Topoisomerase IB poisons induce histone H2A phosphorylation as a response to DNA damage in Leishmania infantum.

DNA topoisomerases are considered consolidated druggable targets against diseases produced by trypanosomatids. Several reports indicated that indenoisoquinolines, a family of non-camptothecinic based topoisomerase poisons, have a strong leishmanicidal effect both in vitro and in vivo in murine models of visceral leishmaniasis. The antileishmanial effect of the indenoisoquinolines implies several mechanisms that include the stabilization of the cleavage complex, histone H2A phosphorylation and DNA fragmentation. A series of 20 compounds with the indenoisoquinoline scaffold and several substituents at positions N6, C3, C8 and C9, were tested both in promastigotes and in intramacrophage splenic amastigotes obtained from an experimental murine infection. The antileishmanial effect of most of these compounds was within the micromolar or submicromolar range. In addition, the introduction of an N atom in the indenoisoquinoline ring (7-azaindenoisoquinolines) produced the highest selectivity index along with strong DNA topoisomerase IB inhibition, histone H2A phosphorylation and DNA-topoisomerase IB complex stabilization. This report shows for the first time the effect of a series of synthetic indenoisoquinolines on histone H2A phosphorylation, which represents a primary signal of double stranded DNA break in genus Leishmania.

Activity and Cell-Death Pathway in Leishmania infantum Induced by Sugiol: Vectorization Using Yeast Cell Wall Particles Obtained From Saccharomyces cerevisiae.

Visceral leishmaniasis, caused by Leishmania infantum, is a neglected tropical disease, to which efforts in the innovation of effective and affordable treatments remain limited, despite the rising incidence in several regions of the world. In this work, the antileishmanial effects of sugiol were investigated in vitro. This compound was isolated from the bark of Cupressus lusitanica and showed promising activity against L. infantum. In spite of the positive results, it is known that the compound is a poorly water-soluble diterpene molecule, which hinders further investigation, especially in preclinical animal studies. Thus, in an alternative delivery method, sugiol was entrapped in glucan-rich particles obtained from Saccharomyces cerevisiae yeast cell walls (YCWPs). To evaluate the activity of sugiol, the experiments were divided into two parts: (i) the in vitro investigation of antileishmanial activity of free sugiol against L. infantum promastigotes after 24, 48, and 72 h of treatment and (ii) the evaluation of antileishmanial activity of sugiol entrapped in glucan-rich particles against intracellular L. infantum amastigotes. Free sugiol induced the cell-death process in promastigotes, which was triggered by enhancing cytosolic calcium level and promoting the autophagy up to the first 24 h. Over time, the presence of autophagic vacuoles became rarer, especially after treatment with lower concentrations of sugiol, but other cellular events intensified, like ROS production, cell shrinkage, and phosphatidylserine exposure. Hyperpolarization of mitochondrial membrane potential was found at 72 h, induced by the mitochondria calcium uptake, causing an increase in ROS production and lipid peroxidation as a consequence. These events resulted in the cell death of promastigotes by secondary necrosis. Sugiol entrapped in glucan-rich particles was specifically recognized by dectin-1 receptor on the plasma membrane of macrophages, the main host cell of Leishmania spp. Electron micrographs revealed particles containing sugiol within the infected macrophages and these particles were active against the intracellular L. infantum amastigotes without affecting the host cell. Therefore, the YCWPs act like a Trojan horse to successfully deliver sugiol into the macrophage, presenting an interesting strategy to deliver water-insoluble drugs to parasitized cells.

A semi-synthetic neolignan derivative from dihydrodieugenol B selectively affects the bioenergetic system of Leishmania infantum and inhibits cell division.

