase: Potential protein biotechnological applications in health.

NTPDases (EC 3.6.1.5) are enzymes belonging to a protein family which have as a common feature the ability to hydrolyze di- and triphosphate nucleotides (ADP and ATP) to monophosphate nucleosides (AMP) in the presence of Ca+2 and Mg+. The potato apyrase has been the first protein of the NTPDase family to be purified. In mammals, these enzymes are involved in physiologic and sick processes as thromboregulation, inflammatory and immunologic responses. In this study, we investigated the in vitro potential of synthetic chalcones on the inhibition of potato apyrase purified from Solanum tuberosum. The protein was purified with high grade purity and its identity was confirmed by electrophoresis, western blot, and LC-MS/MS. Five out of the eight chemically synthetized chalcones analyzed in this study showed significant inhibition of the apyrase activity. The compound with the best rate of inhibition of ATP hydrolytic activity was able to promote 54% inhibition with a concentration of 3.125 µM. Ticlopidine, used as an inhibition drug control, was able to promote inhibitions around 50% of the activity (IC50 = 2.167 µM). Our results with the potato apyrase inhibition with the synthetic chalcones suggest that these compounds may use as potential lead candidates for the treatment of some diseases associated with nucleotides.

Knocking Down TcNTPDase-1 Gene Reduces in vitro Infectivity of Trypanosoma cruzi.

Ecto-Nucleoside Triphosphate Diphosphohydrolases are enzymes that hydrolyze tri- and/or diphosphate nucleosides. Evidences pointed out to their participation in Trypanosoma cruzi virulence, infectivity, and purine acquisition. In this study, recombinant T. cruzi knocking out or overexpressing the TcNTPDase-1 gene were built, and the role of TcNTPDase-1 in the in vitro interaction with VERO cells was investigated. Results show that epimastigote forms of hemi-knockout parasites showed about 50% lower level of TcNTPDase-1 gene expression when compared to the wild type, while the T. cruzi overexpressing this gene reach 20 times higher gene expression. In trypomastigote forms, the same decreasing in TcNTPDase-1 gene expression was observed to the hemi-knockout parasites. The in vitro infection assays showed a reduction to 51.6 and 59.9% at the adhesion and to 25.2 and 26.4% at the endocytic indexes to the parasites knockout to one or other allele (Hygro and Neo hemi-knockouts), respectively. In contrast, the infection assays with T. cruzi overexpressing TcNTPDase-1 from the WT or Neo hemi-knockout parasites showed an opposite result, with the increasing to 287.7 and 271.1% at the adhesion and to 220.4 and 186.7% at the endocytic indexes, respectively. The parasitic load estimated in infected VERO cells by quantitative real time PCR corroborated these findings. Taken together, the partial silencing and overexpression of the TcNTPDase-1 gene generated viable parasites with low and high infectivity rates, respectively, corroborating that the enzyme encoded for this gene plays an important role to the T. cruzi infectivity.

IgE antibodies from schistosomiasis patients to recognize epitopes in potato apyrase.

INTRODUCTION: High percentages of structural identity and cross-immunoreactivity have been reported between potato apyrase and Schistosoma mansoni ATP diphosphohydrolase (SmATPDases) isoforms, showing the existence of particular epitopes shared between these proteins. METHODS: Potato apyrase was employed using ELISA, western blot, and mouse immunization methods to verify IgE reactivity. RESULTS: Most of the schistosomiasis patient's (75%) serum was seropositive for potato apyrase and this protein was recognized using western blotting, suggesting that parasite and plant proteins share IgE-binding epitopes. C57BL/6 mice immunized with potato apyrase showed increased IgE antibody production. CONCLUSIONS: Potato apyrase and SmATPDases have IgE-binding epitopes.

Leishmania infantum nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1) B-domain: Antibody antiproliferative effect on the promastigotes and IgG subclass responses in canine visceral leishmaniasis.

A nucleoside triphosphate diphosphohydrolase-1 (NTPDase 1) was identified on the surface, flagellum and kinetoplast from L. infantum promastigotes by immunocytochemistry and confocal laser scanning microscopy, using immune sera that recognized specifically the B domain of NTPDase 1 and produced against synthetic peptides (LbB1LJ and LbB2LJ) derived from this domain. The polyclonal antibodies had effective antileishmanial effect, reducing significantly in vitro promastigotes growth (21-25%), an antiproliferative effect also demonstrated by immune sera produced against recombinant r-pot B domain, and two other synthetic peptides (potB1LJ and potB2LJ). In addition, using these biomolecules in ELISA technique, IgG1 and IgG2 subclasses reactivities of either healthy dogs or infected by L. infantum and classified clinically as asymptomatic, oligosymptomatic and symptomatic were tested. Analysis of distinct IgG1 and IgG2 seropositivities patterns suggested antibody subclasses binding epitopes along B domain for protection against infection, indicating this domain as a new tool for prophylactic and immunotherapeutic investigations.

