Amastigotes Research Articles

The Kinetoplastid-Specific Protein TcCAL1 Plays Different Roles During In Vitro Differentiation and Host-Cell Invasion in Trypanosoma cruzi.

In the pathogen Typanosoma cruzi, the calcium ion (Ca2+) regulates key processes for parasite survival. However, the mechanisms decoding Ca2+ signals are not fully identified or understood. Here, we investigate the role of a hypothetical Ca2+-binding protein named TcCAL1 in the in vitro life cycle of T. cruzi. Results showed that the overexpression of TcCAL1 fused to a 6X histidine tag (TcCAL1-6xHis) impaired the differentiation of epimastigotes into metacyclic trypomastigotes, significantly decreasing metacyclogenesis rates. When the virulence of transgenic metacyclic trypomastigotes was explored in mammalian cell invasion assays, we found that the percentage of infection was significantly higher in Vero cells incubated with TcCAL1-6xHis-overexpressing parasites than in controls, as well as the number of intracellular amastigotes. Additionally, the percentage of Vero cells with adhered metacyclic trypomastigotes significantly increased in samples incubated with TcCAL1-6xHis-overexpressing parasites compared with controls. In contrast, the differentiation rates from metacyclic trypomastigotes to axenic amastigotes or the epimastigote proliferation in the exponential phase of growth have not been affected by TcCAL1-6xHis overexpression. Based on our findings, we speculate that TcCAL1 exerts its function by sequestering intracellular Ca2+ by its EF-hand motifs (impairing metacyclogenesis) and/or due to an unknown activity which could be amplified by the ion binding (promoting cell invasion). This work underpins the importance of studying the kinetoplastid-specific proteins with unknown functions in pathogen parasites.

Essential Role of Enzymatic Activity in the Leishmanicidal Mechanism of the Eosinophil Cationic Protein (RNase 3).

The recruitment of eosinophils into Leishmania lesions is frequently associated with a favorable evolution. A feasible effector for this process is eosinophil cationic protein (ECP, RNase 3), one of the main human eosinophil granule proteins, endowed with a broad spectrum of antimicrobial activity, including parasites. ECP was active on Leishmania promastigotes and axenic amastigotes (LC50’s = 3 and 16 μM, respectively) but, in contrast to the irreversible membrane damage caused on bacteria and reproduced by its N-terminal peptides, it only induced a mild and transient plasma membrane destabilization on Leishmania donovani promastigotes. To assess the contribution of RNase activity to the overall leishmanicidal activity of ECP, parasites were challenged in parallel with a single-mutant version, ECP-H15A, devoid of RNase activity, that fully preserves the conformation and liposome permeabilization ability. ECP-H15A showed a similar uptake to ECP on promastigotes, but with higher LC50’s (>25 μM) for both parasite stages. ECP-treated promastigotes showed a degraded RNA pattern, absent in ECP-H15A-treated samples. Moreover ECP, but not ECP-H15A, reduced more than 2-fold the parasite burden of infected macrophages. Altogether, our results suggest that ECP enters the Leishmania cytoplasm by an endocytic pathway, ultimately leading to RNA degradation as a key contribution to the leishmanicidal mechanism. Thus, ECP combines both membrane destabilization and enzymatic activities to effect parasite killing. Taken together, our data highlight the microbicidal versatility of ECP as an innate immunity component and support the development of cell-penetrating RNases as putative leishmanicidal agents.

Parasitological and immunological evaluation of a quinoline derivative salt incorporated into a polymeric micelle formulation against Leishmania infantum infection.

Leishmaniasis is a parasitic disease caused by Leishmania protozoa, which presents a large spectrum of clinical manifestations. In the present study, a quinoline derivative salt named N-(2-((7-chloroquinolin-4-yl)amino)ethyl)-N-(prop-2-yn-1-yl)prop-2-yn-1-aminium chloride or QDS3 was in vitro and in vivo tested against L. infantum by means of its incorporation in Poloxamer 407-based polymeric micelles (QDS3/M). The in vitro antileishmanial activity of QDS3 and QDS3/M was investigated in L. infantum promastigotes, axenic amastigotes and infected macrophages. BALB/c mice were infected with L. infantum, and parasitological parameters were evaluated 1 and 15 days post-treatment by determining the parasite load by a limiting dilution assay, besides a quantitative PCR (qPCR) method. Immunological response was assessed based on production of cellular cytokines, as well as by quantification of nitrite levels and specific antibodies. In vitro results showed that QDS3 free or in micelles presented effective antileishmanial action against both parasite stages, being more effective in amastigotes. In vivo data showed that treatment using QDS3 or QDS3/M reduced the parasite load in the livers, spleens, draining lymph nodes (dLN) and bone marrows of the treated animals, 1 and 15 days after treatment, when compared to values found in the control groups. Additionally, treated mice developed a polarized Th1-type immune response, with higher levels of IL-12, IFN-γ, GM-CSF and nitrite, besides high production of specific IgG2a antibodies, when compared to the controls. Parasitological and immunological data obtained using the micellar composition were better than the others. In conclusion, QDS3, mainly when applied in a delivery adjuvant system, could be considered for future studies as therapeutic candidate against VL.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00436-022-07544-1.

