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Sample records for leishmania major dihydroorotate

  1. Crystal structure of dihydroorotate dehydrogenase from Leishmania major.

    PubMed

    Cordeiro, Artur T; Feliciano, Patricia R; Pinheiro, Matheus P; Nonato, M Cristina

    2012-08-01

    Dihydroorotate dehydrogenase (DHODH) is the fourth enzyme in the de novo pyrimidine biosynthetic pathway and has been exploited as the target for therapy against proliferative and parasitic diseases. In this study, we report the crystal structures of DHODH from Leishmania major, the species of Leishmania associated with zoonotic cutaneous leishmaniasis, in its apo form and in complex with orotate and fumarate molecules. Both orotate and fumarate were found to bind to the same active site and exploit similar interactions, consistent with a ping-pong mechanism described for class 1A DHODHs. Analysis of LmDHODH structures reveals that rearrangements in the conformation of the catalytic loop have direct influence on the dimeric interface. This is the first structural evidence of a relationship between the dimeric form and the catalytic mechanism. According to our analysis, the high sequence and structural similarity observed among trypanosomatid DHODH suggest that a single strategy of structure-based inhibitor design can be used to validate DHODH as a druggable target against multiple neglected tropical diseases such as Leishmaniasis, Sleeping sickness and Chagas' diseases.

  2. Crystallization and preliminary X-ray diffraction analysis of Leishmania major dihydroorotate dehydrogenase

    SciTech Connect

    Cordeiro, Artur T.; Feliciano, Patricia R.; Nonato, M. Cristina

    2006-10-01

    Dihydroorotate dehydrogenase from L. major has been crystallized by the vapour-diffusion technique using lithium sulfate as the precipitant agent. A complete data set from a native crystal has been collected to 2.0 Å resolution using an in-house rotating-anode generator. Dihydroorotate dehydrogenases (DHODHs) are flavin-containing enzymes that catalyze the oxidation of l-dihydroorotate to orotate, the fourth step in the de novo pyrimidine nucleotide synthesis pathway. In this study, DHODH from Leishmania major has been crystallized by the vapour-diffusion technique using lithium sulfate as the precipitating agent. The crystals belong to space group P6{sub 1}, with unit-cell parameters a = 143.7, c = 69.8 Å. X-ray diffraction data were collected to 2.0 Å resolution using an in-house rotating-anode generator. Analysis of the solvent content and the self-rotation function indicate the presence of two molecules in the asymmetric unit. The structure has been solved by the molecular-replacement technique.

  3. The mechanistic study of Leishmania major dihydro-orotate dehydrogenase based on steady- and pre-steady-state kinetic analysis.

    PubMed

    Reis, Renata A G; Ferreira, Patricia; Medina, Milagros; Nonato, M Cristina

    2016-03-01

    Leishmania major dihydro-orotate dehydrogenase (DHODHLm) has been considered as a potential therapeutic target against leishmaniasis. DHODHLm, a member of class 1A DHODH, oxidizes dihydro-orotate (DHO) to orotate (ORO) during pyrimidine biosynthesis using fumarate (FUM) as the oxidizing substrate. In the present study, the chemistry of reduction and reoxidation of the flavin mononucleotide (FMN) cofactor in DHODHLm was examined by steady- and pre-steady state kinetics under both aerobic and anaerobic environments. Our results provide for the first time the experimental evidence of co-operative behaviour in class 1A DHODH regulated by DHO binding and reveal that the initial reductive flavin half-reaction follows a mechanism with two steps. The first step is consistent with FMN reduction and shows a hyperbolic dependence on the DHO concentration with a limiting rate (kred) of 110±6 s(-1) and a K(DHO) d of 180±27 μM. Dissociation of the reduced flavin-ORO complex corresponds to the second step, with a limiting rate of 6 s(-1). In the oxidative half-reaction, the oxygen-sensitive reoxidation of the reduced FMN cofactor of DHODHLm by FUM exhibited a hyperbolic saturation profile dependent on FUM concentration allowing estimation of K(FUM) d and the limiting rate (kreox) of 258±53 μM and 35±2 s(-1), respectively. Comparison between steady- and pre-steady-state parameters together with studies of interaction for DHODHLm with both ORO and succinate (SUC), suggests that ORO release is the rate-limiting step in overall catalysis. Our results provide evidence of mechanistic differences between class 1A and class 2 individual half-reactions to be exploited for the development of selective inhibitors.

  4. Plasmenylethanolamine synthesis in Leishmania major.

    PubMed

    Pawlowic, Mattie C; Hsu, Fong-Fu; Moitra, Samrat; Biyani, Neha; Zhang, Kai

    2016-07-01

    Ethanolamine glycerophospholipids are ubiquitous cell membrane components. Trypanosomatid parasites of the genus Leishmania synthesize the majority of their ethanolamine glycerophospholipids as 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine or plasmenylethanolamine (PME) through the Kennedy pathway. PME is a subtype of ether phospholipids also known as ethanolamine plasmalogen whose functions are not well characterized. In this study, we investigated the role of PME synthesis in Leishmania major through the characterization of an ethanolamine phosphotransferase (EPT) mutant. EPT-null parasites are largely devoid of PME and fully viable in regular medium but fail to proliferate in the absence of fetal bovine serum. They exhibit significant abnormalities in the synthesis and localization of GPI-anchored surface molecules. EPT-null mutants also show attenuated virulence in BALB/c mice. Furthermore, in addition to PME synthesis, ethanolamine also contributes to the production of phosphatidylcholine, the most abundant class of lipids in Leishmania. Together, these findings suggest that ethanolamine production is likely required for Leishmania promastigotes to generate bulk phospholipids, to handle stress, and to control the expression of membrane bound virulence factors. © 2016 John Wiley & Sons Ltd.

  5. Functional analysis of Leishmania major cyclophilin

    PubMed Central

    Yurchenko, Vyacheslav; Xue, Zhu; Sherry, Barbara; Bukrinsky, Michael

    2008-01-01

    A potent immunosuppressive drug cyclosporin A (CsA) is known to inhibit human cell infection by the pathogenic protozoan parasite Leishmania major both in vitro and in vivo. The proposed mechanism of action involves CsA binding to Leishmania major-expressed cyclophilin and subsequent down-regulation of signaling events necessary for establishing productive infection. Recently, we identified a ubiquitously expressed membrane protein, CD147, as a signaling receptor for extracellular cyclophilins in mammalian cells. Here we demonstrate that, while being enzymatically active, the Leishmania cyclophilin, unlike its human homologue, does not interact with CD147 on the cell surface of target cells. CD147 facilitates neither Leishmania binding nor infection. Primary structure and biochemical analyses revealed that the parasite’s cyclophilin is defective in heparan binding, an event required for signaling interaction between CD147 and human cyclophilin. When the heparan-binding motif was reconstituted in Leishmania cyclophilin, it regained the CD147-dependent signaling activity. These results underscore a critical role of cyclophilin-heparan interactions in CD147-mediated signaling events and argue against the role of Leishmania cyclophilin in parasite binding to target cells. PMID:17991468

  6. Mixed mucosal leishmaniasis infection caused by Leishmania tropica and Leishmania major.

    PubMed

    Shirian, Sadegh; Oryan, Ahmad; Hatam, Gholam Reza; Daneshbod, Yahya

    2012-11-01

    Mixed infections with different Leishmania species could explain differences in the clinical courses of these infections. On identification of Leishmania parasites from Iranian patients with mucosal leishmaniasis (ML), a patient with both oral and nasal lesions was found to be concomitantly infected with Leishmania tropica and L. major. Mixed infection was identified by PCR amplification of Leishmania kinetoplast DNA on scraping of cytological smears and histopathological sections. L. major and L. tropica were isolated from the nasal and oral lesions, respectively. These species were also confirmed by immunohistochemistry. This seems to be the first reported case of concurrent ML infection with two Leishmania species. It indicates that, at least in this patient, previous infection with one of these Leishmania species did not protect against infection with the other. This result has important implications for the development of vaccines against leishmaniases and implies careful attention in the treatment of this infectious disease.

  7. Structure of Leishmania major cysteine synthase

    PubMed Central

    Fyfe, Paul K.; Westrop, Gareth D.; Ramos, Tania; Müller, Sylke; Coombs, Graham H.; Hunter, William N.

    2012-01-01

    Cysteine biosynthesis is a potential target for drug development against parasitic Leishmania species; these protozoa are responsible for a range of serious diseases. To improve understanding of this aspect of Leishmania biology, a crystallographic and biochemical study of L. major cysteine synthase has been undertaken, seeking to understand its structure, enzyme activity and modes of inhibition. Active enzyme was purified, assayed and crystallized in an orthorhombic form with a dimer in the asymmetric unit. Diffraction data extending to 1.8 Å resolution were measured and the structure was solved by molecular replacement. A fragment of γ-poly-d-glutamic acid, a constituent of the crystallization mixture, was bound in the enzyme active site. Although a d-­glutamate tetrapeptide had insignificant inhibitory activity, the enzyme was competitively inhibited (K i = 4 µM) by DYVI, a peptide based on the C-­terminus of the partner serine acetyltransferase with which the enzyme forms a complex. The structure surprisingly revealed that the cofactor pyridoxal phosphate had been lost during crystallization. PMID:22750854

  8. Cyclic nucleotide specific phosphodiesterases of Leishmania major

    PubMed Central

    Johner, Andrea; Kunz, Stefan; Linder, Markus; Shakur, Yasmin; Seebeck, Thomas

    2006-01-01

    Background Leishmania represent a complex of important human pathogens that belong to the systematic order of the kinetoplastida. They are transmitted between their human and mammalian hosts by different bloodsucking sandfly vectors. In their hosts, the Leishmania undergo several differentiation steps, and their coordination and optimization crucially depend on numerous interactions between the parasites and the physiological environment presented by the fly and human hosts. Little is still known about the signalling networks involved in these functions. In an attempt to better understand the role of cyclic nucleotide signalling in Leishmania differentiation and host-parasite interaction, we here present an initial study on the cyclic nucleotide-specific phosphodiesterases of Leishmania major. Results This paper presents the identification of three class I cyclic-nucleotide-specific phosphodiesterases (PDEs) from L. major, PDEs whose catalytic domains exhibit considerable sequence conservation with, among other, all eleven human PDE families. In contrast to other protozoa such as Dictyostelium, or fungi such as Saccharomyces cerevisiae, Candida ssp or Neurospora, no genes for class II PDEs were found in the Leishmania genomes. LmjPDEA contains a class I catalytic domain at the C-terminus of the polypeptide, with no other discernible functional domains elsewhere. LmjPDEB1 and LmjPDEB2 are coded for by closely related, tandemly linked genes on chromosome 15. Both PDEs contain two GAF domains in their N-terminal region, and their almost identical catalytic domains are located at the C-terminus of the polypeptide. LmjPDEA, LmjPDEB1 and LmjPDEB2 were further characterized by functional complementation in a PDE-deficient S. cerevisiae strain. All three enzymes conferred complementation, demonstrating that all three can hydrolyze cAMP. Recombinant LmjPDEB1 and LmjPDEB2 were shown to be cAMP-specific, with Km values in the low micromolar range. Several PDE inhibitors were

  9. Leishmania major, the predominant Leishmania species responsible for cutaneous leishmaniasis in Mali.

    PubMed

    Paz, Carlos; Samake, Sibiry; Anderson, Jennifer M; Faye, Ousmane; Traore, Pierre; Tall, Koureishi; Cisse, Moumine; Keita, Somita; Valenzuela, Jesus G; Doumbia, Seydou

    2013-03-01

    Leishmania major is the only species of Leishmania known to cause cutaneous leishmanisis (CL) in Mali. We amplified Leishmania DNA stored on archived Giemsa-stained dermal scraping slides obtained from self-referral patients with clinically suspected CL seen in the Center National d'Appui A La Lutte Contre La Maladie (CNAM) in Bamako, Mali, to determine if any other Leishmania species were responsible for CL in Mali and evaluate its geographic distribution. Polymerase chain reaction (PCR) amplification was performed using a Leishmania species-specific primer pair that can amplify DNA from L. major, L. tropica, L. infantum, and L. donovani parasites, possible causative agents of CL in Mali. L. major was the only species detected in 41 microscopically confirmed cases of CL from five regions of Mali (Kayes, Koulikoro, Ségou, Mopti, and Tombouctou). These results implicate L. major as the predominant, possibly exclusive species responsible for CL in Mali.

  10. High density of Leishmania major and rarity of other mammals' Leishmania in zoonotic cutaneous leishmaniasis foci, Iran.

    PubMed

    Bordbar, Ali; Parvizi, Parviz

    2014-03-01

    Only Leishmania major is well known as a causative agent of zoonotic cutaneous leishmaniasis (ZCL) in Iran. Our objective was to find Leishmania parasites circulating in reservoir hosts, sand flies and human simultaneously. Sand flies, rodents and prepared smears of humans were sampled. DNA of Leishmania parasites was extracted, and two fragments of ITS-rDNA gene amplified by PCR. RFLP and sequencing were employed to identify Leishmania parasites. Leishmania major and L. turanica were identified unequivocally by targeting and sequencing ITS-rDNA from humans, rodents and sand flies. The new Leishmania species close to gerbilli (GenBank Accession Nos. EF413076; EF413087) was discovered only in sand flies. Based on parasite detection of ITS-rDNA in main and potential reservoir hosts and vectors and humans, we conclude that at least two Leishmania species are common in the Turkmen Sahra ZCL focus. Phylogenetic analysis proved that the new Leishmania is closely related to Leishmania mammal parasites (Leishmania major, Leishmania turanica, Leishmania gerbilli). Its role as a principal agent of ZCL is unknown because it was found only in sand flies. Our findings shed new light on the transmission cycles of several Leishmania parasites in sand flies, reservoir hosts and humans. © 2014 John Wiley & Sons Ltd.

  11. Fusion between Leishmania amazonensis and Leishmania major Parasitophorous Vacuoles: Live Imaging of Coinfected Macrophages

    PubMed Central

    Real, Fernando; Mortara, Renato A.; Rabinovitch, Michel

    2010-01-01

    Protozoan parasites of the genus Leishmania alternate between flagellated, elongated extracellular promastigotes found in insect vectors, and round-shaped amastigotes enclosed in phagolysosome-like Parasitophorous Vacuoles (PVs) of infected mammalian host cells. Leishmania amazonensis amastigotes occupy large PVs which may contain many parasites; in contrast, single amastigotes of Leishmania major lodge in small, tight PVs, which undergo fission as parasites divide. To determine if PVs of these Leishmania species can fuse with each other, mouse macrophages in culture were infected with non-fluorescent L. amazonensis amastigotes and, 48 h later, superinfected with fluorescent L. major amastigotes or promastigotes. Fusion was investigated by time-lapse image acquisition of living cells and inferred from the colocalization of parasites of the two species in the same PVs. Survival, multiplication and differentiation of parasites that did or did not share the same vacuoles were also investigated. Fusion of PVs containing L. amazonensis and L. major amastigotes was not found. However, PVs containing L. major promastigotes did fuse with pre-established L. amazonensis PVs. In these chimeric vacuoles, L. major promastigotes remained motile and multiplied, but did not differentiate into amastigotes. In contrast, in doubly infected cells, within their own, unfused PVs metacyclic-enriched L. major promastigotes, but not log phase promastigotes - which were destroyed - differentiated into proliferating amastigotes. The results indicate that PVs, presumably customized by L. major amastigotes or promastigotes, differ in their ability to fuse with L. amazonensis PVs. Additionally, a species-specific PV was required for L. major destruction or differentiation – a requirement for which mechanisms remain unknown. The observations reported in this paper should be useful in further studies of the interactions between PVs to different species of Leishmania parasites, and of the

  12. Fusion between Leishmania amazonensis and Leishmania major parasitophorous vacuoles: live imaging of coinfected macrophages.