Leishmaniasis is a neglected disease that affects more than 12 million people, with a limited therapy. Plant-derived natural products represent a useful source of anti-protozoan prototypes. In this work, four derivatives were prepared from neolignans isolated from the Brazilian plant Nectandra leucantha, and their effects against intracellular amastigotes of Leishmania (L.) infantum evaluated in vitro. IC50 values between 6 and 35 µM were observed and in silico predictions suggested good oral bioavailability, no PAINS similarities, and ADMET risks typical of lipophilic compounds. The most selective (SI > 32) compound was chosen for lethal action and immunomodulatory studies. This compound caused a transient depolarization of the plasma membrane potential and induced an imbalance of intracellular Ca2+, possibly resulting in a mitochondrial impairment and leading to a strong depolarization of the membrane potential and decrease of ATP levels. The derivative also interfered with the cell cycle of Leishmania, inducing a programmed cell death-like mechanism and affecting DNA replication. Further immunomodulatory studies demonstrated that the compound eliminates amastigotes via an independent activation of the host cell, with decrease levels of IL-10, TNF and MCP-1. Additionally, this derivative caused no hemolytic effects in murine erythrocytes and could be considered promising for future lead studies.

Selective effects of Euterpe oleracea (açai) on Leishmania (Leishmania) amazonensis and Leishmania infantum.

Leishmania (Leishmania) amazonensis and Leishmania infantum (=Leishmania chagasi) are protozoa that cause American cutaneous and visceral leishmaniasis, respectively. These diseases show a high incidence in developing countries such as Brazil. The treatments used for leishmaniasis are still limited due to their high cost and toxicity. Currently, some natural products are considered an important alternative source of new leishmanicidal agents. Euterpe oleracea Martius, a palm producing black fruits, is frequently consumed in the Amazon region, as a juice, known as açai, with potent antioxidant, anti-inflammatory and anticonvulsant properties. Interestingly, the biological activity of clarified açai juice (EO) on L. (L.) amazonensis and L. infantum (=L. chagasi) is unknown. Therefore, the mechanism of anti-leishmanial action of EO has been evaluated on L. (L.) amazonensis and L. infantum (=L. chagasi). EO reduced the number of promastigotes and caused morphological alterations, increased the production of reactive oxygen species (ROS) and induced cell death phenotypes probably seems by apoptosis in the promastigotes of L. (L.) amazonensis (IC50 = 1:40) and L. infantum (=L. chagasi) (IC50 = 1:38). EO also presented activity against Leishmania amastigotes. Treatment with EO for 72 h strongly reduced IL-17 cytokine levels at all tested concentrations and decreased the number of intracellular amastigotes in macrophages infected with L. (L.) amazonensis (IC50 = 1:30) and L. infantum (=L. chagasi) (IC50 = 1:38). Additionally, no cytotoxic effect was observed in murine macrophages treated with EO (72 h - CC50 > 1:1). Our results demonstrated that EO has leishmanicidal activity against two different species that cause American visceral and cutaneous leishmaniasis without cytotoxic effects for the host cell.

Design, synthesis and biological evaluation of piperazinyl-ß-carbolinederivatives as anti-leishmanial agents.

Molecular hybridization is a ligand based drug design approach is well known recent medicinal chemistry to design anti-parasitic agents. In the present study, we have designed a series of (1-phenyl-9H-pyrido [3,4-b]indol-3-yl) (4-phenylpiperazin-1-yl)methanone derivatives using molecular hybridization approach. Designed analogues were evaluated for cytotoxicity and inhibition activity against Leishmania infantum and Leishmania donovani. Among these reported analogues 7b, 7d, 7e, 7f and 7m displayed potent inhibition of both L. infantum and L. donovani. Compounds 7i and 7k exhibited selective potent inhibition of L. donovani. Especially, compounds 7e and 7k showed most potent anti-leishmanial activity against L. infantum and L. donovani respectively. Anti-leishmanial activity of these compounds is comparable with standard drugs miltefosine and pentamidine. SAR studies revealed that, electron donating group substitution on phenyl ring recommended for potent anti-leishmanial activity.

First example of peptides targeting the dimer interface of Leishmania infantum trypanothione reductase with potent in vitro antileishmanial activity.

