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.

First evidence of a serine arginine protein kinase (SRPK) in leishmania braziliensis and its potential as therapeutic target.

Leishmaniasis is a parasitic disease found in tropical and subtropical regions around the world, caused by parasites of the genus Leishmania. The disease is a public health concern and presents clinical manifestations that can cause death, disability, and mutilation. The parasite has promastigote (vector) and amastigote (vertebrate host) forms and kinase enzymes are involved in this differentiation process. In the present investigation, we show, for the first time, evidence of a serine/arginine protein kinase in Leshmania braziliensis (LbSRPK). Our results show that amastigotes express more LbSRPK than promastigotes.  Analogues of SRPIN340 (a known inhibitor of SRPK) were evaluated for their leishmanicidal activity and two of them, namely SRVIC22 and SRVIC32 showed important leishmanicidal activity in vitro. SRVIC22 and SRVIC32 were able to reduce the infection rate in macrophages and the number of intracellular amastigotes by 55 and 60%, respectively. Bioinformatics analysis revealed the existence of two different amino acid residues in the active site of LbSRPK compared to their human homologue (Tyr/Leu-and Ser/Tyr), which could explain the absence of leishmanicidal activity of SRPIN340 on infected macrophages. In order to enhance leishmanicidal activity of the analogues, optimizations were proposed in the structures of the ligands, suggesting strong interactions with the catalytic site of LbSRPK. Although the evidence on the action of inhibitors upon LbSRPK is only indirect, our studies not only reveal, for the first time, evidence of a SRPK in Leishmania, but also shed light on a new therapeutic target for drug development.

Leishmania infantum NTPDase1 and NTPDase2 play an important role in infection and nitric oxide production in macrophages.

Leishmania infantum, the causative agent of American Visceral Leishmaniasis (VL), is known for its ability to modulate the host immune response to its own favor. Ecto-nucleoside triphosphate diphosphohydrolase (ENTPDase) represents a family of enzymes that hydrolyze nucleotides and are involved in nucleotide-dependent biological processes. L. infantum has two ENTPDases, namely LiNTPDase1 and LiNTPDase2. Here, we used genetic tools to overexpress or abolish the expression of LiNTPDase1 and -2 to assess their role in parasite growth in culture and macrophage infection. While LiNTPDase1 or 2-overexpressing clones showed no morphological or growth changes in promastigotes, LiNTPDase2 overexpression increased macrophage adhesion and infection by 50% and 30%, respectively. The individual LiNTPDase1 and 2 knockout mutants showed lag in growth profile, which was reversed by the addition of adenine and guanine to the culture media. Moreover, the morphology of the knockout mutants even in supplemented media was changed to an amastigote-like form. The double knockout of both genes was lethal and a mechanism of compensation of deletion of one isoform was detected in these mutants. Correspondingly, the absence of LiNTPDase1 or LiNTPDase2 led to a dramatic reduction in in vitro infection (∼90%). Interestingly, nitric oxide production was decreased in both knockout mutants during infection, which suggests that both LiNTPDases can inhibit macrophage responses against the parasite. Overall, our results show important roles of LiNTPDase1 and -2 concerning in vitro macrophage infection and reinforce their use as potential targets to control Leishmania infections.

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 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.

Insights into the full-length SRPK2 structure and its hydrodynamic behavior.

The serine/arginine-rich protein kinase 2 (SRPK2) has been reported as upregulated in several cancer types, with roles in hallmarks such as cell migration, growth, and apoptosis. These findings have indicated that SRPK2 is a promising emerging target in drug discovery initiatives. Although high-resolution models are available for SRPK2 (PDB 2X7G), they have been obtained with a heavily truncated recombinant protein version (~50% of the primary structure), due to the presence of long intrinsically unstructured regions. In the present work, we sought to characterize the structure of a full-length recombinant version of SRPK2 in solution. Low-resolution Small-Angle X-ray Scattering data were obtained for both versions of SRPK2. The truncated ΔNΔS-SRPK2 presented a propensity to dimerize at higher concentrations whereas the full-length SRPK2 was mainly found as dimers. The hydrodynamic behavior of the full-length SRPK2 was further investigated by analytical size exclusion chromatography and sedimentation velocity analytical ultracentrifugation experiments. SRPK2 behaved as a monomer-dimer equilibrium and both forms have an elongated shape in solution, pointing to a stretched-to-closed tendency among the conformational plasticity observed. Taken together, these findings allowed us to define unique structural features of the SRPK2 within SRPK family, characterized by its flexible regions outside the bipartite kinase domain.

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.

Trypanosoma cruzi nucleoside triphosphate diphosphohydrolase 1 (TcNTPDase-1) biochemical characterization, immunolocalization and possible role in host cell adhesion.

Previous work has suggested that Trypanosoma cruzi diphosphohydrolase 1 (TcNTPDase-1) may be involved in the infection of mammalian cells and serve as a potential target for rational drug design. In this work, we produced recombinant TcNTPDase-1 and evaluated its nucleotidase activity, cellular localization and role in parasite adhesion to mammalian host cells. TcNTPDase-1 was able to utilize a broad range of triphosphate and diphosphate nucleosides. The enzyme's Km for ATP (0.096 mM) suggested a capability to influence the host's ATP-dependent purinergic signaling. The use of specific polyclonal antibodies allowed us to confirm the presence of TcNTPDase-1 at the surface of parasites by confocal and electron microscopy. In addition, electron microscopy revealed that TcNTPDase-1 was also found in the flagellum, flagellum insertion region, kinetoplast, nucleus and intracellular vesicles. The presence of this enzyme in the flagellum insertion region and vesicles suggests that it may have a role in nutrient acquisition, and the widespread distribution of TcNTPDase-1 within the parasite suggests that it may be involved in other biological process. Adhesion assays using anti-TcNTPDase-1 polyclonal antibodies as a blocker or purified recombinant TcNTPDase-1 as a competitor revealed that the enzyme has a role in parasite-host cell adhesion. These data open new frontiers to future studies on this specific parasite-host interaction and other unknown functions of TcNTPDase-1 related to its ubiquitous localization.