DC-SIGN receptor is expressed by cells from cutaneous leishmaniasis lesions and differentially binds to Leishmania (Viannia) braziliensis and L. (Leishmania) amazonensis promastigotes.

BACKGROUND: Dendritic cells (DCs) specific intercellular adhesion molecule (ICAM)-3-grabbing non integrin receptor (DC-SIGN) binds to subgenera Leishmania promastigotes mediating its interaction with DC and neutrophils, potentially influencing the infection outcome. OBJECTIVES: In this work, we investigated whether DC-SIGN receptor is expressed in cells from cutaneous leishmaniasis (CL) lesions as well as the in vitro binding pattern of Leishmania (Viannia) braziliensis (Lb) and L. (L.) amazonensis (La) promastigotes. METHODS: DC-SIGN receptor was labeled by immunohistochemistry in cryopreserved CL tissue fragments. In vitro binding assay with CFSE-labeled Lb or La promastigotes and RAJI-transfecting cells expressing DC-SIGN (DC-SIGNPOS) or mock-transfected (DC-SIGNNEG) were monitored by flow cytometry at 2 h, 24 h and 48 h in co-culture. RESULTS: In CL lesion infiltrate, DC-SIGNPOS cells were present in the dermis and near the epidermis. Both Lb and La bind to DC-SIGNPOS cells, while binding to DC-SIGNNEG was low. La showed precocious and higher affinity to DC-SIGNhi population than to DC-SIGNlow, while Lb binding was similar in these populations. CONCLUSION: Our results demonstrate that DC-SIGN receptor is present in L. braziliensis CL lesions and interact with Lb promastigotes. Moreover, the differences in the binding pattern to Lb and La suggest DC-SIGN can influence in a difference way the intake of the parasites at the first hours after Leishmania infection. These results raise the hypothesis that DC-SIGN receptor could participate in the immunopathogenesis of American tegumentary leishmaniasis accounting for the differences in the outcome of the Leishmania spp. infection.

Dihydroartemisinin, an active metabolite of artemisinin, interferes with Leishmania braziliensis mitochondrial bioenergetics and survival.

Leishmaniasis is one of the most neglected parasitic infections of the world and current therapeutic options show several limitations. In the search for more effective drugs, plant compounds represent a powerful natural source. Artemisinin is a sesquiterpene lactone extracted from Artemisia annua L. leaves, from which dihydroartemisinin (DQHS) and artesunic acid (AA)/artesunate are examples of active derivatives. These lactones have been applied successfully on malaria therapy for decades. Herein, we investigated the sensitivity of Leishmania braziliensis, one of the most prevalent Leishmania species that cause cutaneous manifestations in the New World, to artemisinin, DQHS, and AA. L. braziliensis promastigotes and the stage that is targeted for therapy, intracelular amastigotes, were more sensitive to DQHS, showing EC50 of 62.3 ± 1.8 and 8.9 ± 0.9 µM, respectively. Cytotoxicity assays showed that 50% of bone marrow-derived macrophages cultures were inhibited with 292.8 ± 3.8 µM of artemisinin, 236.2 ± 4.0 µM of DQHS, and 396.8 ± 6.7 µM of AA. The control of intracellular infection may not be essentially attributed to the production of nitric oxide. However, direct effects on mitochondrial bioenergetics and H2O2 production appear to be associated with the leishmanicidal effect of DQHS. Our data provide support for further studies of artemisinin and derivatives repositioning for experimental leishmaniasis.

Biochemical changes in Leishmania braziliensis after photodynamic therapy with methylene blue assessed by the Fourier transform infrared spectroscopy.