    PubMed

    Real, Fernando; Mortara, Renato A; Rabinovitch, Michel

    2010-12-07

    Protozoan parasites of the genus Leishmania alternate between flagellated, elongated extracellular promastigotes found in insect vectors, and round-shaped amastigotes enclosed in phagolysosome-like Parasitophorous Vacuoles (PVs) of infected mammalian host cells. Leishmania amazonensis amastigotes occupy large PVs which may contain many parasites; in contrast, single amastigotes of Leishmania major lodge in small, tight PVs, which undergo fission as parasites divide. To determine if PVs of these Leishmania species can fuse with each other, mouse macrophages in culture were infected with non-fluorescent L. amazonensis amastigotes and, 48 h later, superinfected with fluorescent L. major amastigotes or promastigotes. Fusion was investigated by time-lapse image acquisition of living cells and inferred from the colocalization of parasites of the two species in the same PVs. Survival, multiplication and differentiation of parasites that did or did not share the same vacuoles were also investigated. Fusion of PVs containing L. amazonensis and L. major amastigotes was not found. However, PVs containing L. major promastigotes did fuse with pre-established L. amazonensis PVs. In these chimeric vacuoles, L. major promastigotes remained motile and multiplied, but did not differentiate into amastigotes. In contrast, in doubly infected cells, within their own, unfused PVs metacyclic-enriched L. major promastigotes, but not log phase promastigotes--which were destroyed--differentiated into proliferating amastigotes. The results indicate that PVs, presumably customized by L. major amastigotes or promastigotes, differ in their ability to fuse with L. amazonensis PVs. Additionally, a species-specific PV was required for L. major destruction or differentiation--a requirement for which mechanisms remain unknown. The observations reported in this paper should be useful in further studies of the interactions between PVs to different species of Leishmania parasites, and of the mechanisms

  13. In vitro evaluation of photodynamic therapy using curcumin on Leishmania major and Leishmania braziliensis.

    PubMed

    Pinto, Juliana Guerra; Fontana, Letícia Correa; de Oliveira, Marco Antonio; Kurachi, Cristina; Raniero, Leandro José; Ferreira-Strixino, Juliana

    2016-07-01

    Cutaneous leishmaniasis is an infectious disease caused by the Leishmania protozoan. The conventional treatment is long-lasting and aggressive, in addition to causing harmful effect. Photodynamic therapy has emerged as a promising alternative treatment, which allows local administration with fewer side effects. This study investigated the photodynamic activity of curcumin on Leishmania major and Leishmania braziliensis promastigote. Both species were submitted to incubation with curcumin in serial dilutions from 500 μg/ml up to 7.8 μg/ml. Control groups were kept in the dark while PDT groups received a fluency of 10 J/cm(2) at 450 nm. Mitochondrial activity was assessed by MTT assay 18 h after light treatment, and viability was measured by Trypan blue dye exclusion test. Morphological alterations were observed by Giemsa staining. Confocal microscopy showed the uptake of curcumin by both tested Leishmania species. Mitochondrial activity was inconclusive to determine viability; however, Trypan blue test was able to show that curcumin photodynamic treatment had a significant effect on viability of parasites. The morphology of promastigotes was highly affected by the photodynamic therapy. These results indicated that curcumin may be a promising alternative photosensitizer, because it presents no toxicity in the dark; however, further tests in co-culture with macrophages and other species of Leishmania should be conducted to determine better conditions before in vivo tests are performed.

  14. Detection of Leishmania major and Leishmania tropica in domestic cats in the Ege Region of Turkey.

    PubMed

    Paşa, Serdar; Tetik Vardarlı, Aslı; Erol, Nural; Karakuş, Mehmet; Töz, Seray; Atasoy, Abidin; Balcıoğlu, İ Cüneyt; Emek Tuna, Gülten; Ermiş, Özge V; Ertabaklar, Hatice; Özbel, Yusuf

    2015-09-15

    Leishmaniosis is a group of diseases caused by different species of Leishmania parasites in mammalian species. The aim of the present study was to investigate the presence of Leishmania spp. DNA in cats using real time polymerase chain reaction (RT-PCR) assays targeting internal transcribed spacer (ITS1) and heat-shock protein 70 gene (Hsp70) regions with Leishmania species-specific primers and probes. Blood samples were collected from 147 cats (73 female; 74 male) in the endemic regions for zoonotic visceral leishmaniasis in the western provinces of Turkey and analyzed using two RT-PCR assays. Additionally, Hsp70 RT-PCR products were sequenced. ELISA assays for feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) were also carried out for 145 of the 147 samples. Overall, 13/147 (8.84%) cats were positive for Leishmania by RT-PCR (4 L. major and 9 L. tropica). FIV and FeLV antibody and/or antigen was detected in 4 and 5 cats among Leishmania DNA positives, respectively. To the best of our knowledge, this study is the first to investigate and report the presence of L. major and L. tropica infections in a large group of domestic cats in Turkey. The results obtained indicate that species identification of Leishmania is essential for epidemiological understanding and that clinical signs alone are not indicative for leishmaniosis in cats, as it is in dogs. This study suggests that extensive research should be carried out in cat populations in order to fully understand the role of cats in the epidemiology of the disease. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Fitness and Phenotypic Characterization of Miltefosine-Resistant Leishmania major.

    PubMed

    Turner, Kimbra G; Vacchina, Paola; Robles-Murguia, Maricela; Wadsworth, Mariha; McDowell, Mary Ann; Morales, Miguel A

    2015-01-01

    Trypanosomatid parasites of the genus Leishmania are the causative agents of leishmaniasis, a neglected tropical disease with several clinical manifestations. Leishmania major is the causative agent of cutaneous leishmaniasis (CL), which is largely characterized by ulcerative lesions appearing on the skin. Current treatments of leishmaniasis include pentavalent antimonials and amphotericin B, however, the toxic side effects of these drugs and difficulty with distribution makes these options less than ideal. Miltefosine (MIL) is the first oral treatment available for leishmaniasis. Originally developed for cancer chemotherapy, the mechanism of action of MIL in Leishmania spp. is largely unknown. While treatment with MIL has proven effective, higher tolerance to the drug has been observed, and resistance is easily developed in an in vitro environment. Utilizing stepwise selection we generated MIL-resistant cultures of L. major and characterized the fitness of MIL-resistant L. major. Resistant parasites proliferate at a comparable rate to the wild-type (WT) and exhibit similar apoptotic responses. As expected, MIL-resistant parasites demonstrate decreased susceptibility to MIL, which reduces after the drug is withdrawn from culture. Our data demonstrate metacyclogenesis is elevated in MIL-resistant L. major, albeit these parasites display attenuated in vitro and in vivo virulence and standard survival rates in the natural sandfly vector, indicating that development of experimental resistance to miltefosine does not lead to an increased competitive fitness in L. major.

  16. An agent-based model for Leishmania major infection

    NASA Astrophysics Data System (ADS)

    Dancik, Garrett M.; Jones, Douglas E.; Dorman, Karin S.

    Leishmania are protozoan parasites transmitted by bites of infected sandflies. Over 20 species of Leishmania, endemic in 88 countries, are capable of causing human disease. Disease is either cutaneous, where skin ulcers occur on exposed surfaces of the body, or visceral, with near certain mortality if untreated. C3HeB/FeJ mice are resistant to L. major, but develop chronic cutaneous lesions when infected with another species L. amazonensis. The well-characterized mechanism of resistance to L. major depends on a CD4+ Thl immune response, macrophage activation, and elimination of the parasite [Sacks 2002]. The factors that account for host susceptibility to L. Amazonensis, however, are not completely understood, despite being generally attributed to a weakened Th1 response [Vanloubbeck 2004].

  17. An agent-based model for Leishmania major infection

    NASA Astrophysics Data System (ADS)

    Dancik, Garrett M.; Jones, Douglas E.; Dorman, Karin S.

    Leishmania are protozoan parasites transmitted by bites of infected sandflies. Over 20 species of Leishmania, endemic in 88 countries, are capable of causing human disease. Disease is either cutaneous, where skin ulcers occur on exposed surfaces of the body, or visceral, with near certain mortality if left untreated. C3HeB/FeJ mice are resistant to L. major, but develop chronic cutaneous lesions when infected with another species L. amazonensis. The well-characterized mechanism of resistance to L. major depends on a CD4+ Thl immune response, macrophage activation, and elimination of the parasite [Sacks 2002]. The factors that account for host susceptibility to L. Amazonensis, however, are not completely understood, despite being generally attributed to a weakened Th1 response [Vanloubbeck 2004].

  18. Glycosome turnover in Leishmania major is mediated by autophagy

    PubMed Central

    Cull, Benjamin; Prado Godinho, Joseane Lima; Fernandes Rodrigues, Juliany Cola; Frank, Benjamin; Schurigt, Uta; Williams, Roderick AM; Coombs, Graham H; Mottram, Jeremy C

    2015-01-01

    Autophagy is a central process behind the cellular remodeling that occurs during differentiation of Leishmania, yet the cargo of the protozoan parasite's autophagosome is unknown. We have identified glycosomes, peroxisome-like organelles that uniquely compartmentalize glycolytic and other metabolic enzymes in Leishmania and other kinetoplastid parasitic protozoa, as autophagosome cargo. It has been proposed that the number of glycosomes and their content change during the Leishmania life cycle as a key adaptation to the different environments encountered. Quantification of RFP-SQL-labeled glycosomes showed that promastigotes of L. major possess ∼20 glycosomes per cell, whereas amastigotes contain ∼10. Glycosome numbers were significantly greater in promastigotes and amastigotes of autophagy-defective L. major Δatg5 mutants, implicating autophagy in glycosome homeostasis and providing a partial explanation for the previously observed growth and virulence defects of these mutants. Use of GFP-ATG8 to label autophagosomes showed glycosomes to be cargo in ∼15% of them; glycosome-containing autophagosomes were trafficked to the lysosome for degradation. The number of autophagosomes increased 10-fold during differentiation, yet the percentage of glycosome-containing autophagosomes remained constant. This indicates that increased turnover of glycosomes was due to an overall increase in autophagy, rather than an upregulation of autophagosomes containing this cargo. Mitophagy of the single mitochondrion was not observed in L. major during normal growth or differentiation; however, mitochondrial remnants resulting from stress-induced fragmentation colocalized with autophagosomes and lysosomes, indicating that autophagy is used to recycle these damaged organelles. These data show that autophagy in Leishmania has a central role not only in maintaining cellular homeostasis and recycling damaged organelles but crucially in the adaptation to environmental change through the

  19. Phenylalanine hydroxylase (PAH) from the lower eukaryote Leishmania major.

    PubMed

    Lye, Lon-Fye; Kang, Song Ok; Nosanchuk, Joshua D; Casadevall, Arturo; Beverley, Stephen M

    2011-01-01

    Aromatic amino acid hydroxylases (AAAH) typically use tetrahydrobiopterin (H(4)B) as the cofactor. The protozoan parasite Leishmania major requires biopterin for growth and expresses strong salvage and regeneration systems to maintain H(4)B levels. Here we explored the consequences of genetic manipulation of the sole L. major phenylalanine hydroxylase (PAH) to explore whether it could account for the Leishmania H(4)B requirement. L. major PAH resembles AAAHs of other organisms, bearing eukaryotic-type domain organization, and conservation of key catalytic residues including those implicated in pteridine binding. A pah(-) null mutant and an episomal complemented overexpressing derivative (pah-/+PAH) were readily obtained, and metabolic labeling studies established that PAH was required to hydroxylate Phe to Tyr. Neither WT nor overexpressing lines were able to hydroxylate radiolabeled tyrosine or tryptophan, nor to synthesize catecholamines. WT but not pah(-) parasites showed reactivity with an antibody to melanin when grown with l-3,4-dihydroxyphenylalanine (L-DOPA), although the reactive product is unlikely to be melanin sensu strictu. WT was auxotrophic for Phe, Trp and Tyr, suggesting that PAH activity was insufficient to meet normal Tyr requirements. However, pah(-) showed an increased sensitivity to Tyr deprivation, while the pah(-)/+PAH overexpressor showed increased survival and could be adapted to grow well without added Tyr. pah(-) showed no alterations in H(4)B-dependent differentiation, as established by in vitro metacyclogenesis, or survival in mouse or macrophage infections. Thus Leishmania PAH may mitigate but not alleviate Tyr auxotrophy, but plays no essential role in the steps of the parasite infectious cycle. These findings suggest PAH is unlikely to explain the Leishmania requirement for biopterin.

  20. In Vitro and In Vivo Antileishmanial Effects of Pistacia khinjuk against Leishmania tropica and Leishmania major.

    PubMed

    Ezatpour, Behrouz; Saedi Dezaki, Ebrahim; Mahmoudvand, Hossein; Azadpour, Mojgan; Ezzatkhah, Fatemeh

    2015-01-01

    The present study aims to evaluate the in vitro and in vivo antileishmanial activities of Pistacia khinjuk Stocks (Anacardiaceae) alcoholic extract and to compare its efficacy with a reference drug, meglumine antimoniate (MA, Glucantime), against Leishmania tropica and Leishmania major. This extract (0-100 µg/mL) was evaluated in vitro against promastigote and intracellular amastigote forms of L. tropica (MRHO/IR/75/ER) and then tested on cutaneous leishmaniasis (CL) in male BALB/c mice with L. major to reproduce the antileishmanial activity topically. In vitro, P. khinjuk extract significantly (P < 0.05) inhibited the growth rate of promastigote (IC50 58.6 ± 3.2 µg/mL) and intramacrophage amastigotes (37.3 ± 2.5 µg/mL) of L. tropica as a dose-dependent response. In the in vivo assay, after 30 days of treatment, 75% recovery was observed in the infected mice treated with 30% extract. After treatment of the subgroups with the concentration of 20 and 30% of P. khinjuk extract, mean diameter of lesions was significantly (P < 0.05) reduced. To conclude, the present investigation demonstrated that P. vera extract had in vitro and in vivo effectiveness against L. major. Obtained findings also provide the scientific evidences that natural plants could be used in the traditional medicine for the prevention and treatment of CL.

  1. Protective immunity against Leishmania major induced by Leishmania tropica infection of BALB/c mice.

    PubMed

    Mahmoudzadeh-Niknam, Hamid; Kiaei, Simin Sadat; Iravani, Davood

    2011-02-01

    Leishmania (L.) tropica is a causative agent of human cutaneous and viscerotropic leishmaniasis. Immune response to L. tropica in humans and experimental animals are not well understood. We previously established that L. tropica infection induces partial protective immunity against subsequent challenge infection with Leishmania major in BALB/c mice. Aim of the present study was to study immunologic mechanisms of protective immunity induced by L. tropica infection, as a live parasite vaccine, in BALB/c mouse model. Mice were infected by L. tropica, and after establishment of the infection, they were challenged by L. major. Our findings shows that L. tropica infection resulted in protection against L. major challenge in BALB/c mice and this protective immunity is associated with: (1) a DTH response, (2) higher IFN-γ and lower IL-10 response at one week post-challenge, (3) lower percentage of CD4(+) lymphocyte at one month post-challenge, and (4) the source of IFN-γ and IL-10 were mainly CD4(-) lymphocyte up to one month post-challenge suggesting that CD4(-) lymphocytes may be responsible for protection induced by L. tropica infection in the studied intervals.