A series of 9-mer and 13-mer amide-bridged cyclic peptides derived from the linear prototype Ac-PKIIQSVGIS-Nle-K-Nle-NH2 (Toro et al. ChemBioChem2013) has been designed and synthesized by introduction of the lactam between amino acid side chains that are separated by one helical turn (i, i+4). All of these compounds were tested in vitro as both dimerization and enzyme inhibitors of Leishmania infantum trypanothione reductase (Li-TryR). Three of the 13-mer cyclic peptide derivatives (3, 4 and 6) inhibited the oxidoreductase activity of Li-TryR in the low micromolar range and they also disrupted enzyme dimerization. Cyclic analogues 3 and 4 were more resistant to proteases than was the linear prototype. Furthermore, the most potent TryR inhibitors in the linear and cyclic series displayed potent in vitro activity against Leishmania infantum upon conjugation with cationic cell-penetrating peptides. To date, these conjugated peptides can be considered the first example of TryR dimerization inhibitors that are active in cell culture.

The sesquiterpene (-)-α-bisabolol is active against the causative agents of Old World cutaneous leishmaniasis through the induction of mitochondrial-dependent apoptosis.

Cutaneous leishmaniasis treatment remains challenging due to the absence of a satisfactory treatment. The screening of natural compounds is a valuable strategy in the search of new drugs against leishmaniasis. The sesquiterpene (-)-α-bisabolol is effective in vivo against visceral leishmaniasis due to Leishmania infantum, but its mechanism of action remains elusive. The aim of this study is to validate this promising compound against the causative species of Old World cutaneous leishmaniasis and to get an insight into its antileishmanial mode of action. The compound was evaluated on L. tropica promastigotes and intracellular amastigotes using bone marrow-derived macrophages and its cytotoxicity was evaluated on L929 fibroblasts. The reactive oxygen species generation was evaluated using a sensitive probe. Mitochondrial depolarization was assessed evaluating the fluorescence due to rhodamine 123 in a flow cytometer. Apoptosis was investigated by measuring the fluorescence due to annexin V and propidium iodide in a flow cytometer. The ultrastructure of treated promastigotes and intracellular amastigotes was analysed through transmission electron microscopy. (-)-α-Bisabolol was active against L. tropica intracellular amastigotes displaying an inhibitory concentration 50 % of 25.2 µM and showing low cytotoxicity. This compound induced time and dose-dependent oxidative stress, mitochondrial depolarization and phosphatidilserine externalization (a marker of apoptosis). These effects were noticed at a low concentration and short exposure time. In the ultrastructural analyses, the treated parasites showed mitochondrial disruption, presence of electron-dense structures and chromatin condensation. These results suggest that this natural compound induces oxidative stress and mitochondrial-dependent apoptosis on Leishmania without disturbing the plasma membrane.

Functional divergence of SIR2 orthologs between trypanosomatid parasites.

SIR2 proteins are NAD+-dependent deacetylases involved in epigenetic control of gene expression and metabolic regulation through post-translational modification of diverse target proteins. In pathogens, these enzymes are considered as attractive drug targets involved in key aspects of the infectious cycle. Leishmania infantum LiSIR2rp1 was among the first non-nuclear and essential SIR2 deacetylases described in eukaryotes. Here, we show that the two other LiSIR2rp2 and LiSIRrp3 paralogs are both located in mitochondria. Gene deletion experiments show that LiSIR2rp3 is not required for parasite survival. Surprisingly, multiple extrachromosomal amplicons bearing the LiSIR2rp2 gene are constitutively produced in wild type strains. Consequently, a knockout of this gene could not be obtained, even after episomal rescue experiments. We further provide genetic and biochemical evidence showing that SIR2rp2 protein directly affects parasite proliferation in relation to NAD+ bioavailability. Together, these results highlight unexpected genus-specific divergence of the SIR2 machinery among trypanosomatid parasites.

A putative Leishmania DNA polymerase theta protects the parasite against oxidative damage.