Photodynamic therapy (PDT) with photosensitizer methylene blue was applied to Leishmania braziliensis, and Fourier transform infrared (FTIR) spectroscopy was used to study biochemical changes in the parasite after PDT in comparison to untreated (C), only irradiation (I), and only photosensitizer (PS). Spectral analysis suggests increase in lipids, proteins, and protein secondary structures in PDT compared with C and decrease in nucleic acids and carbohydrates. Interestingly, these trends are different from PDT of Leishmania major species, wherein lipids decrease; there are minimal changes in secondary structures and increase in nucleic acids and carbohydrates. The study thus suggests possibility of different biomolecular players/pathways in PDT-induced death of L. braziliensis and L. major.

Identification of the interactomes associated with SCD6 and RBP42 proteins in Leishmania braziliensis.

Leishmania are protozoan parasites responsible for leishmaniasis. These parasites present a precise gene regulation that allows them to survive different environmental conditions during their digenetic life cycle. This adaptation depends on the regulation of the expression of a wide variety of genes, which occurs, mainly at the post-transcriptional level. This differential gene expression is achieved by mechanisms based mainly in RNA binding proteins that regulate the translation and/or stability of mRNA targets by interaction with cis elements principally located in the untranslated regions (UTR). In recent studies, our group identified and characterized two proteins, SCD6 and RBP42, as RNA binding proteins in Leishmania braziliensis. To find clues about the cellular processes in which these proteins are involved, this work was aimed to determine the SCD6- and RBP42-interacting proteins (interactome) in L. braziliensis promastigotes. For this purpose, after an in vivo UV cross-linking, cellular extracts were used to immunoprecipitated, by specific antibodies, protein complexes in which SCD6 or RBP42 were present. Protein mass spectrometry analysis of the immunoprecipitated proteins identified 96 proteins presumably associated with SCD6 and 173 proteins associated with RBP42. Notably, a significant proportion of the identified proteins were shared in both interactomes, indicating a possible functional relationship between SCD6 and RBP42. Remarkably, many of the proteins identified in the SCD6 and RBP42 interactomes are related to RNA metabolism and translation processes, and many of them have been described as components of ribonucleoprotein (RNP) granules in Leishmania and related trypanosomatids. Thus, these results support a role of SCD6 and RBP42 in the assembly and/or function of mRNA-protein complexes, participating in the fate (decay/accumulation/translation) of L. braziliensis transcripts. SIGNIFICANCE: Parasites of the Leishmania genus present a particular regulation of gene expression, operating mainly at the post-transcriptional level, surely aimed to modulate quickly both mRNA and protein levels to survive the sudden environmental changes that occur during a parasite's life cycle as it moves from one host to another. This regulation of gene expression processes would be governed by the interaction of mRNA with RNA binding proteins. Nevertheless, the entirety of protein networks involved in these regulatory processes is far from being understood. In this regard, our work is contributing to stablish protein networks in which the L. braziliensis SCD6 and RBP42 proteins are involved; these proteins, in previous works, have been described as RNA binding proteins and found to participate in gene regulation in different cells and organisms. Additionally, our data point out a possible functional relationship between SCD6 and RBP42 proteins as constituents of mRNA granules, like processing bodies or stress granules, which are essential structures in the regulation of gene expression. This knowledge could provide a new approach for the development of therapeutic targets to control Leishmania infections.

Chemotactic activities of vasoactive intestinal peptide, neuropeptide Y and substance P in Leishmania braziliensis.

Cell-cell interaction and active migration (and invasion) of parasites into skin host-cell(s) are key steps for successful infection by Leishmania. Chemotaxis constitutes a primordial chapter of Leishmania-host cell interaction, potentially modulated by neuropeptides released into the skin due, for example, to the noxious stimuli represented by the insect bite. Herein we have evaluated in vitro the effect of sensory (Substance P, SP) and autonomic (Vasoactive Intestinal Peptide, VIP, and Neuropeptide Y, NPY) neuropeptides on parasite taxis, and investigated the potential modulatory effect of SP on Leishmania (Viannia) braziliensis-macrophage interaction. We demonstrated that VIP (10-10 M) and NPY (10-9 M) are chemorepellent to the parasites, while SP (10-8 M) produces a chemoattractant response. SP did not affect macrophage viability but seems to impair parasite-macrophage interaction as it decreased promastigote adherence to macrophages. As this effect is blocked by ([D-Pro 2, D-Trp7,9]-Substance P (10-6 M), the observed action may be mediated by neurokinin-1 (NK1) transmembrane receptors. VIP and NPY repellent chemotactic effect is impaired by their corresponding receptor antagonists. Additionally, they suggest that SP may be a key molecule to guide promastigote migration towards, and interaction, with dendritic cells and macrophage host cells.