  2. In Vitro and In Vivo Antileishmanial Effects of Pistacia khinjuk against Leishmania tropica and Leishmania major

    PubMed Central

    Saedi Dezaki, Ebrahim; Mahmoudvand, Hossein; Azadpour, Mojgan; Ezzatkhah, Fatemeh

    2015-01-01

    The present study aims to evaluate the in vitro and in vivo antileishmanial activities of Pistacia khinjuk Stocks (Anacardiaceae) alcoholic extract and to compare its efficacy with a reference drug, meglumine antimoniate (MA, Glucantime), against Leishmania tropica and Leishmania major. This extract (0–100 µg/mL) was evaluated in vitro against promastigote and intracellular amastigote forms of L. tropica (MRHO/IR/75/ER) and then tested on cutaneous leishmaniasis (CL) in male BALB/c mice with L. major to reproduce the antileishmanial activity topically. In vitro, P. khinjuk extract significantly (P < 0.05) inhibited the growth rate of promastigote (IC50 58.6 ± 3.2 µg/mL) and intramacrophage amastigotes (37.3 ± 2.5 µg/mL) of L. tropica as a dose-dependent response. In the in vivo assay, after 30 days of treatment, 75% recovery was observed in the infected mice treated with 30% extract. After treatment of the subgroups with the concentration of 20 and 30% of P. khinjuk extract, mean diameter of lesions was significantly (P < 0.05) reduced. To conclude, the present investigation demonstrated that P. vera extract had in vitro and in vivo effectiveness against L. major. Obtained findings also provide the scientific evidences that natural plants could be used in the traditional medicine for the prevention and treatment of CL. PMID:25815025

  3. A proposed role for Leishmania major carboxypeptidase in peptide catabolism

    PubMed Central

    Isaza, Clara E.; Zhong, Xuejun; Rosas, Lucia E.; White, James D.; Chen, Rita P.-Y.; Liang, George F.-C.; Chan, Sunney I.; Satoskar, Abhay R.; Chan, Michael K.

    2008-01-01

    Leishmaniasis is a tropical disease caused by Leishmania, eukaryotic parasites transmitted to humans by sand flies. Towards the development of new chemotherapeutic targets for this disease, biochemical and in vivo expression studies were performed on one of two M32 carboxypeptidases present within the Leishmania major (LmaCP1) genome. Enzymatic studies reveal that like previously studied M32 carboxypeptidases, LmaCP1 cleaves substrates with a variety of C-terminal amino acids - the primary exception being those having C-terminal acidic residues. Cleavage assays with a series of FRET-based peptides suggest that LmaCP1 exhibits a substrate length restriction, preferring peptides shorter than 9–12 amino acids. The in vivo expression of LmaCP1 was analyzed for each major stage of the L. major life cycle. These studies reveal that LmaCP1 expression occurs only in procyclic promastigotes – the stage of life where the organism resides in the abdominal midgut of the insect. The implications of these results are discussed. PMID:18539138

  4. Neolignan Licarin A presents effect against Leishmania (Leishmania) major associated with immunomodulation in vitro.

    PubMed

    Néris, Patrícia L N; Caldas, John P A; Rodrigues, Yara K S; Amorim, Francianne M; Leite, Jacqueline A; Rodrigues-Mascarenhas, Sandra; Barbosa-Filho, José M; Rodrigues, Luis C; Oliveira, Márcia R

    2013-10-01

    Leishmaniasis' treatment is based mostly on pentavalent antimonials or amphotericin B long-term administration, expensive drugs associated with severe side effects. Considering these aforementioned, the search for alternative effective and safe leishmaniasis treatments is a necessity. This work evaluated a neolignan, licarin A anti-leishmanial activity chemically synthesized by our study group. It was observed that licarin A effectively inhibited Leishmania (Leishmania) major promastigotes (IC₅₀ of 9.59 ± 0.94 μg/mL) growth, by inducing in these parasites genomic DNA fragmentation in a typical death pattern by apoptosis. Additionally, the neolignan proved to be even more active against intracellular amastigotes of the parasite (EC₅₀ of 4.71 ± 0.29 μg/mL), and significantly more effective than meglumine antimoniate (EC₅₀ of 216.2 ± 76.7 μg/mL) used as reference drug. The antiamastigote activity is associated with an immunomodulatory activity, since treatment with licarin A of the infected macrophages induced a decrease in the interleukin (IL)-6 and IL-10 production. This study demonstrates for the first time the antileishmanial activity of licarin A and suggests that the compound may be a promising in the development of a new leishmanicidal agent. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Effect of Amphotericin B Nanodisks on Leishmania major Infected Mice

    PubMed Central

    Cole, PA; Bishop, JV; Beckstead, JA; Titus, R; Ryan, RO

    2014-01-01

    Objective To assess the efficacy of a novel formulation of the polyene antibiotic, amphotericin B (AMB), as therapy for cutaneous leishmaniasis in different mouse strains. Methods (AMB), was formulated into water-soluble transport particles, termed nanodisks (ND). Balb/c and CH3 mice infected with Leishmania major on Day 0 were administered vehicle alone, empty ND or AMB-ND on Day 1 and day 7, via the tail vein. Mice were sacrificed 25 or 50 days post inoculation and tissue histology evaluated. Balb/c mice treated with vehicle or empty ND showed signs of severe infection while CH3 mice had less inflammation and fewer parasites. AMB-ND treatment (2 mg/kg) had a marked therapeutic effect on L. major infected Balb/c mice and a discernable therapeutic benefit on CH3 mice. Conclusions AMB-ND is efficacious in the treatment of cutaneous leishmaniasis in both susceptible and resistant mouse strains. It may be inferred that AMB-ND may be useful for prophylactic and/or treatment of early stage Leishmania spp. infection. PMID:25584195

  6. Tamoxifen Induces Apoptosis of Leishmania major Promastigotes in Vitro.

    PubMed

    Doroodgar, Masoud; Delavari, Mahdi; Doroodgar, Moein; Abbasi, Ali; Taherian, Ali Akbar; Doroodgar, Abbas

    2016-02-01

    Tamoxifen is an antagonist of the estrogen receptor and currently used for the treatment of breast cancer. The current treatment of cutaneous leishmaniasis with pentavalent antimony compounds is not satisfactory. Therefore, in this study, due to its antileishmanial activity, effects of tamoxifen on the growth of promastigotes and amastigotes of Leishmania major Iranian strain were evaluated in vitro. Promastigotes and amastigotes were treated with different concentrations (1, 5, 10, 20, and 50 μg/ml) and time periods (24, 48, and 72 hr) of tamoxifen. After tamoxifen treatment, MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5 biphenyl tetrazolium bromide assay) was used to determine the percentage of live parasites and Graph Pad Prism software to calculate IC50. Flow cytometry was applied to investigate the induction of tamoxifen-induced apoptosis in promastigotes. The half maximal inhibitory concentration (IC50) of tamoxifen on promastigotes was 2.6 μg/ml after 24 hr treatment. Flow cytometry analysis showed that tamoxifen induced early and late apoptosis in Leishmania promastigotes. While after 48 hr in control group the apoptosis was 2.0%, the 50 µg/L concentration of tamoxifen increased it to 59.7%. Based on the in vitro antileishmanial effect, tamoxifen might be used for leishmaniasis treatment; however, further researches on in vivo effects of tamoxifen in animal models are needed.

  7. Structure of the SAS-6 cartwheel hub from Leishmania major

    PubMed Central

    van Breugel, Mark; Wilcken, Rainer; McLaughlin, Stephen H; Rutherford, Trevor J; Johnson, Christopher M

    2014-01-01

    Centrioles are cylindrical cell organelles with a ninefold symmetric peripheral microtubule array that is essential to template cilia and flagella. They are built around a central cartwheel assembly that is organized through homo-oligomerization of the centriolar protein SAS-6, but whether SAS-6 self-assembly can dictate cartwheel and thereby centriole symmetry is unclear. Here we show that Leishmania major SAS-6 crystallizes as a 9-fold symmetric cartwheel and provide the X-ray structure of this assembly at a resolution of 3.5 Å. We furthermore demonstrate that oligomerization of Leishmania SAS-6 can be inhibited by a small molecule in vitro and provide indications for its binding site. Our results firmly establish that SAS-6 can impose cartwheel symmetry on its own and indicate how this process might occur mechanistically in vivo. Importantly, our data also provide a proof-of-principle that inhibition of SAS-6 oligomerization by small molecules is feasible. DOI: http://dx.doi.org/10.7554/eLife.01812.001 PMID:24596152

  8. Structure of the SAS-6 cartwheel hub from Leishmania major.

    PubMed

    van Breugel, Mark; Wilcken, Rainer; McLaughlin, Stephen H; Rutherford, Trevor J; Johnson, Christopher M

    2014-01-01

    Centrioles are cylindrical cell organelles with a ninefold symmetric peripheral microtubule array that is essential to template cilia and flagella. They are built around a central cartwheel assembly that is organized through homo-oligomerization of the centriolar protein SAS-6, but whether SAS-6 self-assembly can dictate cartwheel and thereby centriole symmetry is unclear. Here we show that Leishmania major SAS-6 crystallizes as a 9-fold symmetric cartwheel and provide the X-ray structure of this assembly at a resolution of 3.5 Å. We furthermore demonstrate that oligomerization of Leishmania SAS-6 can be inhibited by a small molecule in vitro and provide indications for its binding site. Our results firmly establish that SAS-6 can impose cartwheel symmetry on its own and indicate how this process might occur mechanistically in vivo. Importantly, our data also provide a proof-of-principle that inhibition of SAS-6 oligomerization by small molecules is feasible. DOI: http://dx.doi.org/10.7554/eLife.01812.001.

  9. The first detection of Leishmania major in naturally infected Sergentomyia minuta in Portugal.

    PubMed

    Campino, Lenea; Cortes, Sofia; Dionísio, Lídia; Neto, Luís; Afonso, Maria Odete; Maia, Carla

    2013-06-01

    Phlebotomine sandflies of the genus Sergentomyia are widely distributed throughout the Old World. It has been suggested that Sergentomyia spp are involved in the transmission of Leishmania in India and Africa, whereas Phlebotomus spp are thought to be the sole vectors of Leishmania in the Old World. In this study, Leishmania major DNA was detected in one Sergentomyia minuta specimen that was collected in the southern region of Portugal. This study challenges the dogma that Leishmania is exclusively transmitted by species of the genus Phlebotomus in the Old World.

  10. The first detection of Leishmania major in naturally infected Sergentomyia minuta in Portugal

    PubMed Central

    Campino, Lenea; Cortes, Sofia; Dionísio, Lídia; Neto, Luís; Afonso, Maria Odete; Maia, Carla

    2013-01-01

    Phlebotomine sandflies of the genus Sergentomyia are widely distributed throughout the Old World. It has been suggested that Sergentomyia spp are involved in the transmission of Leishmania in India and Africa, whereas Phlebotomus spp are thought to be the sole vectors of Leishmania in the Old World. In this study, Leishmania major DNA was detected in one Sergentomyia minuta specimen that was collected in the southern region of Portugal. This study challenges the dogma that Leishmania is exclusively transmitted by species of the genus Phlebotomus in the Old World. PMID:23828004

  11. Expression and subcellular localization of ORC1 in Leishmania major

    SciTech Connect

    Kumar, Diwakar; Mukherji, Agnideep; Saha, Swati

    2008-10-10

    The mechanism of DNA replication is highly conserved in eukaryotes, with the process being preceded by the ordered assembly of pre-replication complexes (pre-RCs). Pre-RC formation is triggered by the association of the origin replication complex (ORC) with chromatin. Leishmania major appears to have only one ORC ortholog, ORC1. ORC1 in other eukaryotes is the largest of the ORC subunits and is believed to play a significant role in modulating replication initiation. Here we report for the first time, the cloning of ORC1 from L. major, and the analysis of its expression in L. major promastigotes. In human cells ORC1 levels have been found to be upregulated in G1 and subsequently degraded, thus playing a role in controlling replication initiation. We examine the subcellular localization of L. major ORC1 in relation to the different stages of the cell cycle. Our results show that, unlike what is widely believed to be the case with ORC1 in human cells, ORC1 in L. major is nuclear at all stages of the cell cycle.

  12. Holothuria leucospilota Extract Induces Apoptosis in Leishmania major Promastigotes

    PubMed Central

    FOROUTAN-RAD, Masoud; KHADEMVATAN, Shahram; SAKI, Jasem; HASHEMITABAR, Mahmoud

    2016-01-01

    Background: The present study aimed to survey antileishmanial activity of methanolic Holothuria leucospilota extract against Leishmania major promastigotes in vitro. Methods: Promastigotes were cultured in RPMI 1640 and after reaching the stationary phase, the study was conducted with different concentrations of the extract. Afterwards, MTT colorimetric assay for the obtaining of 50% inhibitory concentration (IC50) was utilized. Furthermore, in order to determine the possible induction of apoptosis in L. major promastigotes, flow cytometry and DNA fragmentation methods were employed using annexin-V FLUOS staining kit and DNA ladder kit, respectively. Results: The IC50 value of H. leucospilota extract at three time points of 24, 48, and 72 h was estimated 2000, 300 and 85 μg/ml, respectively. In addition, the extract revealed a dose and time-dependent antileishmanial activity. Furthermore, various characteristics of apoptosis appeared after L. major promastigotes treatment, which included cell shrinkage, formation of apoptotic bodies, blebbing of the cell membrane, and externalization of phosphatidylserine, although no laddering pattern was observed. Conclusion: The methanolic extract of H. leucospilota possesses lethal effect on L. major promastigotes and induces the apoptosis in parasites. Further studies are required to address the apoptosis mechanism in vivo. PMID:28127339

  13. Systems analysis of metabolism in the pathogenic trypanosomatid Leishmania major

    PubMed Central

    Chavali, Arvind K; Whittemore, Jeffrey D; Eddy, James A; Williams, Kyle T; Papin, Jason A

    2008-01-01

    Systems analyses have facilitated the characterization of metabolic networks of several organisms. We have reconstructed the metabolic network of Leishmania major, a poorly characterized organism that causes cutaneous leishmaniasis in mammalian hosts. This network reconstruction accounts for 560 genes, 1112 reactions, 1101 metabolites and 8 unique subcellular localizations. Using a systems-based approach, we hypothesized a comprehensive set of lethal single and double gene deletions, some of which were validated using published data with approximately 70% accuracy. Additionally, we generated hypothetical annotations to dozens of previously uncharacterized genes in the L. major genome and proposed a minimal medium for growth. We further demonstrated the utility of a network reconstruction with two proof-of-concept examples that yielded insight into robustness of the network in the presence of enzymatic inhibitors and delineation of promastigote/amastigote stage-specific metabolism. This reconstruction and the associated network analyses of L. major is the first of its kind for a protozoan. It can serve as a tool for clarifying discrepancies between data sources, generating hypotheses that can be experimentally validated and identifying ideal therapeutic targets. PMID:18364711

  14. Leishmania tropica infection, in comparison to Leishmania major, induces lower delayed type hypersensitivity in BALB/c mice.

    PubMed

    Mahmoudzadeh-Niknam, Hamid; Kiaei, Simin Sadat; Iravani, Davood

    2007-06-01

    Leishmania tropica and L. major are etiologic agents of human cutaneous leishmaniasis. Delayed type hypersensitivity (DTH) is an immunologic response that has been frequently used as a correlate for protection against or sensitization to leishmania antigen. In BALB/c mice, L. tropica infection results in non-ulcerating disease, whereas L. major infection results in destructive lesions. In order to clarify the immunologic mechanisms of these 2 different outcomes, we compared the ability of these 2 leishmania species in induction of DTH response in this murine model. BALB/c mice were infected with L. major or L. tropica, and disease evolution and DTH responses were determined. The results show that the primary L. major infection can exacerbate the secondary L. major infection and is associated with DTH response. Higher doses of the primary L. major infection result in more disease exacerbation of the secondary L. major infection as well as higher DTH response. L. tropica infection induces lower DTH responses than L. major. We have previously reported that the primary L. tropica infection induces partial protection against the secondary L. major infection in BALB/c mice. Induction of lower DTH response by L. tropica suggests that the protection induced against L. major by prior L. tropica infection may be due to suppression of DTH response.