The alkylaminoalkanethiosulfuric acids exhibit in-vitro antileishmanial activity against Leishmania (Viannia) braziliensis: a new perspective for use of these schistosomicidal agents.

The alkylaminoalkanethiosulfuric acids (AAATs) are amphipathic compounds effective against experimental schistosomiasis, of low toxicity, elevated bioavailability after a single oral dose and prompt tissue absorption. OBJECTIVES: To explore the in-vitro antileishmanial potential of AAATs using five compounds of this series against Leishmania (Viannia) braziliensis. METHODS: Their effects on promastigotes and axenic amastigotes, and cytotoxicity to macrophages were tested by the MTT method, and on Leishmania-infected macrophages by Giemsa stain. Effects on the mitochondrial membrane potential of promastigotes and axenic amastigotes and DNA of intracellular amastigotes were tested using JC-1 and TUNEL assays, respectively. KEY FINDINGS: The 2-(isopropylamino)-1-octanethiosulfuric acid (I) and 2-(sec-butylamino)-1-octanethiosulfuric acid (II) exhibit activity against both promastigotes and intracellular amastigotes (IC50 25-35 µm), being more toxic to intracellular parasites than to the host cell. Compound I induced a loss of viability of axenic amastigotes, significantly reduced (30%) the mitochondrial membrane potential of both promastigotes and axenic amastigotes and promoted selective DNA fragmentation of the nucleus and kinetoplast of intracellular amastigotes. CONCLUSIONS: In this previously unpublished study of trypanosomatids, it is shown that AAATs could also exhibit selective antileishmanial activity, a new possibility to be investigated in oral treatment of leishmaniasis.

A new series of schistosomicide drugs, the alkylaminoalkanethiosulfuric acids, partially inhibit the activity of Schistosoma mansoni ATP diphosphohydrolase.

The effects of the alkylaminoalkanethiosulfuric acids (AAATs), new schistosomicidal drugs, on Schistosoma mansoni ATP diphosphohydrolase isoforms, members of the NTPDase family, were analyzed. Pre-incubation of worm adult tegument with AAATs derivatives, with small apolar alkyl groups and an apolar alkane portion of 6 or 8 carbon atoms linked to the amino group, inhibited ATPase activity with a Ki 100-1000 microM. Little inhibition (20%) was observed when ADP was the substrate. The 2-[(tert-butyl)amino]-1-ethanethiosulfuric acid (100 microM) which has a less lipophilic structure, inhibited 28% ATPase and 12% ADPase activities, suggesting that the lipophilicity, although important, is not the only requisite for enzyme activity inhibition. The N-(sec-butyl)-2-bromo-1-octanaminium bromide, which contains a bromide atom instead of thiosulphate, inhibited <10% of the enzyme activity, suggesting the involvement of cysteine residue(s) from S. mansoni ATP diphosphohydrolase isoforms in a mixed disulfide formation. Treatment of parasite tegument with 5 mM iodoacetamide or 1 mM dithiothreitol protected ATPase and ADPase activities against inhibition by AAATs, corroborating the participation of disulfide interchange in the AAATs mechanism. Since S. mansoni ATP diphosphohydrolase isoforms and potato apyrase share structural similarities, the latter enzyme was also tested. ADPase activity from potato apyrase was inhibited by 55%, showing a higher sensitivity to 1 mM AAATs than that shown by ADPase activity from the tegument, while the ATPase activities from both samples showed similar inhibition levels. Furthermore, sulfhydryl reagents protected potato apyrase activity. Therefore, it is possible that both soluble S. mansoni ATP diphosphohydrolase and membrane-associated isoforms are targets for the AAATs.

IgE antibodies from schistosomiasis patients to recognize epitopes in potato apyrase

Abstract INTRODUCTION: High percentages of structural identity and cross-immunoreactivity have been reported between potato apyrase and Schistosoma mansoni ATP diphosphohydrolase (SmATPDases) isoforms, showing the existence of particular epitopes shared between these proteins. METHODS: Potato apyrase was employed using ELISA, western blot, and mouse immunization methods to verify IgE reactivity. RESULTS: Most of the schistosomiasis patient's (75%) serum was seropositive for potato apyrase and this protein was recognized using western blotting, suggesting that parasite and plant proteins share IgE-binding epitopes. C57BL/6 mice immunized with potato apyrase showed increased IgE antibody production. CONCLUSIONS: Potato apyrase and SmATPDases have IgE-binding epitopes.