Study of the differentially abundant proteins among Leishmania amazonensis, L. braziliensis, and L. infantum.

Leishmaniasis has been considered as emerging and re-emerging disease, and its increasing global incidence has raised concerns. The great clinical diversity of the disease is mainly determined by the species. In several American countries, tegumentary leishmaniasis (TL) is associated with both Leishmania amazonensis and L. braziliensis, while visceral leishmaniasis (VL) is associated with L. (L.) infantum. The major molecules that determine the most diverse biological variations are proteins. In the present study, through a DIGE approach, we identified differentially abundant proteins among the species mentioned above. We observed a variety of proteins with differential abundance among the studied species; and the biological networks predicted for each species showed that many of these proteins interacted with each other. The prominent proteins included the heat shock proteins (HSPs) and the protein network involved in oxide reduction process in L. amazonensis, the protein network of ribosomes in L. braziliensis, and the proteins involved in energy metabolism in L. infantum. The important proteins, as revealed by the PPI network results, enrichment categories, and exclusive proteins analysis, were arginase, HSPs, and trypanothione reductase in L. amazonensis; enolase, peroxidoxin, and tryparedoxin1 in L. braziliensis; and succinyl-CoA ligase [GDP -forming] beta-chain and transaldolase in L. infantum.

Mode of action of a formulation containing hydrazones and saponins against leishmania spp. Role in mitochondria, proteases and reinfection process.

Toxicity and poor adherence to treatment that favors the generation of resistance in the Leishmania parasites highlight the need to develop better alternatives. Here, we evaluated the in vitro effectiveness of hydrazone derived from chromanes 2-(2,3-dihydro-4H-1-benzothiopyran-4-ylidene) hydrazide (TC1) and 2-(2,3-dihydro-4H-1-benzopyran-4-ylidene) hydrazide (TC2) and the mixture of triterpene saponin hederagenin-3-O-(3,4-O-diacetyl-ß-D-xylopyranosyl-(1à3)-a-L- rhamnopyranosyl-(1à2)-a-L-arabinofuranoside, hederagenin-3-O-(3,4-O-diacetyl-a-L- arabinopyranosyl-(1à3)-a-L-rhamnopyranosyl-(1à2)-a-L-arabinofuranoside and, hederagenin-3-O-(4-O-acetyl-ß-D-xylopyranosyl-(1à3)-a-L-rhamnopyranosyl-(1à2)-a-L-arabinofuranoside from Sapindus saponaria (SS) on L. braziliensis and L. pifanoi. Mixtures of TC1 or TC2 with saponin were formulated for topical application and the therapeutic effectiveness was evaluated in the model for cutaneous leishmaniasis (CL) in golden hamster. The mode of action of these compounds was tested on various parasite processes and ultrastructural parasite modifications. TC1, TC2 and SS showed moderate cytotoxicity when tested independently but toxicity was improved when tested in combination. The compounds were more active against intracellular Leishmania amastigotes. In vivo studies showed that combinations of TC1 or TC2 with SS in 1:1 ratio (w/w) cured 100% of hamsters with no signs associated with toxicity. The compounds did cause changes in the mitochondrial activity of the parasite with a decrease in ATP levels and depolarization of membrane potential and overproduction of reactive oxygen species; nevertheless, these effects were not related to alterations in membrane permeability. The phagolysosome ultrastructure was also affected impacting the survival of Leishmania but the function of the lysosome nor the pH inside the phagolysosome did not change. Lastly, there was a protease inhibition which was directly related to the decrease in the ability of Leishmania to infect and multiply inside the macrophage. The results suggest that the combination of TC1 and TC2 with SS in a 1:1 ratio is capable of curing CL in hamsters. This effect may be due to the ability of these compounds to affect parasite survival and the ability to infect new cells.