Subtilisin of Leishmania amazonensis as Potential Druggable Target: Subcellular Localization, In Vitro Leishmanicidal Activity and Molecular Docking of PF-429242, a Subtilisin Inhibitor.

Subtilisin proteases, found in all organisms, are enzymes important in the post-translational steps of protein processing. In Leishmania major and L. donovani, this enzyme has been described as essential to their survival; however, few compounds that target subtilisin have been investigated for their potential as an antileishmanial drug. In this study, we first show, by electron microscopy and flow cytometry, that subtilisin has broad localization throughout the cytoplasm and membrane of the parasite in the promastigote form with foci in the flagellar pocket. Through in silico analysis, the similarity between subtilisin of different Leishmania species and that of humans were determined, and based on molecular docking, we evaluated the interaction capacity of a serine protease inhibitor against both life cycle forms of Leishmania. The selected inhibitor, known as PF-429242, has already been used against the dengue virus, arenaviruses, and the hepatitis C virus. Moreover, it proved to have antilipogenic activity in a mouse model and caused hypolipidemia in human cells in vitro. Here, PF-429242 significantly inhibited the growth of L. amazonensis promastigotes of four different strains (IC50 values = 3.07 ± 0.20; 0.83 ± 0.12; 2.02 ± 0.27 and 5.83 ± 1.2 µM against LTB0016, PH8, Josefa and LV78 strains) whilst having low toxicity in the host macrophages (CC50 = 170.30 µM). We detected by flow cytometry that there is a greater expression of subtilisin in the amastigote form; however, PF-429242 had a low effect against this intracellular form with an IC50 of >100 µM for intracellular amastigotes, as well as against axenic amastigotes (94.12 ± 2.8 µM for the LV78 strain). In conclusion, even though PF-429242 does not affect the intracellular forms, this drug will serve as a tool to explore pharmacological and potentially leishmanicidal targets.

Functional Study of Leishmania braziliensis Protein Arginine Methyltransferases (PRMTs) Reveals That PRMT1 and PRMT5 Are Required for Macrophage Infection.

In trypanosomatids, regulation of gene expression occurs mainly at the posttranscriptional level, and RNA-binding proteins (RBPs) are key players in determining the fates of transcripts. RBPs are targets of protein arginine methyltransferases (PRMTs), which posttranslationally regulate the RNA-binding capacity and other RBP interactions by transferring methyl groups to arginine residues (R-methylation). Herein, we functionally characterized the five predicted PRMTs in Leishmania braziliensis by gene knockout and endogenous protein HA tagging using CRISPR/Cas9 gene editing. We report that R-methylation profiles vary among Leishmania species and across L.braziliensis lifecycle stages, with the peak PRMT expression occurring in promastigotes. A list of PRMT-interacting proteins was obtained in a single coimmunoprecipitation assay using HA-tagged PRMTs, suggesting a network of putative targets of PRMTs and cooperation between the R-methylation writers. Knockout of each L.braziliensis PRMT led to significant changes in global arginine methylation patterns without affecting cell viability. Deletion of either PRMT1 or PRMT3 disrupted most type I PRMT activity, resulting in a global increase in monomethyl arginine levels. Finally, we demonstrate that L.braziliensis PRMT1 and PRMT5 are required for efficient macrophage infection in vitro, and for axenic amastigote proliferation. The results indicate that R-methylation is modulated across lifecycle stages in L.braziliensis and show possible functional overlap and cooperation among the different PRMTs in targeting proteins. Overall, our data suggest important regulatory roles of these proteins throughout the L.braziliensis life cycle, showing that arginine methylation is important for parasite-host cell interactions.