  15. Leishmania tropica infection, in comparison to Leishmania major, induces lower delayed type hypersensitivity in BALB/c mice

    PubMed Central

    Kiaei, Simin Sadat; Iravani, Davood

    2007-01-01

    Leishmania tropica and L. major are etiologic agents of human cutaneous leishmaniasis. Delayed type hypersensitivity (DTH) is an immunologic response that has been frequently used as a correlate for protection against or sensitization to leishmania antigen. In BALB/c mice, L. tropica infection results in non-ulcerating disease, whereas L. major infection results in destructive lesions. In order to clarify the immunologic mechanisms of these 2 different outcomes, we compared the ability of these 2 leishmania species in induction of DTH response in this murine model. BALB/c mice were infected with L. major or L. tropica, and disease evolution and DTH responses were determined. The results show that the primary L. major infection can exacerbate the secondary L. major infection and is associated with DTH response. Higher doses of the primary L. major infection result in more disease exacerbation of the secondary L. major infection as well as higher DTH response. L. tropica infection induces lower DTH responses than L. major. We have previously reported that the primary L. tropica infection induces partial protection against the secondary L. major infection in BALB/c mice. Induction of lower DTH response by L. tropica suggests that the protection induced against L. major by prior L. tropica infection may be due to suppression of DTH response. PMID:17570972

  16. Effects of Sheep and Mouse Urine on the Growth Pattern of Leishmania major Promastigotes

    PubMed Central

    Karimi, Gholamreza; Dalimi, Abdolhossein; Paykari, Habibollah; Ghaffarifar, Fatemeh

    2013-01-01

    The protozoan parasites of the genus Leishmania are the causative agents of different clinical diseases. Fetal calf serum (FCS) is the main part and the most expensive ingredient of the Leishmania culture media. Here, the efficacies of different concentrations (1%, 2.5%, 5%, and 10%) of the filtered and autoclaved sheep and mouse urine were evaluated as a growth stimulator in Leishmania culture procedure. The results indicated that culture media enriched with the filtered sheep and mouse urine supported the growth of the parasites and can be used for cultivation of Leishmania parasites. In conclusion, this study has demonstrated an alternative low-cost medium that could be used in cultivation process of Leishmania major promastigotes. PMID:23957003

  17. The activity of ozonated olive oil against Leishmania major promastigotes

    PubMed Central

    Rajabi, Omid; Sazgarnia, Ameneh; Abbasi, Fatemeh; Layegh, Pouran

    2015-01-01

    Objective(s): Cutaneous Leishmaniasis is a common and endemic disease in Khorasan province in North-East of Iran. The pentavalant antimony (Sb V) is the mainstay of treatment that has many side effects and resistance to the drug has been reported. The microbicidal effect of ozone was proven in different microorganisms. Since there is no study in this respect and to achieve a low cost and effective treatment, we decided to evaluate the efficacy of ozone against promastigotes of Leishmania major, in vitro. Materials and Methods: Ozonated olive oil was prepared after production of ozone by bubbling ozone-oxygen gas produced by ozone generator through olive oil until it solidified. Promastigotes of L. major were cultivated in two phasic media. After calculation of the number of promastigotes, they were incubated with ozonated olive oil (0, 0.626, 0.938, 1.25, 2.5, 5, 10 mcg/ml) at 28 °c for 24 hr. Parasites survival percentage was evaluated using MTS and microscopic assay, and then compared with Glucantime and non-ozonated olive oil. Results: According to the results, there were significant differences in parasites survival percentage between ozonated olive oil and non-ozonated olive oil, at similar concentrations (P<0.001). Ozonated olive oil was more effective than Glucantime. According to MTS results, Glucantime and ozonated olive oil gel concentrations that are required to inhibit the growth of L. major promastigotes by 50% (IC50), were 165 and 0.002 mg/ml, respectively. Conclusion: Ozonated olive oil has in vitro activity against the promastigotes of L. major and this effect is dose dependent. PMID:26523224

  18. Cross-protective efficacy of Leishmania infantum LiHyD protein against tegumentary leishmaniasis caused by Leishmania major and Leishmania braziliensis species.

    PubMed

    Lage, Daniela Pagliara; Martins, Vívian Tamietti; Duarte, Mariana Costa; Costa, Lourena Emanuele; Tavares, Grasiele de Sousa Vieira; Ramos, Fernanda Fonseca; Chávez-Fumagalli, Miguel Angel; Menezes-Souza, Daniel; Roatt, Bruno Mendes; Tavares, Carlos Alberto Pereira; Coelho, Eduardo Antonio Ferraz

    2016-06-01

    Vaccination can be considered the most cost-effective strategy to control neglected diseases, but nowadays there is not an effective vaccine available against leishmaniasis. In the present study, a vaccine based on the combination of the Leishmania-specific hypothetical protein (LiHyD) with saponin was tested in BALB/c mice against infection caused by Leishmania major and Leishmania braziliensis species. This antigen was firstly identified in Leishmania infantum and showed to be protective against infection of BALB/c mice using this parasite species. The immunogenicity of rLiHyD/saponin vaccine was evaluated, and the results showed that immunized mice produced high levels of IFN-γ, IL-12 and GM-CSF after in vitro stimulation with rLiHyD, as well as by using L. major or L. braziliensis protein extracts. After challenge, vaccinated animals showed significant reductions in the infected footpad swellings, as well as in the parasite burden in the infection site, liver, spleen, and infected paws draining lymph nodes, when compared to those that were inoculated with the vaccine diluent (saline) or immunized with saponin. The immunization of rLiHyD without adjuvant was not protective against both challenges. The partial protection obtained by the rLiHyD/saponin vaccine was associated with a parasite-specific IL-12-dependent IFN-γ secretion, which was produced mainly by CD4(+) T cells. In these animals, a decrease in the parasite-mediated IL-4 and IL-10 responses, associated with the presence of high levels of LiHyD- and parasite-specific IgG2a isotype antibodies, were also observed. The present study showed that a hypothetical protein that was firstly identified in L. infantum, when combined to a Th1 adjuvant, was able to confer a cross-protection against highly infective stationary-phase promastigotes of two Leishmania species causing tegumentary leishmaniasis.

  19. Inhibition by Dications of in vitro growth of Leishmania major and Leishmania tropica: causative agents of old world cutaneous leishmaniasis.

    PubMed

    Rosypal, Alexa C; Werbovetz, Karl A; Salem, Manar; Stephens, Chad E; Kumar, Arvind; Boykin, David W; Hall, James E; Tidwell, Richard R

    2008-06-01

    Old World cutaneous leishmaniasis is caused by infection with Leishmania major and Leishmania tropica. Pentamidine and related dications exhibit broad spectrum antiprotozoal activity. Based on the previously reported efficacy of these compounds against related organisms, 18 structural analogs of pentamidine were evaluated for in vitro antileishmanial activity, using pentamidine as the standard reference drug for comparison. Furan analogs and reversed amidine compounds were examined for activity against L. major and L. tropica promastigotes. The most active compounds against both Leishmania species were in the reversed amidine series. DB745 and DB746 exhibited the highest activity against L. major and DB745 was the most active compound against L. tropica. Both of these compounds exhibited 50% inhibitory concentrations (IC50) below 1 nM for L. major. Ten reversed amidines were also tested for their ability to inhibit growth in an axenic amastigote model. Nine of 10 reversed amidine analogs were active at concentrations below 1 nM. These results justify further study of dicationic compounds as potential new agents for treating cutaneous leishmaniasis.

  20. Intracellular Survival of Leishmania major Depends on Uptake and Degradation of Extracellular Matrix Glycosaminoglycans by Macrophages

    PubMed Central

    Saunders, Eleanor C.; Kloehn, Joachim; Rupasinghe, Thusitha W.; Brown, Tracey J.; McConville, Malcolm J.

    2015-01-01

    Abstract Leishmania parasites replicate within the phagolysosome compartment of mammalian macrophages. Although Leishmania depend on sugars as a major carbon source during infections, the nutrient composition of the phagolysosome remains poorly described. To determine the origin of the sugar carbon source in macrophage phagolysosomes, we have generated a N-acetylglucosamine acetyltransferase (GNAT) deficient Leishmania major mutant (∆gnat) that is auxotrophic for the amino sugar, N-acetylglucosamine (GlcNAc). This mutant was unable to grow or survive in ex vivo infected macrophages even when macrophages were cultivated in presence of exogenous GlcNAc. In contrast, the L. major ∆gnat mutant induced normal skin lesions in mice, suggesting that these parasites have access to GlcNAc in tissue macrophages. Intracellular growth of the mutant in ex vivo infected macrophages was restored by supplementation of the macrophage medium with hyaluronan, a GlcNAc-rich extracellular matrix glycosaminoglycan. Hyaluronan is present and constitutively turned-over in Leishmania-induced skin lesions and is efficiently internalized into Leishmania containing phagolysosomes. These findings suggest that the constitutive internalization and degradation of host glycosaminoglycans by macrophages provides Leishmania with essential carbon sources, creating a uniquely favorable niche for these parasites. PMID:26334531

  1. Mycobacterium hsp65 DNA entrapped into TDM-loaded PLGA microspheres induces protection in mice against Leishmania (Leishmania) major infection.

    PubMed

    Coelho, Eduardo Antonio Ferraz; Tavares, Carlos Alberto Pereira; Lima, Karla de Melo; Silva, Célio Lopes; Rodrigues, José Maciel; Fernandes, Ana Paula

    2006-05-01

    Heat shock proteins (HSPs) are highly conserved among different organisms. A mycobacterial HSP65 DNA vaccine was previously shown to have prophylactic and immunotherapeutic effects against Mycobacterium tuberculosis infection in mice. Here, BALB/c mice were immunized with mycobacterial DNA-hsp65 or with DNA-hsp65 and trehalose dymicolate (TDM), both carried by biodegradable microspheres (MHSP/TDM), and challenged with Leishmania (Leishmania) major. MHSP/TDM conferred protection against L. major infection, as indicated by a significant reduction of edema and parasite loads in infected tissues. Although high levels of interferon-gamma and low levels of interleukin (IL)-4 and IL-10 were detected in mice immunized with DNA-hsp65 or MHSP/TDM, only animals immunized with MHSP/TDM displayed a consistent Th1 immune response, i.e., significantly higher levels of anti-soluble Leishmania antigen (SLA) immunoglobulin G (IgG)2a and low anti-SLA IgG1 antibodies. These findings indicate that encapsulated MHSP/TDM is more immunogenic than naked hsp65 DNA, and has great potential to improve vaccine effectiveness against leishmaniasis and tuberculosis.

  2. The interactions and essential effects of intrinsic insulin-like growth factor-I on Leishmania (Leishmania) major growth within macrophages.

    PubMed

    Reis, L C; Ramos-Sanchez, E M; Goto, H

    2013-07-01

    Previously, we showed in Leishmania infections that extrinsic insulin-like growth factor (IGF)-I favored Leishmania proliferation and leishmaniasis development. In this study, the interaction of intrinsically expressed IGF-I and Leishmania (Leishmania) major in macrophages was addressed, and a key finding was the observation, using confocal microscopy, of the co-localization of IGF-I and parasites within macrophages. Following stimulation with interferon-γ (IFN-γ), which is known to inhibit IGF-I production in macrophages, we observed a reduction in the expression of both IGF-I mRNA and protein. This reduced expression was accompanied by a reduction in the cellular parasite load that was completely recovered with the addition of extrinsic IGF-I, which suggests an essential role for IGF-I in Leishmania growth. © 2013 The Authors. Parasite Immunology published by John Wiley & Sons Ltd.

  3. High infection frequency, low diversity of Leishmania major and first detection of Leishmania turanica in human in northern Iran.

    PubMed

    Bordbar, Ali; Parvizi, Parviz

    2014-05-01

    Smears of suspected patients infected with zoonotic cutaneous leishmaniasis (ZCL) were stained and examined under a light microscopic observation. DNA of parasites within human ulcers was extracted directly from their smears. Nested PCR was used to amplify a fragment containing the internal transcribed spacers of the ribosomal RNA genes (ITS-rDNA) of Lesihmania parasites in human from Turkemen Sahara located in the northeastern part of Iran. Based on RFLP method by digesting BsuRI restriction enzyme and more precisely sequencing of DNA ITS-rDNA was shown to be species-specific. The infection rates of Leishmania parasites were high with 154 (93.9%) infections out of 164 suspected patients using microscopic observations. Only from 128 suspected patients out of 164, ITS-rDNA fragments were amplified and 125 samples had enough DNA to digest BsuRI restriction enzyme and do DNA sequencing. The Nested PCR assays detected not only Leishmania major but also Leishmania turanica for the first time, another parasite of the great gerbil in human. The density of L. major was high but the diversity was low with only 2 haplotypes. The overall ratio of L. major (123 infections) to L. turanica (2 infections) was significantly higher (Chi-squared test: p<0.05). Infections of L. turanica are not reported only and/or not known to cause human disease. Our analytical framework conveys a clear understanding of both L. major and L. turanica which can only be approved as causative agents of ZCL by more extensive sampling and followed by standardized molecular diagnosis. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Population genetics of Leishmania (Leishmania) major DNA isolated from cutaneous leishmaniasis patients in Pakistan based on multilocus microsatellite typing

    PubMed Central

    2014-01-01

    Background Cutaneous leishmaniasis (CL) is a major and fast increasing public health problem, both among the local Pakistani populations and the Afghan refugees in camps. Leishmania (Leishmania) major is one of the etiological agents responsible for CL in Pakistan. Genetic variability and population structure have been investigated for 66 DNA samples of L. (L.) major isolated from skin biopsy of CL patients. Methods Multilocus microsatellite typing (MLMT), employing 10 independent genetic markers specific to L. (L.) major, was used to investigate the genetic polymorphisms and population structures of Pakistani L. (L.) major DNA isolated from CL human cases. Their microsatellite profiles were compared to those of 130 previously typed strains of L. (L.) major from various geographical localities. Results All the markers were polymorphic and fifty-one MLMT profiles were recognized among the 66 L. (L.) major DNA samples. The data displayed significant microsatellite polymorphisms with rare allelic heterozygosities. A Bayesian model-based approach and phylogenetic analysis inferred two L. (L.) major populations in Pakistan. Thirty-four samples belonged to one population and the remaining 32 L. (L.) major samples grouped together into another population. The two Pakistani L. (L.) major populations formed separate clusters, which differ genetically from the populations of L. (L.) major from Central Asia, Iran, Middle East and Africa. Conclusions The considerable genetic variability of L. (L.) major might be related to the existence of different species of sand fly and/or rodent reservoir host in Sindh province, Pakistan. A comprehensive study of the epidemiology of CL including the situation or spreading of reservoirs and sand fly vectors in these foci is, therefore, warranted. PMID:25030377

  5. Cluster of zoonotic cutaneous leishmaniasis (Leishmania major) in European travelers returning from Turkmenistan.

    PubMed

    Larréché, Sébastien; Launay, Grégoire; Weibel Galluzzo, Christelle; Bousquet, Aurore; Eperon, Gilles; Pilo, Jean-Etienne; Ravel, Christophe; Chappuis, François; Dupin, Michel; Mérens, Audrey

    2013-01-01

    We report a cluster of cutaneous leishmaniasis due to Leishmania major in four immunocompetent travelers returning from Western Turkmenistan and having atypical and/or multiple lesions. Treatments with pentamidine or fluconazole were effective. Physicians should be aware that some virulent strains of L major currently circulate in Central Asia.

  6. Chromosome and gene copy number variation allow major structural change between species and strains of Leishmania.

    PubMed

    Rogers, Matthew B; Hilley, James D; Dickens, Nicholas J; Wilkes, Jon; Bates, Paul A; Depledge, Daniel P; Harris, David; Her, Yerim; Herzyk, Pawel; Imamura, Hideo; Otto, Thomas D; Sanders, Mandy; Seeger, Kathy; Dujardin, Jean-Claude; Berriman, Matthew; Smith, Deborah F; Hertz-Fowler, Christiane; Mottram, Jeremy C

    2011-12-01

    Leishmania parasites cause a spectrum of clinical pathology in humans ranging from disfiguring cutaneous lesions to fatal visceral leishmaniasis. We have generated a reference genome for Leishmania mexicana and refined the reference genomes for Leishmania major, Leishmania infantum, and Leishmania braziliensis. This has allowed the identification of a remarkably low number of genes or paralog groups (2, 14, 19, and 67, respectively) unique to one species. These were found to be conserved in additional isolates of the same species. We have predicted allelic variation and find that in these isolates, L. major and L. infantum have a surprisingly low number of predicted heterozygous SNPs compared with L. braziliensis and L. mexicana. We used short read coverage to infer ploidy and gene copy numbers, identifying large copy number variations between species, with 200 tandem gene arrays in L. major and 132 in L. mexicana. Chromosome copy number also varied significantly between species, with nine supernumerary chromosomes in L. infantum, four in L. mexicana, two in L. braziliensis, and one in L. major. A significant bias against gene arrays on supernumerary chromosomes was shown to exist, indicating that duplication events occur more frequently on disomic chromosomes. Taken together, our data demonstrate that there is little variation in unique gene content across Leishmania species, but large-scale genetic heterogeneity can result through gene amplification on disomic chromosomes and variation in chromosome number. Increased gene copy number due to chromosome amplification may contribute to alterations in gene expression in response to environmental conditions in the host, providing a genetic basis for disease tropism.