Cellular localization, cloning and expression of Leishmania braziliensis Phospholipase A1.

Leishmaniasis is caused by several species of protozoan parasites of the genus Leishmania and represents an important global health problem. Leishmania braziliensis in particular is responsible of cutaneous and mucocutaneous forms of this parasitosis, with prevalence in Latin America. In the present work, we describe in L. braziliensis promastigotes and amastigotes the presence of a Phospholipase A1 (PLA1) activity, an enzyme that catalyses extensive deacylation of phospholipids like phosphatidylcholine. In order to deepen the knowledge about L. braziliensis PLA1, the cloning and expression of the gene that codifies for this enzyme was carried out in a baculovirus expression system with the obtaintion of a purified recombinant protein that displayed PLA1 activity. Given that this is the first molecular and functional protein characterization of a PLA1 in the Leishmania genus, we also performed a phylogenetic analysis of this gene throughout 12 species whose genome sequences were available. The results presented here will contribute to increase the knowledge about trypanosome phospholipases, which could be novel and valuable as potential targets to fight neglected diseases like Leishmaniasis.

Leishmania braziliensis prostaglandin F2α synthase impacts host infection.

BACKGROUND: Prostaglandins (PG) are lipid mediators derived from arachidonic acid metabolism. They are involved in cellular processes such as inflammation and tissue homeostasis. PG production is not restricted to multicellular organisms. Trypanosomatids also synthesize several metabolites of arachidonic acid. Nevertheless, their biological role in these early-branching parasites and their role in host-parasite interaction are not well elucidated. Prostaglandin F2α synthase (PGF2S) has been observed in the Leishmania braziliensis secreted proteome and in L. donovani extracellular vesicles. Furthermore, we previously reported a positive correlation between L. braziliensis PGF2S (LbrPGF2S) expression and pathogenicity in mice. METHODS: LbrPGF2S gene expression and PGF2α synthesis in promastigotes were detected and quantified by western blotting and EIA assay kit, respectively. To investigate LbrPGF2S localization in amastigotes during bone marrow-derived macrophage infection, parasites expressing mCherry-LbrPGF2S were generated and followed by time-lapse imaging for 48 h post-infection. PGF2S homolog sequences from Leishmania and humans were analyzed in silico using ClustalW on Geneious v6 and EMBOSS Needle. RESULTS: Leishmania braziliensis promastigotes synthesize prostaglandin F2α in the presence of arachidonic acid, with peak production in the stationary growth phase under heat stress. LbrPGF2S is a cytoplasmic protein enriched in the secretory site of the parasite cell body, the flagellar pocket. It is an enzyme constitutively expressed throughout promastigote development, but overexpression of LbrPGF2S leads to an increase of infectivity in vitro. The data suggest that LbrPGF2S may be released from intracellular amastigotes into the cytoplasm of bone marrow-derived macrophages over a 48-hour infection period, using time-lapse microscopy and mCherry-PGF2S (mChPGF2S)-expressing parasites. CONCLUSIONS: LbrPGF2S, a parasite-derived protein, is targeted to the host cell cytoplasm. The putative transfer of this enzyme, involved in pro-inflammatory lipid mediator synthesis, to the host cell suggests a potential role in host-parasite interaction and may partially explain the increased pathogenicity associated with overexpression of LbrPGF2S in L. braziliensis. Our data provide valuable insights to help understand the importance of parasite-derived lipid mediators in pathogenesis.