Synthesis, crystallographic study, molecular docking, ADMET, DFT and biological evaluation of new series of aurone derivatives as anti-leishmanial agents

A new series of aurone derivatives (4a-l) was synthesized and their structures were established on the basis of IR, NMR, mass spectra and elemental analysis. In addition, the identity of the representative compound 4g was successfully characterized with the aid of X-ray crystallography. All the synthesised compounds were docked in the binding pocket of Leishmania L-arginine amidinohydrolase enzyme (PDB ID: 5HJA). Docking score and molecular interaction studies revealed a good picture of compounds’ affinity and fitting inside the binding pocket of enzyme. In silico ADME, toxicity and DFT studies were also performed for the compound 4g, having highest docking score. The compound did not violate Lipinski's rule of five. Results showed good intestinal absorption and human serum binding. Compound was also found non-carcinogenic in acute and chronic rat and mice model studies, predicted using Maestro 9.6 software. All the derivatives were tested for anti-leishmanial activity against Leishmania donovani (L. donovani) promastigotes. Compound 4g exhibited more than 90 percent inhibition (IC50 4.97 ± 0.856 µM) in L. donovani promastigotes inhibition assay and was further evaluated for axenic amastigote study, changes in the cellular morphology and cytotoxicity. Compound 4g demonstrated better anti-leishmanial activity and lesser cytotoxicity as compared to standard drug miltefosine.

A zymographic study of metalloproteinase activities in whole cell extracts and extracellular secretions of Leishmania (L.) , L. major and L. infantum from Iran.

Leishmaniosis encompasses a group of diseases that is transmitted by sand flies and caused by different species of Leishmania. The skin is the initial organ to be infected by the Leishmania in cutaneous, mucocutaneous and visceral forms of leishmaniosis. The matrix metalloproteinases (MMPs) are capable of degrading all kinds of extracellular matrix (ECM) proteins. The aim of this study was to investigate the protease activity through zymography in cell extracts and extracellular secretions of L. major, L. tropica and L. infantum as three prevalent Leishmania spp. in Iran. The three Leishmania spp. were cultured in RPMI-1640 medium supplemented with fetal calf serum. Promastigotes and axenic amastigotes were harvested and lysed at various phases, and extracellular secretions and cell extracts were collected. Leishmania spp. were proved by targeting kDNA gene. Enzymes were characterized according to gelatin zymography and sensitivity to distinct proteinase inhibitors. We observed proteinase bands with molecular weights (MWs) between 66 to 180 kDa in cellular extracts of axenic amastigotes of L. infantum, L. tropica, and L. major, and from 66 to 92 kDa in extracellular secretions of L. infantum. No proteinase activities were observed in extracellular secretions of axenic amastigotes and in cellular extracts of promastigotes in logarithmic and stationary phases of L. major and L. tropica. Using specific inhibitors, we determined that these proteolytic activities are due to metalloproteases. Our study demonstrated that amastigotes of all three Leishmania spp. have distinct amounts of proteinase activities and therefore can cause various types of lesions and outcomes of the disease.

Differential regulation of E-NTPdases during Leishmania amazonensis lifecycle and effect of their overexpression on parasite infectivity and virulence.

Infections caused by Leishmania amazonensis are characterized by a persistent parasitemia due to the ability of the parasite to modulate the immune response of macrophages. It has been proposed that ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDases) could be able to suppress the host immune defense by reducing the ATP and ADP levels. The AMP generated from E-NTPDase activity can be subsequently hydrolyzed by ecto-nucleotidases, increasing the levels of adenosine, which can reduce the inflammatory response. In the present work, we provide new information about the role of E-NTPDases on infectivity and virulence of L. amazonensis. Our data demonstrate that not only the E-NTPDase activity is differentially regulated during the parasite development but also the expression of the genes ntpd1 and ntpd2. E-NTPDase activity increases significantly in axenic amastigotes and metacyclic promastigotes, both infective forms in mammalian host. A similar profile was found for mRNA levels of the ntpd1 and ntpd2 genes. Using parasites overexpressing the genes ntpd1 and ntpd2, we could demonstrate that L. amazonensis promastigotes overexpressing ntpd2 gene show a remarkable increase in their ability to interact with macrophages compared to controls. In addition, both ntpd1 and ntpd2-overexpressing parasites were more infective to macrophages than controls. The kinetics of lesion formation by transfected parasites were similar to controls until the second week. However, twenty days post-infection, mice infected with ntpd1 and ntpd2-overexpressing parasites presented significantly reduced lesions compared to controls. Interestingly, parasite load reached similar levels among the different experimental groups. Thus, our data show a non-linear relationship between higher E-NTPDase activity and lesion formation. Previous studies have correlated increased ecto-NTPDase activity with virulence and infectivity of Leishmania parasites. Based in our results, we are suggesting that the induced overexpression of E-NTPDases in L. amazonensis could increase extracellular adenosine levels, interfering with the balance of the immune response to promote the pathogen clearance and maintain the host protection.