  7. First detection of Leishmania major DNA in Sergentomyia (Spelaeomyia) darlingi from cutaneous leishmaniasis foci in Mali.

    PubMed

    Berdjane-Brouk, Zohra; Koné, Abdoulaye K; Djimdé, Abdoulaye A; Charrel, Rémi N; Ravel, Christophe; Delaunay, Pascal; del Giudice, Pascal; Diarra, Adama Z; Doumbo, Siala; Goita, Siaka; Thera, Mahamadou A; Depaquit, Jérôme; Marty, Pierre; Doumbo, Ogobara K; Izri, Arezki

    2012-01-01

    Leishmania major complex is the main causative agent of zoonotic cutaneous leishmaniasis (ZCL) in the Old World. Phlebotomus papatasi and Phlebotomus duboscqi are recognized vectors of L. major complex in Northern and Southern Sahara, respectively. In Mali, ZCL due to L. major is an emerging public health problem, with several cases reported from different parts of the country. The main objective of the present study was to identify the vectors of Leishmania major in the Bandiagara area, in Mali. An entomological survey was carried out in the ZCL foci of Bandiagara area. Sandflies were collected using CDC miniature light traps and sticky papers. In the field, live female Phlebotomine sandflies were identified and examined for the presence of promastigotes. The remaining sandflies were identified morphologically and tested for Leishmania by PCR in the ITS2 gene. The source of blood meal of the engorged females was determined using the cyt-b sequence. Out of the 3,259 collected sandflies, 1,324 were identified morphologically, and consisted of 20 species, of which four belonged to the genus Phlebotomus and 16 to the genus Sergentomyia. Leishmania major DNA was detected by PCR in 7 of the 446 females (1.6%), specifically 2 out of 115 Phlebotomus duboscqi specimens, and 5 from 198 Sergentomyia darlingi specimens. Human DNA was detected in one blood-fed female S. darlingi positive for L. major DNA. Our data suggest the possible involvement of P. duboscqi and potentially S. darlingi in the transmission of ZCL in Mali.

  8. The route of Leishmania tropica infection determines disease outcome and protection against Leishmania major in BALB/c mice.

    PubMed

    Mahmoudzadeh-Niknam, Hamid; Khalili, Ghader; Abrishami, Firoozeh; Najafy, Ali; Khaze, Vahid

    2013-02-01

    Leishmania tropica is one of the causative agents of leishmaniasis in humans. Routes of infection have been reported to be an important variable for some species of Leishmania parasites. The role of this variable is not clear for L. tropica infection. The aim of this study was to explore the effects of route of L. tropica infection on the disease outcome and immunologic parameters in BALB/c mice. Two routes were used; subcutaneous in the footpad and intradermal in the ear. Mice were challenged by Leishmani major, after establishment of the L. tropica infection, to evaluate the level of protective immunity. Immune responses were assayed at week 1 and week 4 after challenge. The subcutaneous route in the footpad in comparison to the intradermal route in the ear induced significantly more protective immunity against L. major challenge, including higher delayed-type hypersensitivity responses, more rapid lesion resolution, lower parasite loads, and lower levels of IL-10. Our data showed that the route of infection in BALB/c model of L. tropica infection is an important variable and should be considered in developing an appropriate experimental model for L. tropica infections.

  9. Increased glycolytic ATP synthesis is associated with tafenoquine resistance in Leishmania major.

    PubMed

    Manzano, José Ignacio; Carvalho, Luis; Pérez-Victoria, José M; Castanys, Santiago; Gamarro, Francisco

    2011-03-01

    Tafenoquine (TFQ), an 8-aminoquinoline used to treat and prevent Plasmodium infections, could represent an alternative therapy for leishmaniasis. Indeed, TFQ has shown significant leishmanicidal activity both in vitro and in vivo, where it targets Leishmania mitochondria and activates a final apoptosis-like process. In order not to jeopardize the life span of this potential antileishmania drug, it is important to determine the likelihood that Leishmania will develop resistance to TFQ and the mechanisms of resistance induced. To address this issue, a TFQ-resistant Leishmania major promastigote line (R4) was selected. This resistance, which is unstable in a drug-free medium (revertant line), was maintained in intramacrophage amastigote forms, and R4 promastigotes were found to be cross-resistant to other 8-aminoquinolines. A decreased TFQ uptake, which is probably associated with an alkalinization of the intracellular pH rather than drug efflux, was observed for both the R4 and revertant lines. TFQ induces a decrease in ATP synthesis in all Leishmania lines, although total ATP levels were maintained at higher values in R4 parasites. In contrast, ATP synthesis by glycolysis was significantly increased in R4 parasites, whereas mitochondrial ATP synthesis was similar to that in wild-type parasites. We therefore conclude that increased glycolytic ATP synthesis is the main mechanism underlying TFQ resistance in Leishmania.

  10. The associations of Leishmania major and Leishmania tropica aspects by focusing their morphological and molecular features on clinical appearances in Khuzestan Province, Iran.

    PubMed

    Spotin, Adel; Rouhani, Soheila; Parvizi, Parviz

    2014-01-01

    Cutaneous leishmaniasis has various phenotypic aspects consisting of polymorphic amastigotes with different genetic ranges. Samples were collected from suspected patients of Khuzestan province. Prepared smears were stained, scaled, and measured using ocular micrometer. The Cyt b, ITS-rDNA, and microsatellite genes of Leishmania were amplified and Leishmania species were identified by molecular analyses. Of 150 examined suspected patients, 102 were identified to Leishmania species (90 L. major, nine L. tropica, and three unidentified). The amastigotes of 90 L. major had regular and different irregular shapes within three clinical lesions with no and/or low genetic diversity. Three haplotypes of Cyt b of L. major were found but no variation was observed using ITS-rDNA gene. Interesting findings were that all nine L. tropica had regular amastigote shapes with more genetic variations, also a patient which had coinfection of L. major, L. tropica, and Crithidia. At least two L. major and L. tropica were identified in suspected patients of the regions. Different irregular amastigotes' shapes of L. major can be explained by various reservoir hosts and vectors. In contrast, more molecular variations in L. tropica could be justified by genetic characters. Unidentified Leishmania could be mixed pathogens or nonpathogens with mammals' Leishmania or Crithidia.

  11. Lipid Droplet Formation, Their Localization and Dynamics during Leishmania major Macrophage Infection

    PubMed Central

    Rabhi, Sameh; Rabhi, Imen; Trentin, Bernadette; Piquemal, David; Regnault, Béatrice; Goyard, Sophie; Lang, Thierry; Descoteaux, Albert; Enninga, Jost; Guizani-Tabbane, Lamia

    2016-01-01

    Leishmania, the causative agent of vector-borne diseases, known as leishmaniases, is an obligate intracellular parasite within mammalian hosts. The outcome of infection depends largely on the activation status of macrophages, the first line of mammalian defense and the major target cells for parasite replication. Understanding the strategies developed by the parasite to circumvent macrophage defense mechanisms and to survive within those cells help defining novel therapeutic approaches for leishmaniasis. We previously showed the formation of lipid droplets (LDs) in L. major infected macrophages. Here, we provide novel insights on the origin of the formed LDs by determining their cellular distribution and to what extent these high-energy sources are directed to the proximity of Leishmania parasites. We show that the ability of L. major to trigger macrophage LD accumulation is independent of parasite viability and uptake and can also be observed in non-infected cells through paracrine stimuli suggesting that LD formation is from cellular origin. The accumulation of LDs is demonstrated using confocal microscopy and live-cell imagin in parasite-free cytoplasmic region of the host cell, but also promptly recruited to the proximity of Leishmania parasites. Indeed LDs are observed inside parasitophorous vacuole and in parasite cytoplasm suggesting that Leishmania parasites besides producing their own LDs, may take advantage of these high energy sources. Otherwise, these LDs may help cells defending against parasitic infection. These metabolic changes, rising as common features during the last years, occur in host cells infected by a large number of pathogens and seem to play an important role in pathogenesis. Understanding how Leishmania parasites and different pathogens exploit this LD accumulation will help us define the common mechanism used by these different pathogens to manipulate and/or take advantage of this high-energy source. PMID:26871576

  12. Lipid Droplet Formation, Their Localization and Dynamics during Leishmania major Macrophage Infection.

    PubMed

    Rabhi, Sameh; Rabhi, Imen; Trentin, Bernadette; Piquemal, David; Regnault, Béatrice; Goyard, Sophie; Lang, Thierry; Descoteaux, Albert; Enninga, Jost; Guizani-Tabbane, Lamia

    2016-01-01

    Leishmania, the causative agent of vector-borne diseases, known as leishmaniases, is an obligate intracellular parasite within mammalian hosts. The outcome of infection depends largely on the activation status of macrophages, the first line of mammalian defense and the major target cells for parasite replication. Understanding the strategies developed by the parasite to circumvent macrophage defense mechanisms and to survive within those cells help defining novel therapeutic approaches for leishmaniasis. We previously showed the formation of lipid droplets (LDs) in L. major infected macrophages. Here, we provide novel insights on the origin of the formed LDs by determining their cellular distribution and to what extent these high-energy sources are directed to the proximity of Leishmania parasites. We show that the ability of L. major to trigger macrophage LD accumulation is independent of parasite viability and uptake and can also be observed in non-infected cells through paracrine stimuli suggesting that LD formation is from cellular origin. The accumulation of LDs is demonstrated using confocal microscopy and live-cell imagin in parasite-free cytoplasmic region of the host cell, but also promptly recruited to the proximity of Leishmania parasites. Indeed LDs are observed inside parasitophorous vacuole and in parasite cytoplasm suggesting that Leishmania parasites besides producing their own LDs, may take advantage of these high energy sources. Otherwise, these LDs may help cells defending against parasitic infection. These metabolic changes, rising as common features during the last years, occur in host cells infected by a large number of pathogens and seem to play an important role in pathogenesis. Understanding how Leishmania parasites and different pathogens exploit this LD accumulation will help us define the common mechanism used by these different pathogens to manipulate and/or take advantage of this high-energy source.

  13. Vectorial competence of Phlebotomus papatasi (Diptera: Psychodidae) to transmit two old world Leishmania species: Leishmania major and L. Tropica.

    PubMed

    Darwish, A B; Tewfick, M K; Doha, S A; Abo-Ghalia, A H; Soliman, B A

    2011-12-01

    The vectorial competence of Phlebotomus papatasi for two old world Leishmania species, L. major & L. tropica was investigated. Phlebotomus papatasi originally collected from Suez Governorate, were membrane fed on homogenized hamster's lesion infected with L. major, MHOM/EG/06/RTC-63, and L. tropica, MGER/EG/06/RTC-74 identified from patients with suspected CL in Northern Sinai, Egypt. Fed flies were dissected at different time intervals and examined microscopically to determine the infection rate and parasite intensity. The feeding rate of P. papatasi on L. major (58.69%) was found higher than on L. tropica (45.99%). Infection rate with L. major (60.19%) was significantly higher than that with L. tropica (39.73%). Transmission by bites in case of P. papatasi/L. tropica failed. A characteristic L. major lesion was developed on the foot pads region 120 days post infective bites on healthy hamster. It is therefore concluded that P. papatasi is a much more effective vector for L. major than for L. tropica.

  14. Dataset for distribution of SIDER2 elements in the Leishmania major genome and transcriptome.

    PubMed

    Requena, Jose M; Rastrojo, Alberto; Garde, Esther; López, Manuel C; Thomas, M Carmen; Aguado, Begoña

    2017-04-01

    This paper contains data related to the research article entitled "Genomic cartography and proposal of nomenclature for the repeated, interspersed elements of the Leishmania major SIDER2 family and identification of SIDER2-containing transcripts" [1]. SIDER2 elements are repeated sequences, derived from, nowadays, extinct retrotransposons, that populate the genomes of protist of the genera Leishmania. This dataset (Supplementary file 1), an inventory of 1100 SIDER2 elements, was generated by surveying the L. major complete genome using bioinformatics tools with further manual refinements. In addition to the genomic distribution of these elements (summarized in Fig. 1), this dataset contains information regarding their association with specific transcripts, based on the recently established transcriptome for L. major[2].

  15. Enhanced survival of Leishmania major in neutrophil granulocytes in the presence of apoptotic cells

    PubMed Central

    Hellberg, Lars; Köhl, Jörg; Laskay, Tamás

    2017-01-01

    Neutrophil granulocytes are the first leukocytes that encounter and phagocytose Leishmania major (L. major) parasites in the infected skin. The parasites can nonetheless survive within neutrophils. However, the mechanisms enabling the survival of Leishmania within neutrophils are still elusive. Previous findings indicated that human neutrophils can engulf apoptotic cells. Since apoptotic neutrophils are abundant in infected tissues, we hypothesized that the uptake of apoptotic cells results in diminished anti-leishmanial activity and, consequently, contributes to enhanced survival of the parasites at the site of infection. In the present study, we demonstrated that L. major-infected primary human neutrophils acquire enhanced capacity to engulf apoptotic cells. This was associated with increased expression of the complement receptors 1 and 3 involved in phagocytosis of apoptotic cells. Next, we showed that ingestion of apoptotic cells affects neutrophil antimicrobial functions. We observed that phagocytosis of apoptotic cells by neutrophils downregulates the phosphorylation of p38 MAPK and PKCδ, the kinases involved in activation of NADPH oxidase and hence reactive oxygen species (ROS) production. In line, uptake of apoptotic cells inhibits TNF- and L. major-induced ROS production by neutrophils. Importantly, we found that the survival of Leishmania in neutrophils is strongly enhanced in neutrophils exposed to apoptotic cells. Together, our findings reveal that apoptotic cells promote L. major survival within neutrophils by downregulating critical antimicrobial functions. This suggests that the induction of enhanced uptake of apoptotic cells represents a novel evasion mechanism of the parasites that facilitates their survival in neutrophil granulocytes. PMID:28187163

  16. Previous exposure to a low infectious dose of Leishmania major exacerbates infection with Leishmania infantum in the susceptible BALB/c mouse.

    PubMed

    Nation, Catherine S; Dondji, Blaise; Stryker, Gabrielle A

    2012-09-01

    The geographic distribution of Leishmania major overlaps with several other species of Leishmania. This study seeks to examine what effect previous exposure to L. major has on the outcome of infection with Leishmania infantum, the agent of virulent visceral leishmaniasis. The L. major immune response is well characterized by a strong Th1 response leading to resolution and protection against subsequent re-infection. A contrasting Th2 immune response leads to disseminated disease, while the role Th17 cytokines may play in Leishmania infection is still being explored. The cytokine profile, antibody titer, and parasite burden were evaluated in the susceptible BALB/c mouse after L. infantum infection in either naïve mice or those previously infected with a low/self-healing dose of L. major. Only IL-4 expression in mice previously exposed to L. major was found to be significantly increased over controls, a cytokine with an ambiguous role in L. infantum infection. However, disease exacerbation, with a notably higher parasite burden, was observed in the L. major exposed mice compared to the L. infantum only. Cross-reactive antibodies were seen in both groups of infected mice regardless of their immune history. Studies have shown a role for opsonizing antibodies leading to increased disease in visceral leishmaniasis. We speculate that cross-reactive antibodies may be playing a role in augmenting visceral disease in mice with immunological memory to L. major.

  17. Lipophosphoglycan is a virulence factor distinct from related glycoconjugates in the protozoan parasite Leishmania major

    PubMed Central

    Späth, Gerald F.; Epstein, Linda; Leader, Ben; Singer, Steven M.; Avila, Herbert A.; Turco, Salvatore J.; Beverley, Stephen M.