Tracking of quiescence in Leishmania by quantifying the expression of GFP in the ribosomal DNA locus.

Under stressful conditions some microorganisms adopt a quiescent stage characterized by a reversible non or slow proliferative condition that allows their survival. This adaptation was only recently discovered in Leishmania. We developed an in vitro model and a biosensor to track quiescence at population and single cell levels. The biosensor is a GFP reporter gene integrated within the 18S rDNA locus, which allows monitoring the expression of 18S rRNA (rGFP expression). We showed that rGFP expression decreased significantly and rapidly during the transition from extracellular promastigotes to intracellular amastigotes and that it was coupled in vitro with a decrease in replication as measured by BrdU incorporation. rGFP expression was useful to track the reversibility of quiescence in live cells and showed for the first time the heterogeneity of physiological stages among the population of amastigotes in which shallow and deep quiescent stages may coexist. We also validated the use of rGFP expression as a biosensor in animal models of latent infection. Our models and biosensor should allow further characterization of quiescence at metabolic and molecular level.

Calpains of Leishmania braziliensis: genome analysis, differential expression, and functional analysis.

BACKGROUND: Calpains are proteins belonging to the multi-gene family of calcium-dependent cysteine peptidases that undergo tight on/off regulation, and uncontrolled proteolysis of calpains is associated with severe human pathologies. Calpain orthologues are expanded and diversified in the trypanosomatids genome. OBJECTIVES: Here, we characterised calpains in Leishmania braziliensis, the main causative agent of cutaneous leishmaniasis in Brazil. METHODS/FINDINGS: In total, 34 predicted calpain-like genes were identified. After domain structure evaluation, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) during in vitro metacyclogenesis revealed (i) five genes with enhanced expression in the procyclic stage, (ii) one augmented gene in the metacyclic stage, and (iii) one procyclic-exclusive transcript. Western blot analysis revealed that an antibody against a consensus-conserved peptide reacted with multiple calpain-like proteins, which is consistent with the multi-gene family characteristic. Flow cytometry and immunocytochemistry analyses revealed the presence of calpain-like molecules mainly in the cytoplasm, to a lesser extent in the plasma membrane, and negligible levels in the nucleus, which are all consistent with calpain localisation. Eventually, the calpain inhibitor MDL28170 was used for functional studies revealing (i) a leishmaniostatic effect, (ii) a reduction in the association index in mouse macrophages, (iii) ultra-structural alterations conceivable with autophagy, and (iv) an enhanced expression of the virulence factor GP63. CONCLUSION: This report adds novel insights into the domain structure, expression, and localisation of L. braziliensis calpain-like molecules.

Antileishmanial and cytotoxic activities of ionic surfactants compared to those of miltefosine.

Using the electron paramagnetic resonance (EPR) of spin-labeled stearic acid and a spin label chemically attached to the membrane proteins, the interaction of miltefosine (MIL) and the ionic surfactants sodium dodecyl sulfate (SDS, anionic), cetyltrimethylammonium chloride (CTAC, cationic) and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS, zwitterionic) with the plasma membrane of Leishmania (L.) amazonensis promastigotes was studied. The spin-label EPR data indicated that the four compounds studied have the ability to increase the molecular dynamics of membrane proteins to a large extent. Compared to the other compounds, SDS produced the smallest increases in dynamics and demonstrated the lowest antileishmanial activity and cytotoxicity to J774.A1 macrophages. The activities of the other three compounds were not different from each other, but CTAC had a stronger activity against L. amazonensis promastigotes at higher cellular concentrations (> 1 × 109 cells/mL) and was the most effective against L. amazonensis-infected macrophages. However, CTAC was also the most cytotoxic to macrophages. By measuring the IC50/CC50 values for assays of different cell concentrations, we estimated the membrane-water partition coefficient (KM/W) as well as the concentrations in the membrane (cm50) and aqueous phase (cw50) of the compounds at their IC50/CC50. Compared to the other compounds, SDS showed the lowest value of KM/W and the highest value of cm50. In all experiments in this study, the data for the zwitterionic molecules HPS and MIL were not significantly different.