Comparative efficacy of amphotericin B-loaded chitosan nanoparticles and free amphotericin B drug against Leishmania tropica

BackgroundThe preparation of an effective drug delivery formulation is an urgent need to treat cutaneous leishmaniasis (CL). Pentavalent antimonials and Amphotericin B (AmB) are considered to treat leishmaniasis; however, their clinical usage is hampered by poor solubility, high cost, toxicity, and the emergence of drug-resistant Leishmania spp. The drug delivery systems (DDS) could be used as an alternative treatment option for the treatment of CL to circumvent these problems. We tested the antileishmanial efficacies of free AmB and amphotericin B-loaded chitosan nanoparticles (AmB-CNPs) under in vitro conditions.ResultsChitosan nanoparticles (CNPs) were synthesized using the ionic gelation method with negatively charged tripolyphosphate (TPP). During the synthesis of CNPs, AmB was incorporated into the nanoparticles (NPs). The NPs were characterized for their size, surface morphology, encapsulation efficacy (EE), drug loading content (DLC), and surface charge using different techniques. Their efficacy was evaluated against promastigotes and axenic amastigotes forms of Leishmania tropica using MTT assay. The synthesized AmB-CNPs displayed a spherical shape with a mean particle size of 118 nm, a positive zeta potential of (+ 6.21 ± 2.02 mV), and an encapsulation efficacy of 88%. Dynamic light scattering technique (DLS) shows that the average size of prepared AmB-CNPs was 95.5 nm. Free AmB presented very low efficacy (only 65% and 67% inhibition of the promastigotes and axenic amastigotes parasite load), whereas AmB-CNPs exhibited 90% and 84% parasite inhibition after 72 h incubation. The AmB-CNPs exhibited significantly higher efficacy than free AmB in terms of reduction in parasite viability. Half-maximal inhibitory concentration (IC50) measured values of the AmB-CNPs were significant lowers than free AmB.ConclusionsThe present data indicated that AmB-CNPs exhibited vigorous anti-leishmanial activity than free AmB by dose and time-dependent manner. This formulation can be used for local therapy of CL after in vivo efficacy conformational studies.

In vitro efficacy of polymer coated miltefosine drug against leishmania tropica.

Polymer based nanoparticles for drug delivery is an alternative approach to overcome drug resistance and drug toxicity especially for cutaneous leishmaniasis treatment. The present study shows synthesis and characterization of Miltefosine loaded chitosan nanoparticles (MFS-CNPs). The synthesized MFS-CNPs were experimented to evaluate the in vitro cytotoxicity and efficacy of the synthesized drug loaded nanoparticles by hemolysis assay and 3-(4, 5- dimethylthiazol-2-yl)-2,5-diphenyletetrazolium bromide (MTT) assay. MFS-CNPs were synthesized by ionic gelation method with sodium tripolyphosphate. The characterization of synthesized NPs was performed to observe the surface morphology, encapsulation efficacy, drug loading content, average size, and zeta potential. In vitro MTT assay was performed to calculate half maximal inhibitory concentration value of synthesized nanoparticles against promastigotes and axenic amastigotes of L. tropica. By using Scanning electron microscope, MFS-CNPs displayed spherical shape having a mean size of 70nm along with high EE (97%), DLC (91%) and negative surface charge (-28.0mV). Dynamic light scattering shows the average size of NPs was 91.4nm. Moreover, less than 5% hemolytic activity was observed in MFS-CNPs as compared to free MFS in different concentrations (100μg/ml, 125μg/ml, 150μg/ml).It was observed that the effect of MFS-CNPs and free MFS on both forms of the parasite was dose and time dependent. However, the cytotoxic effects of MFS-CNPs were more salient than free MFS on both forms of L. tropica. Using MTT assay, free MFS presented low efficacy at higher concentrations (30µg/ml) with 21.4 ± 1.3 and 20.5 ± 1.4 mean viability rate of the promastigotes and axenic amastigotes, respectively after 72h incubation. While MFS-CNPs showed strong antileishmanial effects on both forms of L. tropica (11 ± 0.3 and 14 ± 0.8) mean viability rate after 72h incubation at (30µg/ml). When analyzed statistically by the software, Graph Pad Prism version 5, the IC50 value of MFS-CNPs (0.0218 ± 0.01µg/ml) against promastigotes was effective than free MFS (0.3548 ± 0.17µg/ml). Similarly, MFS-CNPs activity against axenic amastigotes (0.1008 ± 0.02µg/ml) was potent than free MFS (0.5320 ± 0.21µg/ml). Hence, MFS-CNPs exhibited significant antileishmanial activity in vitro. In conclusion, MFS-CNPs manifested enhanced in vitro Leishmanicidal and less hemolytic activity; however more studies are needed to support its efficacy in both animal and human cutaneous leishmaniasis.