Leishmania infantum is a protozoan parasite that is phagocytized by human macrophages. The host macrophages kill the parasite by generating oxidative compounds that induce DNA damage. We have identified, purified and biochemically characterized a DNA polymerase θ from L. infantum (LiPolθ), demonstrating that it is a DNA-dependent DNA polymerase involved in translesion synthesis of 8oxoG, abasic sites and thymine glycol lesions. Stably transfected L. infantum parasites expressing LiPolθ were significantly more resistant to oxidative and interstrand cross-linking agents, e.g. hydrogen peroxide, cisplatin and mitomycin C. Moreover, LiPolθ-overexpressing parasites showed an increased infectivity toward its natural macrophage host. Therefore, we propose that LiPolθ is a translesion synthesis polymerase involved in parasite DNA damage tolerance, to confer resistance against macrophage aggression.

Lutzomyia migonei is a permissive vector competent for Leishmania infantum.

BACKGROUND: Leishmania infantum is the most widespread etiological agent of visceral leishmaniasis (VL) in the world, with significant mortality rates in human cases. In Latin America, this parasite is primarily transmitted by Lutzomyia longipalpis, but the role of Lutzomyia migonei as a potential vector for this protozoan has been discussed. Laboratory and field investigations have contributed to this hypothesis; however, proof of the vector competence of L. migonei has not yet been provided. In this study, we evaluate for the first time the susceptibility of L. migonei to L. infantum. METHODS: Females of laboratory-reared L. migonei were fed through a chick-skin membrane on rabbit blood containing L. infantum promastigotes, dissected at 1, 5 and 8 days post-infection (PI) and checked microscopically for the presence, intensity and localisation of Leishmania infections. In addition, morphometric analysis of L. infantum promastigotes was performed. RESULTS: High infection rates of both L. infantum strains tested were observed in L. migonei, with colonisation of the stomodeal valve already on day 5 PI. At the late-stage infection, most L. migonei females had their cardia and stomodeal valve colonised by high numbers of parasites, and no significant differences were found compared to the development in L. longipalpis. Metacyclic forms were found in all parasite-vector combinations since day 5 PI. CONCLUSIONS: We propose that Lutzomyia migonei belongs to sand fly species permissive to various Leishmania spp. Here we demonstrate that L. migonei is highly susceptible to the development of L. infantum. This, together with its known anthropophily, abundance in VL foci and natural infection by L. infantum, constitute important evidence that L. migonei is another vector of this parasite in Latin America.

In vitro antileishmanial activity of trans-stilbene and terphenyl compounds.

Leishmaniasis are globally widespread parasitic diseases which often leads to death if left untreated. Currently available drugs present different drawbacks, so there is an urgent need to develop new, safe and cost-effective drugs against leishmaniasis. In this study we tested a small library of trans-stilbene and terphenyl derivatives against promastigote, amastigotes and intramacrophage amastigote forms of Leishmania infantum. Two compounds of the series, the trans-stilbene 3 and the terphenyl 11, presented the best activity and safety profiles. Terphenyl 11 showed a leshmanicidal activity higher than pentostam and the ability to induce apoptosis selectively in Leishmania infantum while saving macrophages and primary epithelial cells. Our data indicate that terphenyl compounds, as well as stilbenes, are endowed with leishmanicidal activity, showing potential for further studies in the context of leishmanial therapy.

Serum Removal from Culture Induces Growth Arrest, Ploidy Alteration, Decrease in Infectivity and Differential Expression of Crucial Genes in Leishmania infantum Promastigotes.