Major changes in chromosomal somy, gene expression and gene dosage driven by SbIII in Leishmania braziliensis and Leishmania panamensis.

Leishmania braziliensis and Leishmania panamensis are two species clinically and epidemiologically important, among others because of their relative resistance to first-line drugs (antimonials). The precise mechanism underlying the ability of these species to survive antimony treatment remains unknown. Therefore, elucidating the pathways mediating drug resistance is essential. We herein experimentally selected resistance to trivalent antimony (SbIII) in the reference strains of L. braziliensis (MHOM/BR75/M2904) and L. panamensis (MHOM/COL/81L13) and compared whole genome and transcriptome alterations in the culture promastigote stage. The results allowed us to identify differences in somy, copy number variations in some genes related to antimony resistance and large-scale copy number variations (deletions and duplications) in chromosomes with no somy changes. We found mainly in L. braziliensis, a direct relation between the chromosomal/local copy number variation and the gene expression. We identified differentially expressed genes in the resistant lines that are involved in antimony resistance, virulence, and vital biological processes in parasites. The results of this study may be useful for characterizing the genetic mechanisms of these Leishmania species under antimonial pressure, and for clarifying why the parasites are resistant to first-line drug treatments.

Intraspecies Polymorphisms in the Lipophosphoglycan of L. braziliensis Differentially Modulate Macrophage Activation via TLR4.

Lipophosphoglycan (LPG) is the major Leishmania surface glycoconjugate having importance during the host-parasite interface. Leishmania (Viannia) braziliensis displays a spectrum of clinical forms including: typical cutaneous leishmaniasis (TL), mucocutaneous (ML), and atypical lesions (AL). Those variations in the immunopathology may be a result of intraspecies polymorphisms in the parasite's virulence factors. In this context, we evaluated the role of LPG of strains originated from patients with different clinical manifestations and the sandfly vector. Six isolates of L. braziliensis were used: M2903, RR051 and RR418 (TL), RR410 (AL), M15991 (ML), and M8401 (vector). LPGs were extracted and purified by hydrophobic interaction. Peritoneal macrophages from C57BL/6 and respective knock-outs (TLR2-/- and TLR-4-/-) were primed with IFN-γ and exposed to different LPGs for nitric oxide (NO) and cytokine production (IL-1ß, IL-6, IL-12, and TNF-α). LPGs differentially activated the production of NO and cytokines via TLR4. In order to ascertain if such functional variations were related to intraspecies polymorphisms in the LPG, the purified glycoconjugates were subjected to western blot with specific LPG antibodies (CA7AE and LT22). Based on antibody reactivity preliminary variations in the repeat units were detected. To confirm these findings, LPGs were depolymerized for purification of repeat units. After thin layer chromatography, intraspecies polymorphisms were confirmed especially in the type and/size of sugars branching-off the repeat units motif. In conclusion, different isolates of L. braziliensis from different clinical forms and hosts possess polymorphisms in their LPGs that functionally affected macrophage responses.

Antileismanial activity, mechanism of action study and molecular docking of 1,4-bis(substituted benzalhydrazino)phthalazines.