In-vitro evaluation of nano-liposomal formulation of Fluconazole and Amphotericin B against visceral leishmaniasis

Objectives: The aim of the present study was to compare the efficacy of a dual and single drug loaded nano-liposomal formulation of Amphotericin B and Fluconazole for the treatment of visceral leishmaniasis with plain drugs. Methods: We have formulated nano-liposomes (200-250 nm) from Amphotericin B and Fluconazole using dry film hydration method and have tested their efficacy on promastigotes and amastigotes of Leishmania donovani strain. Physicochemical characterization, entrapment study, stability study, in-vitro release study, in-vitro macrophagic uptake studies (Confocal microscopy) and in-vitro antileishmanial activity were evaluated for various formulations containing Amphotericin B and Fluconazole. Results: The in-vitro cellular uptake confocal studies revealed that NR-loaded AmpB + Flu nanoliposomes have enhanced cellular uptake of formulation. The in-vitro inhibition of promastigotes and amastigotes with liposome containing both Amphotericin B and Fluconazole was significantly more than with liposomes containing individual drugs. The IC50 and CC50 of AmpB + Flu nanoliposomes against promastigotes was found to be 3.308μg/mL and 73.48μg/mL respectively, while the IC50 against axenic and intramacrophagic amastigotes was found to be 3.412 and 3.7028μg/mL respectively. Conclusion: In conclusion, Liposomal formulation containing both Amphotericin B and Fluconazole had significantly greater efficacy than conventional combination and other formulation with individual drugs. Current dual drug loaded formulation may have a favourable safety profile, and if production costs are low, it may prove to be a feasible alternative to currently available therapy after in-vivo testing.

Curzerene antileishmania activity: Effects on Leishmania amazonensis and possible action mechanisms

Leishmaniasis is a set of infectious diseases with high rates of morbidity and mortality, it affects millions of people around the world. Treatment, mainly with pentavalent antimonials, presents significant toxicity and many cases of resistance. In previous works we have demonstrated the effective and selective antileishmanial activity of Eugenia uniflora L. essential oil, being constituted (47.3%) by the sesquiterpene curzerene. Considering the high rate of parasite inhibition demonstrated for E. uniflora essential oil, and the significant presence of curzerene in the oil, this study aimed to evaluate its antileishmania activity and possible mechanisms of action. Curzerene was effective in inhibiting the growth of promastigotes (IC50 3.09 ± 0.14 µM) and axenic amastigotes (EC50 2.56 ± 0.12 µM), with low cytotoxicity to RAW 264.7 macrophages (CC50 83.87 ± 4.63 µM). It was observed that curzerene has direct effects on the parasite, inducing cell death by apoptosis with secondary necrotic effects (producing pores in the plasma membrane). Curzerene proved to be even more effective against intra-macrophage amastigote forms, with an EC50 of 0.46 ± 0.02 µM. The selectivity index demonstrated by curzerene on these parasite forms was 182.32, being respectively 44.15 and 8.47 times more selective than meglumine antimoniate and amphotericin B. The antiamastigote activity of curzerene was associated with immunomodulatory activity, as it increased TNF-α, IL-12, and NO levels, and lysosomal activity, and decreased IL-10 and IL-6 cytokine levels detected in macrophages infected and treated. In conclusion, our results demonstrate that curzerene is an effective and selective antileishmanial agent, a candidate for in vivo investigation in models of antileishmanial activity.

