Heterologous expression and biochemical characterization of the recombinant nucleoside triphosphate diphosphohydrolase 2 (LbNTPDase2) from Leishmania (Viannia) braziliensis.

Leishmania braziliensis is a pathogenic protozoan parasite that causes American Tegumentary Leishmaniasis (ATL), an important tropical neglected disease. ENTPDases are nucleotidases that hydrolyze intracellular and/or extracellular nucleotides. ENTPDases are known as regulators of purinergic signalling induced by extracellular nucleotides. Leishmania species have two isoforms of ENTPDase, and, particularly, ENTPDase2 seems to be involved in infectivity and virulence. In this study, we conducted the heterologous expression and biochemical characterization of the recombinant ENTPDase2 of L. braziliensis (rLbNTPDase2). Our results show that this enzyme is a canonical ENTPDase with apyrase activity, capable of hydrolysing triphosphate and diphosphate nucleotides, and it is dependent on divalent cations (calcium or magnesium). Substrate specificity was characterized as UDP>GDP>ADP>GTP>ATP=UTP. The enzyme showed optimal activity at a neutral to basic pH and was partially inhibited by suramin and DIDS. Furthermore, the low apparent Km for ADP suggests that the enzyme may play a role in adenosine-mediated signalling. The biochemical characterization of this enzyme can open new avenues for using LbNTPDase2 as a drug target.

Urea, salts, and Tween 20 influence on adsorption of IgG and Leishmania rNTPDase2 to nitrocellulose.

Lateral flow immunochromatography is a widely used technique for immunological assays. Construction of test and control lines is mostly done by antigen adsorption to nitrocellulose membranes, a process not fully understood. This study aimed to evaluate the influence of urea, salts, and Tween 20, on adsorption. The performance of canine IgG in water and in buffer containing urea and salts (pH 8.3) were compared to observe if the interferents would lead to protein stripping when challenged with increasing concentrations of Tween 20 in the lateral flow buffer. Immobilization of the rLiNTPDase2, an antigen for Canine Leishmaniasis diagnosis, was evaluated and compared to the rLbNTPDase2 by the same method. There were no differences between adsorption coefficients of IgG in water and in buffer, but high salt and urea concentrations seems to stabilize and enhance IgG immobilization. Adsorption performance between canine IgG and rNTPDases had different patterns, but was highly similar between rNTPDases, indicating that protein identity may have an important role. Also, low concentrations of Tween 20 in the flow solution may aid the maintenance of rNTPDase2 on the strips. Our results bring insights about protein adsorption and perspectives about the influence of urea, salts and Tween 20 on this process.

High-resolution mapping of linear epitopes from LiNTPDase2: Advancing leishmaniasis detection using optimized protein and peptide antigens.

Visceral Leishmaniasis, caused by Leishmania infantum, is a tropical neglected disease and the most dangerous form of Leishmaniasis. It occurs zoonotically, with domestic transmission posing risks to humans as dogs have high susceptibility and are natural reservoirs of the parasite. Given their epidemiological role, improvements are needed in diagnosing Canine Visceral Leishmaniasis (CVL). Thus, we mapped linear epitopes from the rLiNTPDase2 antigen through peptide microarray and identified six positive epitopes. Validation through peptide ELISA revealed three promising peptides with accuracies of 78.6%, 85.92%, and 79.59%. Their combination yielded 97.58% accuracy. Negative epitopes were also found, which interacted with CVL-negative and Chagas Disease positive samples. Their removal from the rLiNTPDase2 sequence resulted in the rNT2.neg, which obtained enhanced specificity over rLiNTPDase2. The rNT2.neg validation achieved 87.50% sensitivity, 90.55% specificity, and 93.5% accuracy within 127 CVL-positive and 96 CVL-negative samples. Therefore, three peptides and rNT2.neg show significant promise for CVL diagnosis.

ENTPDases from Pathogenic Trypanosomatids and Purinergic Signaling: Shedding Light towards Biotechnological Applications.

ENTPDases are enzymes known for hydrolyzing extracellular nucleotides and playing an essential role in controlling the nucleotide signaling via nucleotide/purinergic receptors P2. Moreover, ENTPDases, together with Ecto-5´-nucleotidase activity, affect the adenosine signaling via P1 receptors. These signals control many biological processes, including the immune system. In this context, ATP is considered as a trigger to inflammatory signaling, while adenosine (Ado) induces anti-inflammatory response. The trypanosomatids Leishmania and Trypanosoma cruzi, pathogenic agents of Leishmaniasis and Chagas Disease, respectively, have their own ENTPDases named "TpENTPDases," which can affect the nucleotide signaling, adhesion and infection, in order to favor the parasite. Besides, TpENTPDases are essential for the parasite nutrition, since the Purine De Novo synthesis pathway is absent in them, which makes these pathogens dependent on the intake of purines and nucleopurines for the Salvage Pathway, in which TpENTPDases also take place. Here, we review information regarding TpNTPDases, including their known biological roles and their effect on the purinergic signaling. We also highlight the roles of these enzymes in parasite infection and their biotechnological applications, while pointing to future developments.