To identify new agents for the treatment of American cutaneous leishmaniasis, a series of eight 1,4-bis(substituted benzalhydrazino)phthalazines was evaluated against Leishmania braziliensis and Leishmania mexicana parasites. These compounds represent a disubstituted version of the 1-chloro-4-(monoaryl/heteroarylhydranizyl)phthalazine that exhibited a significant response against L. braziliensis according to our previous findings. Two disubstituted phthalazines 3b and 3f were identified as potential antileishmanial agents against L. braziliensis parasites, exhibiting a submicromolar IC50 response of 2.37 and 7.90 µM on the promastigote form, and of 1.82 and 4.56 µM against intracellular amastigotes, respectively. In particular, compound 3b showed interesting responses against amastigote isolates from reference, glucantime-resistant and clinical human strains, which were by far superior to the biological response found for the glucantime drug. With regard to the toxicity results, both 3b and 3f exhibited moderate LD50 values against murine macrophages (BMDM), with good selectivity indexes on promastigotes and intracellular amastigotes of L. braziliensis. A comparison of biological response was established between the monosubstituted and disubstituted versions of these benzalhydrazino-phthalazines. Easy synthetic procedure and significant response against amastigote strains including against resistant lines made compound 3b a potential candidate for further pharmacokinetic and in vivo experiments as antileishmanial agent, and as a platform for further structural optimization. Mechanism-of-action studies and molecular docking simulations discarded to inhibition of superoxide dismutase as possible mode of action.

Protein-protein interactions of the nicotinamide/nicotinate mononucleotide adenylyltransferase of Leishmania braziliensis.

BACKGROUND: Nicotinamide adenine dinucleotide (NAD) plays a central role in energy metabolism and integrates cellular metabolism with signalling and gene expression. NAD biosynthesis depends on the enzyme nicotinamide/nicotinate mononucleotide adenylyltransferase (NMNAT; EC: 2.7.7.1/18), in which converge the de novo and salvage pathways. OBJECTIVE: The purpose of this study was to analyse the protein-protein interactions (PPI) of NMNAT of Leishmania braziliensis (LbNMNAT) in promastigotes. METHODS: Transgenic lines of L. braziliensis promastigotes were established by transfection with the pSP72αneoαLbNMNAT-GFP vector. Soluble protein extracts were prepared, co-immunoprecipitation assays were performed, and the co-immunoprecipitates were analysed by mass spectrometry. Furthermore, bioinformatics tools such as network analysis were applied to generate a PPI network. FINDINGS: Proteins involved in protein folding, redox homeostasis, and translation were found to interact with the LbNMNAT protein. The PPI network indicated enzymes of the nicotinate and nicotinamide metabolic routes, as well as RNA-binding proteins, the latter being the point of convergence between our experimental and computational results. MAIN CONCLUSION: We constructed a model of PPI of LbNMNAT and showed its association with proteins involved in various functions such as protein folding, redox homeostasis, translation, and NAD synthesis.

Anti-leishmanial effect of spiro dihydroquinoline-oxindoles on volume regulation decrease and sterol biosynthesis of Leishmania braziliensis.

Diverse spiro dihydroquinoline-oxindoles (JS series) were prepared using the BF3•OEt2-catalyzed imino Diels-Alder reaction between ketimine-isatin derivatives and trans-isoeugenol. Ten spiro-oxiindole derivatives were selected and evaluated at different stages of the life cycle of Leishmania braziliensis parasites, responsible for cutaneous leishmaniasis in South America. Among them, the 8'-ethyl-4'-(4-hydroxy-3-methoxyphenyl)-3'-methyl-3',4'-dihydro-1'H-spiro[indoline-3,2'-quinolin]-2-one called JS87 was able to inhibit the growth of promastigotes without affecting the mammalian cells viability, and to decrease the number of intracellular amastigotes of L. braziliensis. This spiro compound was found to act through the alteration of parasite internal regulation by disrupting the regulatory volume decrease (RVD), and to affect the sterol biosynthetic pathway at level of squalene epoxidase (SE) enzyme. These results revealed that the spiro annulation between quinoline and oxindole scaffolds enhances the anti-leishmanial activity, and could assist in the development of potent quinoline-oxindole hybrids against Leishmania braziliensis, the main etiological agent of cutaneous leishmaniasis in South America.

Generation and Characterization of a Dual-Reporter Transgenic Leishmania braziliensis Line Expressing eGFP and Luciferase.