Serine proteases profiles of Leishmania (Viannia) braziliensis clinical isolates with distinct susceptibilities to antimony

Glucantime (SbV) is the first-line treatment against American Tegumentary Leishmaniasis. Resistance cases to this drug have been reported and related to host characteristics and parasite phenotypes. In this study, 12 Leishmania (Viannia) braziliensis isolates from patients that presented clinical cure (Responders—R) and relapse or therapeutic failure (Non-responders—NR) after treatment with antimony, were analyzed. These parasites were assessed by in vitro susceptibility to SbIII and SbV, serine proteases activity measured with substrate (z-FR-AMC) and specific inhibitors (TLCK, AEBSF and PMSF). In vitro susceptibility of axenic amastigotes to SbIII showed a significant difference between R and NR groups. The protease assays showed that TLCK inhibited almost 100% of activity in both axenic amastigotes and promastigotes while AEBSF inhibited around 70%, and PMSF showed lower inhibition of some isolates. Principal component and clustering analysis performed with these data yielded one homogeneous cluster with only NR isolates and three heterogeneous clusters with R and NR isolates. Additionally, differential expression of subtilisins (LbrM.13.0860 and LbrM.28.2570) and TXNPx (LbrM.15.1080) was evaluated in promastigotes and axenic amastigotes from both groups. The results showed a higher expression of LbrM.13.0860 and LbrM.15.1080 genes in axenic amastigotes, while LbrM.28.2570 gene had the lowest expression in all isolates, regardless of the parasite form. The data presented here show a phenotypic heterogeneity among the parasites, suggesting that exploration of in vitro phenotypes based on SbIII and serine proteases profiles can aid in the characterization of L. (V.) braziliensis clinical isolates.

Boswellic Acids Show In Vitro Activity against Leishmania donovani.

In continuation of our search for leads from medicinal plants against protozoal pathogens, we detected antileishmanial activity in polar fractions of a dichloromethane extract from Boswellia serrata resin. 11-keto-β-boswellic acid (KBA) could be isolated from these fractions and was tested in vitro against Leishmania donovani axenic amastigotes along with five further boswellic acid derivatives. 3-O-acetyl-11-keto-β-boswellic acid (AKBA) showed the strongest activity with an IC50 value of 0.88 µM against axenic amastigotes but was inactive against intracellular amastigotes in murine macrophages

Selective in vitro antileishmanial activity of Mimosa caesalpiniifolia stem barks and its main constituent betulinic acid against Leishmania amazonensis

IntroductionThe need of novel antileishmanial compounds with a wide therapeutic index has been a challenge for the pharmaceutical industry in reducing the side effects resulted from treatment with chemotherapeutics. The present study aims to investigate the antileishmanial and cytotoxic activities of the ethanol extract (EtOH), the dichloromethane fraction (DCM) and its isolated constituent betulinic acid (BA) obtained from the stem bark of Mimosa caesalpiniifolia Benth. (Fabaceae). MethodsThe antiproliferative effect on Leishmania amazonensis promastigotes was assessed by the Alamar Blue® colorimetric assay. The intramacrophagic amastigotes were exposed to different concentrations of EtOH, DCM and BA to determine the survival index. Selectivity indexes towards parasites were calculated for each treatment performed. The modulation of macrophage activation state was investigated by observing structural (phagocytic and lysosomal activities) and cellular (nitrite production) changes. Results and discussionThe EtOH, DCM and BA were effective against Leishmania amazonensis promastigote forms (IC50: 4.63 ± 0.69, 0.38 ± 0.42 and 29.63 ± 4.63 µg•mL−1, respectively). The same was observed for axenic amastigotes forms (IC50: 4.8 ± 1.27, 0.56 ± 0.39 and 14.2 ± 2.01 µg•mL−1), with selectivity index higher than 20 for DCM and BA. The macrophages infection and the number of intracellular amastigotes were reduced to 54.6%, 52.7% and 27.5%, respectively, after treatments. On the other hand, both DCM and BA were not able to promote macrophages activation, but only EtOH. Since DCM have shown highest potency as antileishmanial agent, BA presented a highest selectivity index. ConclusionThese results demonstrate the importance of M. caesalpiniifolia as a source of promising compounds since both DCM and BA might be investigated as promising and safe candidates for treatment of leishmaniasis. This work opens for further studies with M. caesalpiniifolia and betulinic acid focused on elucidation of possible involved mechanisms of antileishmanial action, as well as the potential in vivo treatment in experimental models of leishmaniasis.

Clustering of adenosine A2B receptors with ectonucleotidases in caveolin-rich lipid rafts underlies immunomodulation by Leishmania amazonensis.