Leishmania infantum is one of the species responsible for visceral leishmaniasis. This species is distributed basically in the Mediterranean basin. A recent outbreak in humans has been reported in Spain. Axenic cultures are performed for most procedures with Leishmania spp. promastigotes. This model is stable and reproducible and mimics the conditions of the gut of the sand fly host, which is the natural environment of promastigote development. Culture media are undefined because they contain mammalian serum, which is a rich source of complex lipids and proteins. Serum deprivation slows down the growth kinetics and therefore, yield in biomass. In fact, we have confirmed that the growth rate decreases, as well as infectivity. Ploidy is also affected. Regarding the transcriptome, a high-throughput approach has revealed a low differential expression rate but important differentially regulated genes. The most remarkable profiles are: up-regulation of the GINS Psf3, the fatty acyl-CoA synthase (FAS1), the glyoxylase I (GLO1), the hydrophilic surface protein B (HASPB), the methylmalonyl-CoA epimerase (MMCE) and an amastin gene; and down-regulation of the gPEPCK and the arginase. Implications for metabolic adaptations, differentiation and infectivity are discussed herein.

Differential Subcellular Localization of Leishmania Alba-Domain Proteins throughout the Parasite Development.

Alba-domain proteins are RNA-binding proteins found in archaea and eukaryotes and recently studied in protozoan parasites where they play a role in the regulation of virulence factors and stage-specific proteins. This work describes in silico structural characterization, cellular localization and biochemical analyses of Alba-domain proteins in Leishmania infantum. We show that in contrast to other protozoa, Leishmania have two Alba-domain proteins, LiAlba1 and LiAlba3, representative of the Rpp20- and the Rpp25-like eukaryotic subfamilies, respectively, which share several sequence and structural similarities but also important differences with orthologs in other protozoa, especially in sequences targeted for post-translational modifications. LiAlba1 and LiAlba3 proteins form a complex interacting with other RNA-binding proteins, ribosomal subunits, and translation factors as supported by co-immunoprecipitation and sucrose gradient sedimentation analysis. A higher co-sedimentation of Alba proteins with ribosomal subunits was seen upon conditions of decreased translation, suggesting a role of these proteins in translational repression. The Leishmania Alba-domain proteins display differential cellular localization throughout the parasite development. In the insect promastigote stage, Alba proteins co-localize predominantly to the cytoplasm but they translocate to the nucleolus and the flagellum upon amastigote differentiation in the mammalian host and are found back to the cytoplasm once amastigote differentiation is completed. Heat-shock, a major signal of amastigote differentiation, triggers Alba translocation to the nucleolus and the flagellum. Purification of the Leishmania flagellum confirmed LiAlba3 enrichment in this organelle during amastigote differentiation. Moreover, partial characterization of the Leishmania flagellum proteome of promastigotes and differentiating amastigotes revealed the presence of other RNA-binding proteins, as well as differences in the flagellum composition between these two parasite lifestages. Shuttling of Alba-domain proteins between the cytoplasm and the nucleolus or the flagellum throughout the parasite life cycle suggests that these RNA-binding proteins participate in several distinct regulatory pathways controlling developmental gene expression in Leishmania.

Cytotoxic Screening and In Vitro Evaluation of Pentadecane Against Leishmania infantum Promastigotes and Amastigotes.

Pentadecane is an organic compound that is made up of primarily carbon and hydrogen atoms. Pentadecane is a floral volatile found in many different plants and essential oils and also in crude extracts of some plants, and it shows antimicrobial activity. This study investigated in vitro effects of pentadecane in Leishmania infantum parasites and found that it decreases growth by 86 ± 2% both in promastigotes (half maximal inhibitory concentration [IC50] = 65.3 µM) and in amastigotes (IC50 = 60.5 µM), resulting in a reduction of macrophage infection; growth inhibition was 77% at 300 µM. Analysis of propidium iodide incorporation in L. infantum , treated with pentadecane at 48 hr, suggested that cells were arresting in the sub-G0/G1 and G1 phases of the cell cycle, whereas cytotoxicity assay of pentadecane in immortalized cells lines DH82 and U937 and in primary epithelial cells of Cercopiteco showed that it caused negligible cytotoxic effect. This study shows that pentadecane has antimicrobial activity against L. infantum parasites in in vitro culture.

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.