    2000-01-01

    Protozoan parasites of the genus Leishmania undergo a complex life cycle involving transmission by biting sand flies and replication within mammalian macrophage phagolysosomes. A major component of the Leishmania surface coat is the glycosylphosphatidylinositol (GPI)-anchored polysaccharide called lipophosphoglycan (LPG). LPG has been proposed to play many roles in the infectious cycle, including protection against complement and oxidants, serving as the major ligand for macrophage adhesion, and as a key factor mitigating host responses by deactivation of macrophage signaling pathways. However, all structural domains of LPG are shared by other major surface or secretory products, providing a biochemical redundancy that compromises the ability of in vitro tests to establish whether LPG itself is a virulence factor. To study truly lpg− parasites, we generated Leishmania major lacking the gene LPG1 [encoding a putative galactofuranosyl (Galf) transferase] by targeted gene disruption. The lpg1− parasites lacked LPG but contained normal levels of related glycoconjugates and GPI-anchored proteins. Infections of susceptible mice and macrophages in vitro showed that these lpg− Leishmania were highly attenuated. Significantly and in contrast to previous LPG mutants, reintroduction of LPG1 into the lpg− parasites restored virulence. Thus, genetic approaches allow dissection of the roles of this complex family of interrelated parasite virulence factors, and definitively establish the role of LPG itself as a parasite virulence factor. Because the lpg1− mutant continue to synthesize bulk GPI-anchored Galf-containing glycolipids other than LPG, a second pathway distinct from the Golgi-associated LPG synthetic compartment must exist. PMID:10908670

  18. Expression of a hydrophilic surface protein in infective stages of Leishmania major.

    PubMed

    Flinn, H M; Rangarajan, D; Smith, D F

    1994-06-01

    A family of differentially expressed genes from Leishmania major contains one sequence (Gene B) that encodes a novel, hydrophilic protein found on the surface of infective parasite stages. The 177-residue, acidic Gene B protein is characterised by an amino acid repetitive element, comprising 45% of the total molecule, that is related to the cell-wall binding domain of protein A from Staphylococcus aureus. No identifiable signal peptide, membrane-spanning domain or consensus for glycosylphosphatidylinositol anchor attachment to the cell surface is found elsewhere in the deduced protein sequence. In vitro, the Gene B protein fractionates with the parasite cell surface glycoconjugates, lipophosphoglycan and the glycoinositolphospholipids. This protein is the first characterised surface peptide marker for infective stages of the Leishmania life cycle.

  19. Fumarate hydratase isoforms of Leishmania major: subcellular localization, structural and kinetic properties.

    PubMed

    Feliciano, Patrícia R; Gupta, Shreedhara; Dyszy, Fabio; Dias-Baruffi, Marcelo; Costa-Filho, Antonio J; Michels, Paul A M; Nonato, M Cristina

    2012-01-01

    Fumarate hydratases (FHs; EC 4.2.1.2) are enzymes that catalyze the reversible hydration of fumarate to S-malate. Parasitic protists that belong to the genus Leishmania and are responsible for a complex of vector-borne diseases named leishmaniases possess two genes that encode distinct putative FH enzymes. Genome sequence analysis of Leishmania major Friedlin reveals the existence of genes LmjF24.0320 and LmjF29.1960 encoding the putative enzymes LmFH-1 and LmFH-2, respectively. In the present work, the FH activity of both L. major enzymes has been confirmed. Circular dichroism studies suggest important differences in terms of secondary structure content when comparing LmFH isoforms and even larger differences when comparing them to the homologous human enzyme. CD melting experiments revealed that both LmFH isoforms are thermolabile enzymes. The catalytic efficiency under aerobic and anaerobic environments suggests that they are both highly sensitive to oxidation and damaged by oxygen. Intracellular localization studies located LmFH-1 in the mitochondrion, whereas LmFH-2 was found predominantly in the cytosol with possibly also some in glycosomes. The high degree of sequence conservation in different Leishmania species, together with the relevance of FH activity for the energy metabolism in these parasites suggest that FHs might be exploited as targets for broad-spectrum antileishmanial drugs.

  20. Coadministration of the Three Antigenic Leishmania infantum Poly (A) Binding Proteins as a DNA Vaccine Induces Protection against Leishmania major Infection in BALB/c Mice.

    PubMed

    Soto, Manuel; Corvo, Laura; Garde, Esther; Ramírez, Laura; Iniesta, Virginia; Bonay, Pedro; Gómez-Nieto, Carlos; González, Víctor M; Martín, M Elena; Alonso, Carlos; Coelho, Eduardo A F; Barral, Aldina; Barral-Netto, Manoel; Iborra, Salvador

    2015-05-01

    Highly conserved intracellular proteins from Leishmania have been described as antigens in natural and experimental infected mammals. The present study aimed to evaluate the antigenicity and prophylactic properties of the Leishmania infantum Poly (A) binding proteins (LiPABPs). Three different members of the LiPABP family have been described. Recombinant tools based on these proteins were constructed: recombinant proteins and DNA vaccines. The three recombinant proteins were employed for coating ELISA plates. Sera from human and canine patients of visceral leishmaniasis and human patients of mucosal leishmaniasis recognized the three LiPABPs. In addition, the protective efficacy of a DNA vaccine based on the combination of the three Leishmania PABPs has been tested in a model of progressive murine leishmaniasis: BALB/c mice infected with Leishmania major. The induction of a Th1-like response against the LiPABP family by genetic vaccination was able to down-regulate the IL-10 predominant responses elicited by parasite LiPABPs after infection in this murine model. This modulation resulted in a partial protection against L. major infection. LiPABP vaccinated mice showed a reduction on the pathology that was accompanied by a decrease in parasite burdens, in antibody titers against Leishmania antigens and in the IL-4 and IL-10 parasite-specific mediated responses in comparison to control mice groups immunized with saline or with the non-recombinant plasmid. The results presented here demonstrate for the first time the prophylactic properties of a new family of Leishmania antigenic intracellular proteins, the LiPABPs. The redirection of the immune response elicited against the LiPABP family (from IL-10 towards IFN-γ mediated responses) by genetic vaccination was able to induce a partial protection against the development of the disease in a highly susceptible murine model of leishmaniasis.

  1. Coadministration of the Three Antigenic Leishmania infantum Poly (A) Binding Proteins as a DNA Vaccine Induces Protection against Leishmania major Infection in BALB/c Mice

    PubMed Central

    Corvo, Laura; Garde, Esther; Ramírez, Laura; Iniesta, Virginia; Bonay, Pedro; Gómez-Nieto, Carlos; González, Víctor M.; Martín, M. Elena; Alonso, Carlos; Coelho, Eduardo A. F.; Barral, Aldina; Barral-Netto, Manoel

    2015-01-01

    Background Highly conserved intracellular proteins from Leishmania have been described as antigens in natural and experimental infected mammals. The present study aimed to evaluate the antigenicity and prophylactic properties of the Leishmania infantum Poly (A) binding proteins (LiPABPs). Methodology/Principal Findings Three different members of the LiPABP family have been described. Recombinant tools based on these proteins were constructed: recombinant proteins and DNA vaccines. The three recombinant proteins were employed for coating ELISA plates. Sera from human and canine patients of visceral leishmaniasis and human patients of mucosal leishmaniasis recognized the three LiPABPs. In addition, the protective efficacy of a DNA vaccine based on the combination of the three Leishmania PABPs has been tested in a model of progressive murine leishmaniasis: BALB/c mice infected with Leishmania major. The induction of a Th1-like response against the LiPABP family by genetic vaccination was able to down-regulate the IL-10 predominant responses elicited by parasite LiPABPs after infection in this murine model. This modulation resulted in a partial protection against L. major infection. LiPABP vaccinated mice showed a reduction on the pathology that was accompanied by a decrease in parasite burdens, in antibody titers against Leishmania antigens and in the IL-4 and IL-10 parasite-specific mediated responses in comparison to control mice groups immunized with saline or with the non-recombinant plasmid. Conclusion/Significance The results presented here demonstrate for the first time the prophylactic properties of a new family of Leishmania antigenic intracellular proteins, the LiPABPs. The redirection of the immune response elicited against the LiPABP family (from IL-10 towards IFN-γ mediated responses) by genetic vaccination was able to induce a partial protection against the development of the disease in a highly susceptible murine model of leishmaniasis. PMID:25955652

  2. The major surface protease (MSP or GP63) in the intracellular amastigote stage of Leishmania chagasi.

    PubMed

    Hsiao, Chia-Hung Christine; Yao, Chaoqun; Storlie, Patricia; Donelson, John E; Wilson, Mary E

    2008-02-01

    The Leishmania spp. protozoa have an abundant surface metalloprotease called MSP (major surface protease), which in Leishmania chagasi is encoded by three distinct gene classes (MSPS, MSPL, MSPC). Although MSP has been characterized primarily in extracellular promastigotes, it also facilitates survival of intracellular amastigotes. Promastigotes express MSPS, MSPL, and two forms of MSPC RNAs, whereas amastigotes express only MSPL RNA and one MSPC transcript. We confirmed the presence of MSPC protein in both promastigotes and amastigotes by liquid chromatography-tandem mass spectrometry (LC-MS/MS). More than 10 MSP isoforms were visualized in both amastigotes and promastigotes using two-dimensional immunoblots, but amastigote MSPs migrated at a more acidic pI. Promastigote MSPs were N-glycosylated, whereas most amastigote MSPs were not. Immuno-electron microscopy showed that two-thirds of the promastigote MSP is distributed along the cell surface. In contrast, most amastigote MSP localized at the flagellar pocket, the major site of leishmania endocytosis/exocytosis. Biochemical analyses indicated that most amastigote MSP is soluble in the cytosol, vesicles or organelles, whereas most promastigote MSP is membrane-associated and GPI anchored. Activity gels and immunoblots confirmed the presence of a novel proteolytically active amastigote MSP of higher Mr than the promastigote MSPs. Furthermore, promastigote MSP is shed extracellularly whereas MSP is not shed from axenic amastigotes. We conclude that amastigotes and promastigotes both express multiple MSP isoforms, but these MSPs differ biochemically and localize differently in the two parasite stages. We hypothesize that MSP plays different roles in the extracellular versus intracellular forms of Leishmania spp.

  3. Leishmania major UDP-sugar pyrophosphorylase salvages galactose for glycoconjugate biosynthesis.

    PubMed

    Damerow, Sebastian; Hoppe, Carolin; Bandini, Giulia; Zarnovican, Patricia; Buettner, Falk F R; Ferguson, Michael A J; Routier, Françoise H

    2015-10-01

    Leishmaniases are a set of tropical and sub-tropical diseases caused by protozoan parasites of the genus Leishmania whose severity ranges from self-healing cutaneous lesions to fatal visceral infections. Leishmania parasites synthesise a wide array of cell surface and secreted glycoconjugates that play important roles in infection. These glycoconjugates are particularly abundant in the promastigote form and known to be essential for establishment of infection in the insect midgut and effective transmission to the mammalian host. Since they are rich in galactose, their biosynthesis requires an ample supply of UDP-galactose. This nucleotide-sugar arises from epimerisation of UDP-glucose but also from an uncharacterised galactose salvage pathway. In this study, we evaluated the role of the newly characterised UDP-sugar pyrophosphorylase (USP) of Leishmania major in UDP-galactose biosynthesis. Upon deletion of the USP encoding gene, L. major lost the ability to synthesise UDP-galactose from galactose-1-phosphate but its ability to convert glucose-1-phosphate into UDP-glucose was fully maintained. Thus USP plays a role in UDP-galactose activation but does not significantly contribute to the de novo synthesis of UDP-glucose. Accordingly, USP was shown to be dispensable for growth and glycoconjugate biosynthesis under standard growth conditions. However, in a mutant seriously impaired in the de novo synthesis of UDP-galactose (due to deficiency of the UDP-glucose pyrophosphorylase) addition of extracellular galactose increased biosynthesis of the cell surface lipophosphoglycan. Thus under restrictive conditions, such as those encountered by Leishmania in its natural habitat, galactose salvage by USP may play a substantial role in biosynthesis of the UDP-galactose pool. We hypothesise that USP recycles galactose from the blood meal within the midgut of the insect for synthesis of the promastigote glycocalyx and thereby contributes to successful vector infection.

  4. Natural infection of Nesokia indica with Leishmania major and Leishmania infantum parasites in Damghan city, Northern Iran.

    PubMed

    Pourmohammadi, Behrad; Mohammadi-Azni, Sadegh; Kalantari, Mohsen

    2017-03-04

    Various species of rodents are proven reservoir hosts of zoonotic cutaneous leishmaniasis in different provinces of Iran and potential reservoir hosts of zoonotic visceral leishmaniasis. Therefore, this study was conducted to determine the leishmanial infection of rodents in Damghan city from April to September, 2015. Sum of 100 rodents of three species; Nesokia indica (95), Mus musculus (3), and Microtus socialis (2), were trapped alive and their tissue samples were examined using parasitological and molecular (nested-PCR) methods. A total of 71% (71/100) of examined rodents were parasitological positive for Leishmania spp. amastigotes. The highest rate (72.6%; 69/95) of infection was related to the N. indica species. The microscopic observations showed that 42% of ear samples were positive. Additionally, 12% of rodents with negative ear result were positive in liver. 16 out of 41 (39%) parasitological positive samples, belonging to the N. indica, were shown molecularly positive. Of which, 15 were L. major (13 of ear and 2 of spleen samples) and one of spleen samples was L. infantum. This is the first report of N. indica natural infection with L. infantum parasite. To understand the role of this rodent as reservoir host of L. infantum, extant ecological and epidemiological studies are needed.

  5. Simultaneous gene expression profiling in human macrophages infected with Leishmania major parasites using SAGE

    PubMed Central

    Guerfali, Fatma Z; Laouini, Dhafer; Guizani-Tabbane, Lamia; Ottones, Florence; Ben-Aissa, Khadija; Benkahla, Alia; Manchon, Laurent; Piquemal, David; Smandi, Sondos; Mghirbi, Ons; Commes, Thérèse; Marti, Jacques; Dellagi, Koussay

    2008-01-01

    Background Leishmania (L) are intracellular protozoan parasites that are able to survive and replicate within the harsh and potentially hostile phagolysosomal environment of mammalian mononuclear phagocytes. A complex interplay then takes place between the macrophage (MΦ) striving to eliminate the pathogen and the parasite struggling for its own survival. To investigate this host-parasite conflict at the transcriptional level, in the context of monocyte-derived human MΦs (MDM) infection by L. major metacyclic promastigotes, the quantitative technique of serial analysis of gene expression (SAGE) was used. Results After extracting mRNA from resting human MΦs, Leishmania-infected human MΦs and L. major parasites, three SAGE libraries were constructed and sequenced generating up to 28,173; 57,514 and 33,906 tags respectively (corresponding to 12,946; 23,442 and 9,530 unique tags). Using computational data analysis and direct comparison to 357,888 publicly available experimental human tags, the parasite and the host cell transcriptomes were then simultaneously characterized from the mixed cellular extract, confidently discriminating host from parasite transcripts. This procedure led us to reliably assign 3,814 tags to MΦs' and 3,666 tags to L. major parasites transcripts. We focused on these, showing significant changes in their expression that are likely to be relevant to the pathogenesis of parasite infection: (i) human MΦs genes, belonging to key immune response proteins (e.g., IFNγ pathway, S100 and chemokine families) and (ii) a group of Leishmania genes showing a preferential expression at the parasite's intra-cellular developing stage. Conclusion Dual SAGE transcriptome analysis provided a useful, powerful and accurate approach to discriminating genes of human or parasitic origin in Leishmania-infected human MΦs. The findings presented in this work suggest that the Leishmania parasite modulates key transcripts in human MΦs that may be beneficial for its

  6. Continual renewal and replication of persistent Leishmania major parasites in concomitantly immune hosts

    PubMed Central

    Mandell, Michael A.