In this study, we generated a transgenic strain of Leishmania braziliensis, an etiological agent associated with a diversity of clinical manifestations of leishmaniasis ranging from localized cutaneous to mucocutaneous to disseminated disease. Transgenic parasites expressing reporter proteins are valuable tools for studies of parasite biology, host-pathogen interactions, and anti-parasitic drug development. To this end, we constructed an L. braziliensis line stably expressing the reporters eGFP and luciferase (eGFP-LUC L. braziliensis). The integration cassette co-expressing the two reporters was targeted to the ribosomal locus (SSU) of the parasite genome. Transgenic parasites were characterized for their infectivity and stability both in vitro and in vivo. Parasite maintenance in axenic long-term culture in the absence of selective drugs did not alter expression of the two reporters or infection of BALB/c mice, indicating stability of the integrated cassette. Infectivity of eGFP-LUC, L. braziliensis, both in vivo and in vitro was similar to that obtained with the parental wild type strain. The possibility of L. braziliensis tracking and quantification using fluorescence and luminescence broadens the scope of research involving this neglected species, despite its importance in terms of public health concerning the leishmaniasis burden.

Calpains of Leishmania braziliensis: genome analysis, differential expression, and functional analysis

BACKGROUND Calpains are proteins belonging to the multi-gene family of calcium-dependent cysteine peptidases that undergo tight on/off regulation, and uncontrolled proteolysis of calpains is associated with severe human pathologies. Calpain orthologues are expanded and diversified in the trypanosomatids genome. OBJECTIVES Here, we characterised calpains in Leishmania braziliensis, the main causative agent of cutaneous leishmaniasis in Brazil. METHODS/FINDINGS In total, 34 predicted calpain-like genes were identified. After domain structure evaluation, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) during in vitro metacyclogenesis revealed (i) five genes with enhanced expression in the procyclic stage, (ii) one augmented gene in the metacyclic stage, and (iii) one procyclic-exclusive transcript. Western blot analysis revealed that an antibody against a consensus-conserved peptide reacted with multiple calpain-like proteins, which is consistent with the multi-gene family characteristic. Flow cytometry and immunocytochemistry analyses revealed the presence of calpain-like molecules mainly in the cytoplasm, to a lesser extent in the plasma membrane, and negligible levels in the nucleus, which are all consistent with calpain localisation. Eventually, the calpain inhibitor MDL28170 was used for functional studies revealing (i) a leishmaniostatic effect, (ii) a reduction in the association index in mouse macrophages, (iii) ultra-structural alterations conceivable with autophagy, and (iv) an enhanced expression of the virulence factor GP63. CONCLUSION This report adds novel insights into the domain structure, expression, and localisation of L. braziliensis calpain-like molecules.

Promastigote parasites cultured from the lesions of patients with mucosal leishmaniasis are more resistant to oxidative stress than promastigotes from a cutaneous lesion.

Human leishmaniasis caused by Leishmania (Viannia) braziliensis can be presented as localized cutaneous leishmaniasis (LCL) or mucosal leishmaniasis (ML). Macrophages kill parasites using nitric oxide (NO) and reactive oxygen species (ROS). The aim of this study was to evaluate the ability of parasites obtained from patients with LCL or ML to produce and resist NO or ROS. Promastigotes and amastigotes from LCL or ML isolates produced similar amounts of NO in culture. Promastigotes from ML isolates were more resistant to NO and H2O2 than LCL parasites in a stationary phase, whereas amastigotes from LCL isolates were more resistant to NO. In addition, in the stationary phase, promastigote isolates from patients with ML expressed more thiol-specific antioxidant protein (TSA) than LCL isolates. Therefore it is suggested that infective promastigotes from ML isolates are more resistant to microbicidal mechanisms in the initial phase of infection. Subsequently, amastigotes lose this resistance. This behavior of ML parasites can decrease the number of parasites capable of stimulating the host immune response shortly after the infection establishment.