Papular dermatitis due to Leishmania infantum infection in seventeen dogs: diagnostic features, extent of the infection and treatment outcome.

UNLABELLED: : BACKGROUND: This study describes immunological responses, diagnostic features, follow up and treatment outcomes from seventeen dogs with papular dermatitis due to Leishmania infection diagnosed by cytology or real time-PCR. METHODS: Specific Leishmania humoral and cellular immune responses were evaluated by means of an immunofluorescence antibody test in all cases and a delayed-type hypersensitivity (DTH) reaction to leishmanin in eight cases. The extent of infection was studied in several tissues including blood, lymph node, conjunctival and oral swabs, by means of PCR, at the time of diagnosis and during follow-up. Culture was performed on nine dogs from cutaneous lesions and lymph node aspirates and molecular typing was carried out on isolates based on ITS-1, ITS-2 and Haspb gene sequencing analysis. RESULTS: Cytological and molecular results from fine needle aspirates of papules were diagnostic in 8 out of 13 (61.5%) cases and in 14 out of 15 dogs (93.3%), respectively. In all dogs, specific anti-Leishmania antibody levels were low or absent. Blood and lymph node PCRs and lymph node culture were negative in all dogs. Three out of the nine dogs (33%) were positive by culture from cutaneous lesions. The three isolates were identified as ITS type A, however, polymorphism was observed in the Haspb gene (PCR products of 626 bp, 962 bp and 371 bp). DTH response was positive in all tested dogs at the time of diagnosis. The majority of dogs were successfully treated with only N-methylglucamine antimoniate, after which cutaneous lesions disappeared or were reduced to depigmented, flattened scars. All dogs remained seronegative and the majority of dogs were negative by PCR in several tissues during follow-up. CONCLUSIONS: This study points out that papular dermatitis due to L. infantum is probably an underestimated benign cutaneous problem, associated with a parasite specific cell mediated immunity and a poor humoral immune response. Papular dermatitis is seen in young dogs, and appears to be a mild disease with restricted parasite dissemination and a good prognosis. PCR can be used as a non-invasive method to routinely evaluate papules if Leishmania infection is suspected in cases in which parasites are not visualized by cytology.

In silico and in vitro comparative activity of novel experimental derivatives against Leishmania major and Leishmania infantum promastigotes.

This in silico and in vitro comparative study was designed to evaluate the effectiveness of some biurets (K1 to K8) and glucantime against Leishmania major and Leishmania infantum promastigotes. Overall, eight experimental ligands and glucantime were docked using AutoDock 4.3 program into the active sites of Leishmania major and Leishmania infantum pteridine reductase 1, which were modeled using homology modeling programs. The colorimetric MTT assay was used to find L. major and L. infantum promastigotes viability at different concentrations of biuret derivatives in a concentration and time-dependent manner and the obtained results were expressed as 50% and 90% of inhibitory concentration (IC50 and IC90). In silico method showed that out of eight experimental ligands, four compounds were more active on pteridine reductase 1. K3 was the most active against L. major promastigotes with an IC50 of 6.8 µM and an IC90 of 40.2 µM, whereas for L. infantum promastigotes was K8 with IC50 of 7.8 µM. The phenylethyl derivative (K7) showed less toxicity (IC50s>60 µM) in both Leishmania strains. Glucantime displayed less growth inhibition in concentration of about 20 µM. In silico and especially docking results in a recent study were in accordance with the in vitro activity of these compounds in presented study and compound K3, K2 and K8 showed reasonable levels of selectivity for the Leishmania pteridine reductase 1.

TTAS a new stilbene derivative that induces apoptosis in Leishmania infantum.