Extracellular adenosine plays important roles in modulating the immune responses. We have previously demonstrated that infection of dendritic cells (DC) by Leishmania amazonensis leads to increased expression of CD39 and CD73 and to the selective activation of the low affinity A2B receptors (A2B R), which contributes to DC inhibition, without involvement of the high affinity A2A R. To understand this apparent paradox, we now characterized the alterations of both adenosine receptors in infected cells. With this aim, bone marrow-derived DC from C57BL/6J mice were infected with metacyclic promastigotes of L. amazonensis. Fluorescence microscopy revealed that L. amazonensis infection stimulates the recruitment of A2B R, but not of A2A R, to the surface of infected DC, without altering the amount of mRNA or the total A2B R density, an effect dependent on lipophosphoglycan (LPG). Log-phase promastigotes or axenic amastigotes of L. amazonensis do not stimulate A2B R recruitment. A2B R clusters are localized in caveolin-rich lipid rafts and the disruption of these membrane domains impairs A2B R recruitment and activation. More importantly, our results show that A2B R co-localize with CD39 and CD73 forming a "purinergic cluster" that allows for the production of extracellular adenosine in close proximity with these receptors. We conclude that A2B R activation by locally produced adenosine constitutes an elegant and powerful evasion mechanism used by L. amazonensis to down-modulate the DC activation.

The Leishmania donovani LDBPK_220120.1 Gene Encodes for an Atypical Dual Specificity Lipid-Like Phosphatase Expressed in Promastigotes and Amastigotes; Substrate Specificity, Intracellular Localizations, and Putative Role(s).

The intracellular protozoan parasites of the Leishmania genus are responsible for Leishmaniases, vector borne diseases with a wide range of clinical manifestations. Leishmania (L.) donovani causes visceral leishmaniasis (kala azar), the most severe of these diseases. Along their biological cycle, Leishmania parasites undergo distinct developmental transitions including metacyclogenesis and differentiation of metacyclic promastigotes (MPs) to amastigotes. Metacyclogenesis inside the phlebotomine sandfly host’s midgut converts the procyclic dividing promastigotes to non-dividing infective MPs eventually injected into the skin of mammalian hosts and phagocytosed by macrophages where the MPs are converted inside modified phagolysosomes to the intracellular amastigotes. These developmental transitions involve dramatic changes in cell size and shape and reformatting of the flagellum requiring thus membrane and cytoskeleton remodeling in which phosphoinositide (PI) signaling and metabolism must play central roles. This study reports on the LDBPK_220120.1 gene, the L. donovani ortholog of LmjF.22.0250 from L. major that encodes a phosphatase from the “Atypical Lipid Phosphatases” (ALPs) enzyme family. We confirmed the expression of the LDBPK_220120.1 gene product in both L. donovani promastigotes and axenic amastigotes and showed that it behaves in vitro as a Dual Specificity P-Tyr and monophosphorylated [PI(3)P and PI(4)P] PI phosphatase and therefore named it LdTyrPIP_22 (Leishmaniad onovani Tyrosine PI Phosphatase, gene locus at chromosome 22). By immunofluorescence confocal microscopy we localized the LdTyrPIP_22 in several intracellular sites in the cell body of L. donovani promastigotes and amastigotes and in the flagellum. A temperature and pH shift from 25°C to 37°C and from pH 7 to 5.5, induced a pronounced recruitment of LdTyrPIP_22 epitopes to the flagellar pocket and a redistribution around the nucleus. These results suggest possible role(s) for this P-Tyr/PI phosphatase in the regulation of processes initiated or upregulated by this temperature/pH shift that contribute to the developmental transition from MPs to amastigotes inside the mammalian host macrophages.

Alkyl-Resorcinol Derivatives as Inhibitors of GDP-Mannose Pyrophosphorylase with Antileishmanial Activities.