    2017-01-01

    In most natural infections or after recovery, small numbers of Leishmania parasites remain indefinitely in the host. Persistent parasites play a vital role in protective immunity against disease pathology upon reinfection through the process of concomitant immunity, as well as in transmission and reactivation, yet are poorly understood. A key question is whether persistent parasites undergo replication, and we devised several approaches to probe the small numbers in persistent infections. We find two populations of persistent Leishmania major: one rapidly replicating, similar to parasites in acute infections, and another showing little evidence of replication. Persistent Leishmania were not found in “safe” immunoprivileged cell types, instead residing in macrophages and DCs, ∼60% of which expressed inducible nitric oxide synthase (iNOS). Remarkably, parasites within iNOS+ cells showed normal morphology and genome integrity and labeled comparably with BrdU to parasites within iNOS− cells, suggesting that these parasites may be unexpectedly resistant to NO. Nonetheless, because persistent parasite numbers remain roughly constant over time, their replication implies that ongoing destruction likewise occurs. Similar results were obtained with the attenuated lpg2− mutant, a convenient model that rapidly enters a persistent state without inducing pathology due to loss of the Golgi GDP mannose transporter. These data shed light on Leishmania persistence and concomitant immunity, suggesting a model wherein a parasite reservoir repopulates itself indefinitely, whereas some progeny are terminated in antigen-presenting cells, thereby stimulating immunity. This model may be relevant to understanding immunity to other persistent pathogen infections. PMID:28096392

  7. Antigen-presenting cells in human cutaneous leishmaniasis due to Leishmania major.

    PubMed Central

    ElHassan, A M; Gaafar, A; Theander, T G

    1995-01-01

    In this study biopsies from skin lesions and draining lymph nodes of patients suffering from cutaneous leishmaniasis caused by Leishmania major were examined by immunohistochemistry, and by light and electron microscopy to identify the types of antigen-presenting cells (APC) and their location. APC, identified morphologically and by their expression of specific cell markers, included Langerhans cells, macrophages, follicular dendritic cells, and interdigitating reticulum cells of the paracortex of lymph nodes. These cells expressed MHC class II antigens and contained Leishmania antigen. Since some keratinocytes and endothelial cells also showed these characteristics, they may also act as APC. By examining tissue samples from skin lesions and draining lymph nodes it was possible to follow the probable route of trafficking of various inflammatory cells between the skin lesion and lymph nodes. Leishmania antigen containing Langerhans cells were found in the epidermis, dermis and the regional lymph nodes. We believe these cells translocate from the epidermis to the dermis, where they take up antigen and migrate to the paracortex of the regional lymph nodes. There they are intimately associated with cells of the paracortex, and could be involved in the generation of Leishmania-specific T memory cells. LFA-1-positive T cells of the CD45RO phenotype were found in the skin lesion. Venular endothelium in the skin lesions expressed intercellular adhesion molecule-1 (ICAM-1), which is the ligand for LFA-1. The migration of lymphocytes from the vascular lumen to the site of inflammation is possibly a result of the interaction of these two adhesion molecules. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7882568

  8. An integrated pipeline for the development of novel panels of mapped microsatellite markers for Leishmania donovani complex, Leishmania braziliensis and Leishmania major.

    PubMed

    Fakhar, M; Motazedian, M H; Daly, D; Lowe, C D; Kemp, S J; Noyes, H A

    2008-04-01

    A panel of microsatellites mapped to the Leishmania genome might make it possible to find associations between specific loci and phenotypic traits. To identify such loci, a Perl programme was written that scans the sequence of a genome and writes all loci containing microsatellites to a MySQL database. The programme was applied to the sequences of the L. braziliensis, L. infantum and L. major genomes. The database is publicly available over the internet: http://www.genomics.liv.ac.uk/tryps/resources.html 'Microsatellite Locus Extractor', and allows the selection of mapped microsatellites that meet user-defined criteria from a specified region of the selected genome. The website also incorporates a primer design pipeline that will design primers to amplify the selected loci. Using this pipeline 12 out of 17 primer sets designed against the L. infantum genome generated polymorphic PCR products. A tailed primer protocol was used to label all microsatellite primers with a single set of labelled primers. To avoid the culture of parasites prior to genotyping, sets of nested PCR primers were developed to amplify parasite DNA eluted from microscope slides. The limit of detection was approximately 1.6 parasite equivalents. However, only 6/56 DNA from slides stored at ambient temperature for over 6 months gave positive PCR results.

  9. Gerbillus nanus (Rodentia: Muridae): a new reservoir host of Leishmania major.

    PubMed

    Azizi, K; Moemenbellah-Fard, M D; Fakoorziba, M R; Fekri, S

    2011-09-01

    Gerbillus nanus Blanford, 1875 known as Baluchistan gerbil, is a granivorous solitary naked-footed species. No evidence of its natural infection with the protozoan parasite, Leishmania, has so far been provided. Cutaneous leishmaniasis (CL) is a major public health problem in many parts of the world, including Iran. The annual nationwide incidence of human CL due to Leishmania major (CLM) in endemic rural areas was above 18,000 cases in 2008. The detection of L. major in rodents is of fundamental importance for incriminating them as potential reservoirs of CLM infection. Between April 2007 and April 2008, following detection of 245 clinical cases in Jask region of south-east Iran, wild rodents were captured and checked by the microscopic slide smears for leishmanial infections. Overall, 106 gerbilline rodents were captured from which 17 were identified as Gerbillus nanus. Females of Meriones hurrianae, Tatera indica and G. nanus were found to be naturally infected with L. MAJOR. The presence of these parasites in G. nanus has never been reported before. All the amastigote-infected rodents came from the eastern plain of this region, except one T. indica from the western plain which was found to be smear-positive or kinetoplast DNA-positive by PCR. The highest (11·8%) prevalence of infection among rodents confirmed by PCR to be infected with L. major was attributed to Baluchistan gerbil, G. nanus, which is thus incriminated as a potential reservoir host of L. major in Iran.

  10. Gerbillus nanus (Rodentia: Muridae): a new reservoir host of Leishmania major

    PubMed Central

    AZIZI, K; MOEMENBELLAH-FARD, M D; FAKOORZIBA, M R; FEKRI, S

    2011-01-01

    Gerbillus nanus Blanford, 1875 known as Baluchistan gerbil, is a granivorous solitary naked-footed species. No evidence of its natural infection with the protozoan parasite, Leishmania, has so far been provided. Cutaneous leishmaniasis (CL) is a major public health problem in many parts of the world, including Iran. The annual nationwide incidence of human CL due to Leishmania major (CLM) in endemic rural areas was above 18 000 cases in 2008. The detection of L. major in rodents is of fundamental importance for incriminating them as potential reservoirs of CLM infection. Between April 2007 and April 2008, following detection of 245 clinical cases in Jask region of south-east Iran, wild rodents were captured and checked by the microscopic slide smears for leishmanial infections. Overall, 106 gerbilline rodents were captured from which 17 were identified as Gerbillus nanus. Females of Meriones hurrianae, Tatera indica and G. nanus were found to be naturally infected with L. major. The presence of these parasites in G. nanus has never been reported before. All the amastigote-infected rodents came from the eastern plain of this region, except one T. indica from the western plain which was found to be smear-positive or kinetoplast DNA-positive by PCR. The highest (11.8%) prevalence of infection among rodents confirmed by PCR to be infected with L. major was attributed to Baluchistan gerbil, G. nanus, which is thus incriminated as a potential reservoir host of L. major in Iran. PMID:22117852

  11. First report of natural infection in hedgehogs with Leishmania major, a possible reservoir of zoonotic cutaneous leishmaniasis in Algeria.

    PubMed

    Tomás-Pérez, Míriam; Khaldi, Mourad; Riera, Cristina; Mozo-León, Denis; Ribas, Alexis; Hide, Mallorie; Barech, Ghania; Benyettou, Meryam; Seghiri, Kamel; Doudou, Souad; Fisa, Roser

    2014-07-01

    We report here the first known cases of natural infection of hedgehogs with Leishmania major. Cutaneous leishmaniasis is an important public health problem in the area of M'sila, a semi-arid province in Algeria's northern Sahara, where two species of hedgehog live, Atelerix algirus and Paraechinus aethiopicus. The aim of this research was to survey Leishmania infection in these hedgehogs and evaluate whether they were reservoir hosts of Leishmania in an endemic zoonotic focus of leishmaniasis. Serological and molecular methods were used to determine the presence of Leishmania in 24 hedgehogs caught directly by hand and identified at species level as 19 A. algirus and 5 P. aethiopicus. Specific anti-Leishmania antibodies were detected in 29.2% of individuals by Western blot and in 26.3% by ELISA. The real-time PCR performed in spleen, ear and blood samples detected Leishmania spp. DNA in 12.5% of the individuals, one A. algirus and two P. aethiopicus. Three skin and two spleen samples of these animals were found to be parasitized and were identified by molecular test as L. major. Considering our results, it is suggested that hedgehogs have a potential epidemiological role as reservoir hosts of L. major. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Genetic differences between two Leishmania major-like strains revealed by suppression subtractive hybridization.

    PubMed

    Wu, Ângela C A; Freitas, Michelle A R; Silva, Soraia de O; Nogueira, Paula M; Soares, Rodrigo P; Pesquero, João Bosco; Gomes, Maria A; Pesquero, Jorge L; Melo, Maria N

    2015-01-01

    Leishmania major, the causative agent of zoonotic leishmaniasis, is restricted to Old World countries. Molecular and biochemical techniques have been used to identify some L. major-like isolated in South America including Brazil. Here, two L. major-like strains, one virulent (BH49) and one non-virulent (BH121), were subjected to suppression subtractive hybridization (SSH) technique in order to identify differentially expressed genes. SSH technique identified nine cDNA fragments exhibiting high homology to previously sequenced L. major genes. Five cDNAs (four specific for BH49 and one for BH121) were confirmed by RT-PCR. Among those differentially expressed subtracted genes, some were involved in physiological processes including metabolism, translation and destination of proteins, production of energy, virulence factors and unknown functions. Western-blot analysis confirmed a higher expression level of β-1,3-galactosyl residues in L. major-like lipophosphoglycan (LPG). This molecular analysis opens the possibility for identification of potential virulence factors not only in different strains, but also in others species of Leishmania.

  13. Comparison Between in Vitro Effects of Aqueous Extract of Artemisia seiberi and Artemisinin on Leishmania major

    PubMed Central

    Esavand Heydari, Farzad; Ghaffarifar, Fatemeh; Soflaei, Saied; Dalimi, Abdolhosein

    2013-01-01

    Background It is necessary to develop novel, affordable, and accessible drugs with few side effects as alternatives of the currently available chemical agents for leishmaniasis. Objectives The main purpose of this study was to evaluate the effects of these drugs on L. major under in vitro conditions. Materials and Methods In the current study, 5, 10, 25, 50, and 100 µg/mL concentrations of aqueous extract of Artemisia sieberi and chemical artemisinin were tested on promastigotes of Leishmania major (L. major), uninfected macrophages, and infected macrophages with intracellular amastigotes of L. major, by direct counting and 3-(4 5-dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bromid methods. Results The results obtained for each drug were compared with other drugs and also with the results of the control groups. The results related to promastigote and amastigote assays showed that when the dose of both drugs increased, the parasite number is reduced in comparison with the control groups. Moreover, the parasitic burden in the test cultures decreased significantly. Macrophage assay results showed that the effects of both drugs on uninfected and healthy macrophages were very low. Conclusions These results indicate that both drugs have anti-Leishmania effects, which was higher in Artemisia sieberi compared with artemisinin. Thus, carrying out further studies on the effects of Artemisia sieberi in infected animals with L. major is recommended. PMID:24624191

  14. Comparative evaluation of two vaccine candidates against experimental leishmaniasis due to Leishmania major infection in four inbred mouse strains.

    PubMed

    Benhnini, Fouad; Chenik, Mehdi; Laouini, Dhafer; Louzir, Hechmi; Cazenave, Pierre André; Dellagi, Koussay

    2009-11-01

    Experimental leishmaniasis in BALB/c and C57BL/6 mice are the most investigated murine models that were used for the preclinical evaluation of Leishmania vaccine candidates. We have previously described two new inbred mouse strains named PWK and MAI issued from feral founders that also support the development of experimental leishmaniasis due to L. major. In this study, we sought to determine whether different mouse inbred strains generate concordant or discordant results when used to evaluate the potential of Leishmania proteins to protect against experimental leishmaniasis. To this end, two Leishmania proteins, namely, LACK (for Leishmania homolog of receptor for activated C kinase) and LmPDI (for L. major protein disulfide isomerase) were compared for their capacity to protect against experimental leishmaniasis in PWK, MAI, BALB/c, and C57BL/6 inbred mouse strains. Our data show that the capacity of Leishmania proteins to confer protection depends on the mouse strain used, stressing the important role played by the genetic background in shaping the immune response against the pathogen. These results may have important implications for the preclinical evaluation of candidate Leishmania vaccines: rather than using a single mouse strain, a panel of different inbred strains of various genetic backgrounds should be tested in parallel. The antigen that confers protection in the larger range of inbred strains may have better chances to be also protective in outbred human populations and should be selected for clinical trials.

  15. Molecular characterization of 5S ribosomal RNA genes and transcripts in the protozoan parasite Leishmania major.

    PubMed

    Moreno-Campos, Rodrigo; Florencio-Martínez, Luis E; Nepomuceno-Mejía, Tomás; Rojas-Sánchez, Saúl; Vélez-Ramírez, Daniel E; Padilla-Mejía, Norma E; Figueroa-Angulo, Elisa; Manning-Cela, Rebeca; Martínez-Calvillo, Santiago

    2016-12-01

    Eukaryotic 5S rRNA, synthesized by RNA polymerase III (Pol III), is an essential component of the large ribosomal subunit. Most organisms contain hundreds of 5S rRNA genes organized into tandem arrays. However, the genome of the protozoan parasite Leishmania major contains only 11 copies of the 5S rRNA gene, which are interspersed and associated with other Pol III-transcribed genes. Here we report that, in general, the number and order of the 5S rRNA genes is conserved between different species of Leishmania. While in most organisms 5S rRNA genes are normally associated with the nucleolus, combined fluorescent in situ hybridization and indirect immunofluorescence experiments showed that 5S rRNA genes are mainly located at the nuclear periphery in L. major. Similarly, the tandemly repeated 5S rRNA genes in Trypanosoma cruzi are dispersed throughout the nucleus. In contrast, 5S rRNA transcripts in L. major were localized within the nucleolus, and scattered throughout the cytoplasm, where mature ribosomes are located. Unlike other rRNA species, stable antisense RNA complementary to 5S rRNA is not detected in L. major.

  16. Spatio-temporal Genetic Structuring of Leishmania major in Tunisia by Microsatellite Analysis

    PubMed Central

    Harrabi, Myriam; Bettaieb, Jihène; Ghawar, Wissem; Toumi, Amine; Zaâtour, Amor; Yazidi, Rihab; Chaâbane, Sana; Chalghaf, Bilel; Hide, Mallorie; Bañuls, Anne-Laure; Ben Salah, Afif

    2015-01-01

    In Tunisia, cases of zoonotic cutaneous leishmaniasis caused by Leishmania major are increasing and spreading from the south-west to new areas in the center. To improve the current knowledge on L. major evolution and population dynamics, we performed multi-locus microsatellite typing of human isolates from Tunisian governorates where the disease is endemic (Gafsa, Kairouan and Sidi Bouzid governorates) and collected during two periods: 1991–1992 and 2008–2012. Analysis (F-statistics and Bayesian model-based approach) of the genotyping results of isolates collected in Sidi Bouzid in 1991–1992 and 2008–2012 shows that, over two decades, in the same area, Leishmania parasites evolved by generating genetically differentiated populations. The genetic patterns of 2008–2012 isolates from the three governorates indicate that L. major populations did not spread gradually from the south to the center of Tunisia, according to a geographical gradient, suggesting that human activities might be the source of the disease expansion. The genotype analysis also suggests previous (Bayesian model-based approach) and current (F-statistics) flows of genotypes between governorates and districts. Human activities as well as reservoir dynamics and the effects of environmental changes could explain how the disease progresses. This study provides new insights into the evolution and spread of L. major in Tunisia that might improve our understanding of the parasite flow between geographically and temporally distinct populations. PMID:26302440

  17. Establishment of Stable, Cell-Mediated Immunity that Makes "Susceptible" Mice Resistant to Leishmania major

    NASA Astrophysics Data System (ADS)

    Bretscher, Peter A.; Wei, Guojian; Menon, Juthika N.; Bielefeldt-Ohmann, Helle

    1992-07-01

    Cell-mediated, but not antibody-mediated, immune responses protect humans against certain pathogens that produce chronic diseases such as leishmaniasis. Effective vaccination against such pathogens must therefore produce an immunological "imprint" so that stable, cell-mediated immunity is induced in all individuals after natural infection. BALB/c mice "innately susceptible" to Leishmania major produce antibodies after substantial infection. In the present study, "susceptible" mice injected with a small number of parasites mounted a cell-mediated response and acquired resistance to a larger, normally pathogenic, challenge. This vaccination strategy may be applicable in diseases in which protection is dependent on cell-mediated immunity.