High Performance of ELISA test using recombinant rLiNTPDase2 from Leishmania infantum: a Phase II diagnosis of Canine Visceral Leishmaniasis.

Canine visceral leishmaniasis (CVL) has been the theme of several studies given the importance of dog as natural reservoir of the pathogen Leishmania infantum in endemic regions and its role on dissemination of CVL and human visceral Lesihmaniasis (VL). The current immunodiagnosis of CVL has limitations concerning accuracy, specificity and sensitivity. Therefore, improvements are required. rLiNTPDase2 has been previously highlighted as a new recombinant antigen from L. infantum to the CVL diagnosis by ELISA assay (rLiNTPDase2-ELISA). In this study, we aimed to evaluate rLiNTPDase2-ELISA in a Phase II study with 651 dog sera samples, also comparing it with methodologies previously established and used in epidemiology surveillance in Brazil, an endemic country of CVL and VL. The rLiNTPDase2-ELISA using standard control sera showed high capability to distinguish between positive and negative sera, sensitivity of 92.6% and specificity of 88.5%. The test was reproductive and the kappa statistics judgement "substantial agreement". rLiNTPDase2-ELISA does not show cross-reactivity with ehrlichiosis-reagent sera. However, we verified 15.3% of cross-reactivity with Chagas disease-reagent sera. The performance of rLiNTPDase2-ELISA was evaluated using sera samples from vaccinated dogs (Leish-Tec®). The results showed high agreement with parasitological and PCR results (sensitivity of 100.0% and specificity of 91.7%). Furthermore, we compared the performance of rLiNTPDase2-ELISA in CVL-reagent sera samples from endemic areas, which were previously diagnosed using other tests for CVL: immunofluorescent (IFI-LVC-Bio-Manguinhos), IFI-LVC-Bio-Manguinhos coupled to ELISA (EIE-LVC-Bio-Manguinhos) and the Rapid Dual Path Platform® (TR-DPP®-Bio-Manguinhos) coupled to EIE-LVC-Bio-Manguinhos. rLiNTPDase2-ELISA showed high level of concordance with IFI-LVC-Bio-Manguinhos (88.6%) and with IFI-LVC-Bio-Manguinhos coupled to EIE-LVC-Bio-Manguinhos (82.9%) but not with TR-DPP® -Bio-Manguinhos coupled to EIE-LVC-Bio-Manguinhos (33.3%), which casts doubts on the effectiveness of this latest test. In addition, the rLiNTPDase2 antigen adsorbed in 96-well plate was stable enough to be used at least for three months. Taken together, our data confirmed, by Phase II study using hundreds samples, the good potential of rLiNTPDase2-ELISA to be used in the field as a new diagnostic assay for CVL.

Leishmania infantum ecto-nucleoside triphosphate diphosphohydrolase-2 is an apyrase involved in macrophage infection and expressed in infected dogs.

BACKGROUND: Visceral leishmaniasis is an important tropical disease, and Leishmania infantum chagasi (synonym of Leishmania infantum) is the main pathogenic agent of visceral leishmaniasis in the New World. Recently, ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) were identified as enablers of infection and virulence factors in many pathogens. Two putative E-NTPDases (∼70 kDa and ∼45 kDa) have been found in the L. infantum genome. Here, we studied the ∼45 kDa E-NTPDase from L. infantum chagasi to describe its natural occurrence, biochemical characteristics and influence on macrophage infection. METHODOLOGY/PRINCIPAL FINDINGS: We used live L. infantum chagasi to demonstrate its natural ecto-nucleotidase activity. We then isolated, cloned and expressed recombinant rLicNTPDase-2 in bacterial system. The recombinant rLicNTPDase-2 hydrolyzed a wide variety of triphosphate and diphosphate nucleotides (GTP> GDP  =  UDP> ADP> UTP  =  ATP) in the presence of calcium or magnesium. In addition, rLicNTPDase-2 showed stable activity over a pH range of 6.0 to 9.0 and was partially inhibited by ARL67156 and suramin. Microscopic analyses revealed the presence of this protein on cell surfaces, vesicles, flagellae, flagellar pockets, kinetoplasts, mitochondria and nuclei. The blockade of E-NTPDases using antibodies and competition led to lower levels of parasite adhesion and infection of macrophages. Furthermore, immunohistochemistry showed the expression of E-NTPDases in amastigotes in the lymph nodes of naturally infected dogs from an area of endemic visceral leishmaniasis. CONCLUSIONS/SIGNIFICANCE: In this work, we cloned, expressed and characterized the NTPDase-2 from L. infantum chagasi and demonstrated that it functions as a genuine enzyme from the E-NTPDase/CD39 family. We showed that E-NTPDases are present on the surface of promastigotes and in other intracellular locations. We showed, for the first time, the broad expression of LicNTPDases in naturally infected dogs. Additionally, the blockade of NTPDases led to lower levels of in vitro adhesion and infection, suggesting that these proteins are possible targets for rational drug design.