Chromatographic fingerprint analysis and effects of the medicinal plant species Mitracarpus frigidus on adult Schistosoma mansoni worms.

The aims of this work were to evaluate the in vitro and in vivo schistosomicidal properties of the methanolic extract of the aerial parts of Mitracarpus frigidus (MFM) and to determine its HPLC profile. For the in vitro experiment, four pairs of adult worms, obtained from infected mice, were exposed to different concentrations of MFM (100 to 400 µg/mL) for 24 and 48 h and analyzed under an inverted microscope. For the in vivo experiment, mice were inoculated with cercariae and, 20 days after infection, MFM (100 and 300 mg/kg) was administered orally for the following 25 days. Mice were euthanized after 60 days. MFM showed in vitro schistosomicidal activity, exhibiting the opening of the gynaecophoral canal of some male schistosomes, the presence of contorted muscles, vesicles, and the darkening of the paired worms skin. In vivo experiments showed that MFM treatments significantly reduced total worm count, as praziquantel, showing a decrease in liver and spleen weight. Also, a significant reduction in granuloma density was observed. MFM treatment did not cause alterations in the liver function of either infected or noninfected mice. The HPLC chromatogram profile showed the presence of kaempferol-O-rutinoside, rutin, kaempferol, psychorubrin, and ursolic acid.

An antigenic domain within a catalytically active Leishmania infantum nucleoside triphosphate diphosphohydrolase (NTPDase 1) is a target of inhibitory antibodies.

We identified a shared B domain within nucleoside triphosphate diphosphohydrolases (NTPDases) of plants and parasites. Now, an NTPDase activity not affected by inhibitors of adenylate kinase and ATPases was detected in Leishmania infantum promastigotes. By non-denaturing gel electrophoresis of detergent-homogenized promastigote preparation, an active band hydrolyzing nucleosides di- and triphosphate was visualized and, following SDS-PAGE and silver staining was identified as a single polypeptide of 50kDa. By Western blots, it was recognized by immune sera raised against potato apyrase (SA), r-pot B domain (SB), a recombinant polypeptide derived from the potato apyrase, and LbB1LJ (SC) or LbB2LJ (SD), synthetic peptides derived from the Leishmania NTPDase 1, and by serum samples from dogs with visceral leishmaniasis, identifying the antigenic L. infantum NTPDase 1 and, also, its conserved B domain (r83-122). By immunoprecipitation assays and Western blots, immune sera SA and SB identified the catalytically active NTPDase 1 in promastigote preparation. In addition, the immune sera SB (44%) and SC or SD (87-99%) inhibited its activity, suggesting a direct effect on the B domain. By ELISA, 37%, 45% or 50% of 38 infected dogs were seropositive for r-pot B domain, LbB1LJ and LbB2LJ, respectively, confirming the B domain antigenicity.

Antimicrobial activity of recombinant Pg-AMP1, a glycine-rich peptide from guava seeds.

Antimicrobial peptides (AMPs) are compounds that act in a wide range of physiological defensive mechanisms developed to counteract bacteria, fungi, parasites and viruses. These molecules have become increasingly important as a consequence of remarkable microorganism resistance to common antibiotics. This report shows Escherichia coli expressing the recombinant antimicrobial peptide Pg-AMP1 previously isolated from Psidium guajava seeds. The deduced Pg-AMP1 open reading frame consists in a 168 bp long plus methionine also containing a His6 tag, encoding a predicted 62 amino acid residue peptide with related molecular mass calculated to be 6.98 kDa as a monomer and 13.96 kDa at the dimer form. The recombinant Pg-AMP1 peptide showed inhibitory activity against multiple Gram-negative (E. coli, Klebsiella pneumonia and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus and Staphylococcus epidermides) bacteria. Moreover, theoretical structure analyses were performed in order to understand the functional differences between natural and recombinant Pg-AMP1 forms. Data here reported suggest that Pg-AMP1 is a promising peptide to be used as a biotechnological tool for control of human infectious diseases.

Occurrence of a conserved domain in ATP diphosphohydrolases from pathogenic organisms associated to antigenicity in human parasitic diseases.