Leishmania parasites are able to undergo apoptosis (programmed cell death), similarly to mammalian cells. Recently it was demonstrated in vitro the anti-leishmanial effect of some natural and synthetic stilbenoids including resveratrol and piceatannol. In this study we evaluated the Leishmanicidal activity of a pool of stilbene derivatives which had previously shown high apoptotic efficacy against neoplastic cells. All the compounds tested were capable to decrease the parasite viability in a dose-dependent manner. Trans-stilbenes proved to be markedly more effective than cis-isomers. This was different from that observed in tumor cells in which cis-stilbenes were more potent cytotoxic agents. Trans-3,4',5-trimethoxy-3'-amino-stilbene (TTAS) was the most active stilbene showing in Leishmania infantum a LD(50) value of 2.6 µg/mL. In contrast TTAS showed a low toxicity when tested on normal hemopoietic cells. This compound induced apoptosis in parasites by disrupting the mitochondrial membrane potential. Moreover it shows the ability to block Leishmania parasites in G(2)-M phase of cell cycle in agreement with the data obtained by affinity chromatography that identify tubulin as the putative target of TTAS. In conclusion, our results indicate that some stilbene derivatives are highly effective as anti-leishmanial agents and TTAS represents a pro-apoptotic agent in Leishmania parasites that merit further in vivo investigation.

Translational control through eIF2alpha phosphorylation during the Leishmania differentiation process.

The parasitic protozoan Leishmania alternates between an invertebrate and a mammalian host. Upon their entry to mammalian macrophages, Leishmania promastigotes differentiate into amastigote forms within the harsh environment of the phagolysosomal compartment. Here, we provide evidence for the importance of translational control during the Leishmania differentiation process. We find that exposure of promastigotes to a combined elevated temperature and acidic pH stress, a key signal triggering amastigote differentiation, leads to a marked decrease in global translation initiation, which is associated with eIF2α phosphorylation. Interestingly, we show that amastigotes adapted to grow in a cell-free medium exhibit lower levels of protein synthesis in comparison to promastigotes, suggesting that amastigotes have to enter a slow growth state to adapt to the stressful conditions encountered inside macrophages. Reconversion of amastigotes back to promastigote growth results in upregulation of global translation and a decrease in eIF2α phosphorylation. In addition, we show that while general translation is reduced during amastigote differentiation, translation of amastigote-specific transcripts such as A2 is preferentially upregulated. We find that A2 developmental gene regulation is triggered by temperature changes in the environment and that occurs mainly at the level of translation. Upon elevated temperature, the A2 transcript is stabilized through its association with polyribosomes leading to high levels of translation. When temperature decreases during amastigote to promastigote differentiation, the A2 transcript is not longer associated with translating polyribosomes and is being gradually degraded. Overall, these findings contribute to our better understanding of the adaptive responses of Leishmania to stress during its development and highlight the importance of translational control in promastigote to amastigote differentiation and vice-versa.

Genome-wide gene expression profile induced by exposure to cadmium acetate in Leishmania infantum promastigotes.

Leishmania infantum is the etiological agent of visceral leishmaniasis in Mediterranean areas. The life cycle of the protist is dimorphic and heteroxene, as promastigotes develop inside the gut of sand-fly vectors and amastigotes multiply inside mammalian phagocytic cells. In previous studies, we analyzed the expression profiles of these stages and the modulation of gene expression triggered by temperature increase and acidification, both of which are crucial in the differentiation of promastigotes to amastigotes. Differential expression profiles of translation initiation and elongation factors were detected. Here we report that the presence of 1 mM cadmium acetate in the culture medium leads to a shock response consisting of growth arrest, morphological changes, the absence of motility, and the up-regulation of genes that code for: a heavy metal transporter, trypanothione reductase, a haloacid-dehalogenase-like hydrolase, and a metalloexopeptidase from the M20 family, among others. This response is probably controlled by the differential expression of regulatory genes such as those encoding initiation factors 4E, eukaryotic translation initiation factor 3 subunits 8 and 2α, and elongation factor 1ß. The initiation factor 2α gene is induced in anomalous environments, i.e., those outside of the protist's normal life-cycle progression, for example, in response to the presence of cadmium ions, acidification without temperature increase, and vice versa. Our results suggest that the regulation of gene expression is a key component of the shock response.