Leishmaniasis is a vector-borne disease caused by the protozoan parasite Leishmania found in tropical and sub-tropical areas, affecting 12 million people around the world. Only few treatments are available against this disease and all of them present issues of toxicity and/or resistance. In this context, the development of new antileishmanial drugs specifically directed against a therapeutic target appears to be a promising strategy. The GDP-Mannose Pyrophosphorylase (GDP-MP) has been previously shown to be an attractive therapeutic target in Leishmania. In this study, a chemical library of 5000 compounds was screened on both L. infantum (LiGDP-MP) and human (hGDP-MP) GDP-MPs. From this screening, oncostemonol D was found to be active on both GDP-MPs at the micromolar level. Ten alkyl-resorcinol derivatives, of which oncostemonols E and J (2 and 3) were described for the first time from nature, were then evaluated on both enzymes as well as on L. infantum axenic and intramacrophage amastigotes. From this evaluation, compounds 1 and 3 inhibited both GDP-MPs at the micromolar level, and compound 9 displayed a three-times lower IC50 on LiGDP-MP, at 11 µM, than on hGDP-MP. As they displayed mild activities on the parasite, these compounds need to be further pharmacomodulated in order to improve their affinity and specificity to the target as well as their antileishmanial activity.

Proposed Mechanism for the Antitrypanosomal Activity of Quercetin and Myricetin Isolated from Hypericum afrum Lam.: Phytochemistry, In Vitro Testing and Modeling Studies.

The in vitro activity of L. donovani (promastigotes, axenic amastigotes and intracellular amastigotes in THP1 cells) and T. brucei, from the fractions obtained from the hydroalcoholic extract of the aerial part of Hypericum afrum and the isolated compounds, has been evaluated. The chloroform, ethyl acetate and n-butanol extracts showed significant antitrypanosomal activity towards T. brucei, with IC50 values of 12.35, 13.53 and 12.93 µg/mL and with IC90 values of 14.94, 19.31 and 18.67 µg/mL, respectively. The phytochemical investigation of the fractions led to the isolation and identification of quercetin (1), myricitrin (2), biapigenin (3), myricetin (4), hyperoside (5), myricetin-3-O-β-d-galactopyranoside (6) and myricetin-3’-O-β-d-glucopyranoside (7). Myricetin-3’-O-β-d-glucopyranoside (7) has been isolated for the first time from this genus. The chemical structures were elucidated by using comprehensive one- and two-dimensional nuclear magnetic resonance (1D and 2D NMR) spectroscopic data, as well as high-resolution electrospray ionization mass spectrometry (HR-ESI–MS). These compounds have also been evaluated for their antiprotozoal activity. Quercetin (1) and myricetin (4) showed noteworthy activity against T. brucei, with IC50 and IC90 values of 7.52 and 5.71 µM, and 9.76 and 7.97 µM, respectively. The T. brucei hexokinase (TbHK1) enzyme was further explored as a potential target of quercetin and myricetin, using molecular modeling studies. This proposed mechanism assists in the exploration of new candidates for novel antitrypanosomal drugs.

Antitrypanosomal and antileishmanial effects of the hydroalcoholic extract of Croton cajucara benth and its 19-nor-clerodane chromatographic fractions

Context: Croton cajucara Benth has been widely used in folk medicine, especially in the Amazonian region of Brazil, to treat several illnesses. Objectives: The objective of the study is to evaluate the stem bark hydroalcoholic extract (CC-EHA) of C. cajucara and their clerodane-type diterpene fractions (F1-7, F25-27, and F28) on promastigotes and axenic amastigotes of Leishmania amazonensis and trypomastigotes and epimastigotes of Trypanosoma cruzi. Materials and Methods: The extract was obtained in ethanol: water and the fractions with solvents of increasing polarity. The antiparasitic activities were assessed by 3,4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide method against promastigotes and axenic amastigotes from L. amazonensis in 24-h cultures and trypomastigotes and epimastigotes of T. cruzi in 72-h cultures. The experiments in triplicate were made in quadruplicate way in each time. The statistical tests used were t-Students and ANOVA. Results: Among those evaluated samples, the CC-EHA extract showed the higher antileishmanial activity of promastigote cultures (IC50 = 18.00 ± 0.01 μg/mL at 24 h). However, against axenic amastigotes, the polar fraction (F28), rich in diterpene transdehydrocrotonin (t-DCTN), showed the highest effect with an IC50 = 6.18 ± 0.02 μg/mL in culture of 24 h. In the T. cruzi assays, F28 also showed the greatest effect against trypomastigotes and epimastigotes, IC50 = 0.43 ± 0.02 μg/mL and 0.27 ± 0.02 μg/mL, respectively, at 72 h of culture. The results showed that the diterpene t-DCTN is the most important antiparasitic component in the hydroalcoholic extract obtained from C. cajucara, specifically against L. amazonensis and T. cruzi. Conclusion: Our results contribute to knowledge of these folk medicinal species as a promising antiparasitic phytotherapeutic alternative.