A polypeptide (r78-117) belonging to the potato apyrase was identified as a conserved domain shared with apyrase-like proteins from distinct pathogenic organisms, and was obtained as a 6xHis tag polypeptide (r-Domain B). By ELISA, high IgG, and IgG1 and IgG2a subtypes levels were detected in BALB/c mice pre-inoculated with r-Domain B. In Schistosoma mansoni adult worm or Leishmania (V.) braziliensis promastigote preparation, anti-r-Domain B antibodies inhibit 22-72% of the phosphohydrolytic activities and when immobilized on Protein A-Sepharose immunoprecipitate 42-91% of them. Western blots of the immunoprecipitated resin-antibody-antigen complexes identified bands of mw similar to those predicted for parasite proteins. Total IgG and subclasses of patients with leishmaniasis or schistosomiasis exhibited cross-immunoreactivity with r-Domain B. Therefore, the domain B within both S. mansoni SmATPDase 2 (r156-195) and L. (V.) braziliensis NDPase (r83-122) are potentially involved in the host immune response, and also seem to be conserved during host and parasites co-evolution.

Immunostimulatory property of a synthetic peptide belonging to the soluble ATP diphosphohydro-lase isoform (SmATPDase 2) and immunolocalisation of this protein in the Schistosoma mansoni egg

A peptide (SmB2LJ; r175-194) that belongs to a conserved domain from Schistosoma mansoni SmATPDase 2 and is shared with potato apyrase, as predicted by in silico analysis as antigenic, was synthesised and its immunostimulatory property was analysed. When inoculated in BALB/c mice, this peptide induced high levels of SmB2LJ-specific IgG1 and IgG2a subtypes, as detected by enzyme linked immunosorbent assay. In addition, dot blots were found to be positive for immune sera against potato apyrase and SmB2LJ. These results suggest that the conserved domain r175-194 from the S. mansoni SmATPDase 2 is antigenic. Western blots were performed and the anti-SmB2LJ antibody recognised in adult worm (soluble worm antigen preparation) or soluble egg antigen antigenic preparations two bands of approximately 63 and 55 kDa, molecular masses similar to those predicted for adult worm SmATPDase 2. This finding strongly suggests the expression of this same isoform in S. mansoni eggs. To assess localisation of SmATPDase 2, confocal fluorescence microscopy was performed using cryostat sections of infected mouse liver and polyclonal antiserum against SmB2LJ. Positive reactions were identified on the external surface from the miracidium in von Lichtenberg's envelope and, in the outer side of the egg-shell, showing that this soluble isoform is secreted from the S. mansoni eggs.

Detection of IgG1 and IgG4 subtypes reactive against potato apyrase in schistosomiasis patients

In this paper, we showed for the first time that the conserved domains within Schistosoma mansoni ATP diphosphohydrolase isoforms, shared with potato apyrase, possess epitopes for the IgG1 and IgG4 subtypes, as 24 (80 percent) of the 30 schistosomiasis patients were seropositive for this vegetable protein. The analyses for each patient cured (n = 14) after treatment (AT) with praziquantel revealed variable IgG1 and IgG4 reactivity against potato apyrase. Different antigenic epitopes shared between the vegetable and parasite proteins could be involved in susceptibility or resistance to S. mansoni AT with praziquantel and these possibilities should be explored.

ATP-Difosfohidrolase do tegumento do schistosoma mansoni: caracterização e localização / ATP-diphosphohydrolase from Schistosoma mansoni tegument: characterization and localization

Nesta tese caracterizamos uma nova enzima na superfície do tegumento do Schistosma mansoni, uma ATP difosfohidrolase. Controles apropriados mostraram que as atividades ATPásica, ADPásica, pirofosfatásica e adenilato quinásica possivelmente contaminantes não são significativamente importantes. Foi mostrado também dependência para íons divalentes e um provável mecanismo de hidrólise sequencial. Experimentos anticorpos com sugerem que sua massa molecular esteja em torno de 50 KDa. É uma ecto-enzima, foi o que comprovado citoquímica por em microscopia eletrônica e pela capacidade do esquistossomo de hidrolisar ATP ou ADP adicionados no lado externo de vermes intactos. Parece ser sintetizada pelo próprio verme, já que esquistossômulos obtidos in vitro apresentaram atividades ATPásica ADPásica e nas condições mesmas usadas para medidas tegumento de vermes adultos. Esta ecto-enzima pode estar envolvida nos mecanismos de escape do parasita ao sistema hemostático e/ou imune do hospedeiro, bloqueando os sistemas que usam ADP e/ou ATP como intermediários.