  18. Overview of DNA Repair in Trypanosoma cruzi, Trypanosoma brucei, and Leishmania major

    PubMed Central

    Passos-Silva, Danielle Gomes; Rajão, Matheus Andrade; Nascimento de Aguiar, Pedro Henrique; Vieira-da-Rocha, João Pedro; Machado, Carlos Renato; Furtado, Carolina

    2010-01-01

    A wide variety of DNA lesions arise due to environmental agents, normal cellular metabolism, or intrinsic weaknesses in the chemical bonds of DNA. Diverse cellular mechanisms have evolved to maintain genome stability, including mechanisms to repair damaged DNA, to avoid the incorporation of modified nucleotides, and to tolerate lesions (translesion synthesis). Studies of the mechanisms related to DNA metabolism in trypanosomatids have been very limited. Together with recent experimental studies, the genome sequencing of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major, three related pathogens with different life cycles and disease pathology, has revealed interesting features of the DNA repair mechanism in these protozoan parasites, which will be reviewed here. PMID:20976268

  19. Transcriptomic profiling of gene expression and RNA processing during Leishmania major differentiation

    PubMed Central

    Dillon, Laura A. L.; Okrah, Kwame; Hughitt, V. Keith; Suresh, Rahul; Li, Yuan; Fernandes, Maria Cecilia; Belew, A. Trey; Corrada Bravo, Hector; Mosser, David M.; El-Sayed, Najib M.

    2015-01-01

    Protozoan parasites of the genus Leishmania are the etiological agents of leishmaniasis, a group of diseases with a worldwide incidence of 0.9–1.6 million cases per year. We used RNA-seq to conduct a high-resolution transcriptomic analysis of the global changes in gene expression and RNA processing events that occur as L. major transforms from non-infective procyclic promastigotes to infective metacyclic promastigotes. Careful statistical analysis across multiple biological replicates and the removal of batch effects provided a high quality framework for comprehensively analyzing differential gene expression and transcriptome remodeling in this pathogen as it acquires its infectivity. We also identified precise 5′ and 3′ UTR boundaries for a majority of Leishmania genes and detected widespread alternative trans-splicing and polyadenylation. An investigation of possible correlations between stage-specific preferential trans-splicing or polyadenylation sites and differentially expressed genes revealed a lack of systematic association, establishing that differences in expression levels cannot be attributed to stage-regulated alternative RNA processing. Our findings build on and improve existing expression datasets and provide a substantially more detailed view of L. major biology that will inform the field and potentially provide a stronger basis for drug discovery and vaccine development efforts. PMID:26150419

  20. The dihydroorotate dehydrogenases: Past and present.

    PubMed

    Reis, Renata A G; Calil, Felipe Antunes; Feliciano, Patricia Rosa; Pinheiro, Matheus Pinto; Nonato, M Cristina

    2017-06-27

    The flavoenzyme dihydroorotate dehydrogenase catalyzes the stereoselective oxidation of (S)-dihydroorotate to orotate in the fourth of the six conserved enzymatic reactions involved in the de novo pyrimidine biosynthetic pathway. Inhibition of pyrimidine metabolism by selectively targeting DHODHs has been exploited in the development of new therapies against cancer, immunological disorders, bacterial and viral infections, and parasitic diseases. Through a chronological narrative, this review summarizes the efforts of the scientific community to achieve our current understanding of structural and biochemical properties of DHODHs. It also attempts to describe the latest advances in medicinal chemistry for therapeutic development based on the selective inhibition of DHODH, including an overview of the experimental techniques used for ligand screening during the process of drug discovery. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. The Transcriptome of Leishmania major Developmental Stages in Their Natural Sand Fly Vector.

    PubMed

    Inbar, Ehud; Hughitt, V Keith; Dillon, Laura A L; Ghosh, Kashinath; El-Sayed, Najib M; Sacks, David L

    2017-04-04

    The life cycle of the Leishmania parasite in the sand fly vector involves differentiation into several distinctive forms, each thought to represent an adaptation to specific microenvironments in the midgut of the fly. Based on transcriptome sequencing (RNA-Seq) results, we describe the first high-resolution analysis of the transcriptome dynamics of four distinct stages of Leishmania major as they develop in a natural vector, Phlebotomus duboscqi The early transformation from tissue amastigotes to procyclic promastigotes in the blood-fed midgut was accompanied by the greatest number of differentially expressed genes, including the downregulation of amastins, and upregulation of multiple cell surface proteins, sugar and amino acid transporters, and genes related to glucose metabolism and cell cycle progression. The global changes accompanying post-blood meal differentiation of procyclic promastigotes to the nectomonad and metacyclic stages were less extensive, though each displayed a unique signature. The transcriptome of nectomonads, which has not been studied previously, revealed changes consistent with cell cycle arrest and the upregulation of genes associated with starvation and stress, including autophagic pathways of protein recycling. Maturation to the infective, metacyclic stage was accompanied by changes suggesting preadaptation to the intracellular environment of the mammalian host, demonstrated by the amastigote-like profiles of surface proteins and metabolism-related genes. Finally, a direct comparison between sand fly-derived and culture-derived metacyclics revealed a reassuring similarity between the two forms, with the in vivo forms distinguished mainly by a stronger upregulation of transcripts associated with nutrient stress.IMPORTANCE The life cycle of Leishmania parasites in the sand fly vector includes their growth and development as morphologically distinct forms of extracellular promastigotes found within the different microenvironments of the

  2. From genome to vaccines for leishmaniasis: screening 100 novel vaccine candidates against murine Leishmania major infection.

    PubMed

    Stober, Carmel B; Lange, Uta G; Roberts, Mark T M; Gilmartin, Brian; Francis, Richard; Almeida, Renata; Peacock, Christopher S; McCann, Sharon; Blackwell, Jenefer M

    2006-03-24

    The genomic sequence of Leishmania major provides a rich source of vaccine candidates. One hundred randomly selected amastigote-expressed genes were screened as DNA vaccines, and efficacy determined following high-dose L. major footpad challenge in BALB/c mice. Fourteen protective novel vaccine candidates were identified; seven vaccines exacerbated disease. There were no differences in the number of predicted MHC H-2d class I or II epitopes mapping to protective versus exacerbatory antigens. A proportion of both protective (7/14; 50%) and exacerbatory (4/7; 57%) proteins showed short (8- to 18-mer) 100% amino acid sequence identities to human, mouse or gut flora proteins. A high proportion of these (4/7 protective; 3/4 exacerbatory) showed full or partial overlap with RANKPEP-predicted H-2d classes I and II epitopes. Our data suggest, therefore, that there may be little difference between antigens/epitopes that drive regulatory versus effector CD4 T cell populations. The best novel protective antigen was an amastin-like gene that maps to a 17-gene tandem array on Leishmania chromosome 8 and is closely related to 37 other amastin-like genes. Two ribosomal proteins, a V-ATPase subunit, and a dynein light chain orthologue were the only other protective genes with putative functions.

  3. Immunization Against Cutaneous Leishmaniasis by Alginate Microspheres Loaded With Autoclaved Leishmania Major (ALM) and Quillaja Saponins.

    PubMed

    Tafaghodi, Mohsen; Eskandari, Maryam; Khamesipour, Ali; Jaafari, Mahmoud Reza

    2016-01-01

    Leishmania antigens are weak immunogens and need to be potentiated by various adjuvants and delivery systems. Alginate microspheres as antigen delivery system and Quillaja saponins (QS) as immunoadjuvant have been used to enhance the immune response against Autoclaved Leishmania major (ALM). Microspheres were prepared by an emulsification technique and characterized for size, encapsulation efficiency and release profile of encapsulates. BALB/c mice were immunized three times in 3-weeks intervals using ALM plus QS loaded microspheres [(ALM+QS)ALG], ALM encapsulated with alginate microspheres [(ALM)ALG], (ALM)ALG + QS, ALM + QS, ALM alone or PBS. The intensity of infection induced by L. major challenge was assessed by measuring size of footpad swelling. The strongest protection, showed by significantly (P < 0.05) smaller footpad, were observed in mice immunized with (ALM)ALG+QS. The (ALM+QS)ALG, ALM and PBS groups showed the least protection and highest swelling, while the (ALM)ALG and ALM+QS showed an intermediate protection with no significant difference. The mice immunized with (ALM+QS)ALG showed the highest IgG2a/IgG1 ratio (P<0.05). The highest IFN-γ and IL-4 production was seen in ALM+QS (P<0.01). It is concluded that QS adjuvant has a mixed Th1/Th2 effect and has increased both humoral and cellular immune responses.

  4. STRUCTURAL CHARACTERISATION OF THE LEISHMANIA MAJOR ORTHOLOGUES OF MACROPHAGE MIGRATION INHIBITORY FACTOR (MIF)

    PubMed Central

    Richardson, Julia M.; Morrison, Lesley S.; Bland, Nicholas D.; Bruce, Sandra; Coombs, Graham H.; Mottram, Jeremy C.; Walkinshaw, Malcolm D.

    2011-01-01

    Leishmania major, an intracellular parasitic protozoon that infects, differentiates and replicates within macrophages, encodes two closely related MIF-like proteins, which have only ~20% amino acid identity with mammalian MIF. Recombinant L. major MIF1 and MIF2 have been expressed and the structures, resolved by X-ray crystallography, show a trimeric ring architecture similar to mammalian MIF but with some structurally distinct features. LmjMIF1, but not LmjMIF2, has tautomerase activity, indicating that the LmjMIFs have evolved potentially different biological roles. This is further demonstrated by the differential life cycle expression of the proteins. LmjMIF2 is found in all life cycle stages whereas LmjMIF1 is found exclusively in amastigotes, the intracellular stage responsible for mammalian disease. The findings are consistent with parasite MIFs modulating or circumventing the host macrophage response and thereby promoting parasite survival, however analysis of the L. braziliensis genome showed that this species lacks intact MIF genes - highlighting that MIF is not a virulence factor in all species of Leishmania. PMID:19187777

  5. Leishmania major: Genetic Profiles of the Parasites Isolated from Chabahar, Southeastern Iran by PPIP-PCR.

    PubMed

    Sharifi-Rad, Mehdi; Dabirzadeh, Mansour; Sharifi, Iraj; Babaei, Zahra

    2016-01-01

    Leishmaniasis is important vector-borne parasitic disease worldwide, caused by the genus Leishmania. The objective of the current study was to identify genetic polymorphism in L. major, one of the species causing cutaneous leishmaniasis (CL), isolated from southeastern Iran, using Permissively Primed Intergenic Polymorphic-Polymerase Chain Reaction (PPIP-PCR) method. Overall, 340 patients with suspected CL were examined. They referred to the Central Laboratory in Chabahar, Iran during Apr 2013 to Feb 2014. Microscopic examination of Giemsa-stained slides from lesions as well as aspirates cultured in Novy- Mac Neal-Nicolle (NNN) Media was employed in order to diagnose CL in these patients. Our analyses detected 86 suspected subjects as having CL from which 35 isolates were cultured successfully. PPIP-PCR method was performed on extracted genomic DNA from selected isolates in order to determine the genetic polymorphism among L. major isolates. The electrophoresis patterns demonstrated two genetic profiles including A or A1 patterns between all samples tested. Frequency of A and A1 sub-types were 33 (94.3%) and two (5.7%), respectively. Both host and parasite factors may contribute to the clinical profile of human leishmaniasis in the endemic foci of the disease. Here we showed that genetic variations pertaining to the Leishmania parasites might determine, in part, the clinical outcomes of human leishmaniasis.

  6. Leishmania major: Genetic Profiles of the Parasites Isolated from Chabahar, Southeastern Iran by PPIP-PCR

    PubMed Central

    SHARIFI-RAD, Mehdi; DABIRZADEH, Mansour; SHARIFI, Iraj; BABAEI, Zahra

    2016-01-01

    Background: Leishmaniasis is important vector-borne parasitic disease worldwide, caused by the genus Leishmania. The objective of the current study was to identify genetic polymorphism in L. major, one of the species causing cutaneous leishmaniasis (CL), isolated from southeastern Iran, using Permissively Primed Intergenic Polymorphic-Polymerase Chain Reaction (PPIP-PCR) method. Methods: Overall, 340 patients with suspected CL were examined. They referred to the Central Laboratory in Chabahar, Iran during Apr 2013 to Feb 2014. Microscopic examination of Giemsa-stained slides from lesions as well as aspirates cultured in Novy- Mac Neal-Nicolle (NNN) Media was employed in order to diagnose CL in these patients. Our analyses detected 86 suspected subjects as having CL from which 35 isolates were cultured successfully. PPIP-PCR method was performed on extracted genomic DNA from selected isolates in order to determine the genetic polymorphism among L. major isolates. Results: The electrophoresis patterns demonstrated two genetic profiles including A or A1 patterns between all samples tested. Frequency of A and A1 sub-types were 33 (94.3%) and two (5.7%), respectively. Conclusion: Both host and parasite factors may contribute to the clinical profile of human leishmaniasis in the endemic foci of the disease. Here we showed that genetic variations pertaining to the Leishmania parasites might determine, in part, the clinical outcomes of human leishmaniasis. PMID:28127333

  7. Immunomodulatory properties of borage (Echium amoenum) on BALB/c mice infected with Leishmania major.

    PubMed

    Hosseini, Nahid; Abolhassani, Mohsen

    2011-06-01

    Leishmaniasis is caused by parasitic protozoa transmitted by the bite of a female sand fly and is currently endemic in 88 countries. BALB/c mice are highly susceptible to the infection with the parasite Leishmania major, and this susceptibility has been attributed, in part, to the expansion of Th2 cells, production of their cytokines, and downregulation of Th1 cytokine, interferon gamma (IFN-γ). In this report, we used both aqueous and alcoholic extracts of Iranian borage (Echium amoenum Fisch & C.A. Mey) for treatment of L. major infection in BALB/c mice. We found that both extracts had immunomodulatory properties and increased the level of IFN-γ and lowered the parasite burden in the proximal lymph nodes and prevented the necrosis of the footpad as compared with the untreated infected mice. These results may provide a basis for further studies directed toward the use of the Iranian borage against L. major infection.

  8. Crystallization and preliminary X-ray analysis of Leishmania major glyoxalase I

    SciTech Connect

    Ariza, Antonio; Vickers, Tim J.; Greig, Neil; Fairlamb, Alan H.; Bond, Charles S.

    2005-08-01

    The detoxification enzyme glyoxalase I from L. major has been crystallized. Preliminary molecular-replacement calculations indicate the presence of three glyoxalase I dimers in the asymmetric unit. Glyoxalase I (GLO1) is a putative drug target for trypanosomatids, which are pathogenic protozoa that include the causative agents of leishmaniasis. Significant sequence and functional differences between Leishmania major and human GLO1 suggest that it may make a suitable template for rational inhibitor design. L. major GLO1 was crystallized in two forms: the first is extremely disordered and does not diffract, while the second, an orthorhombic form, produces diffraction to 2.0 Å. Molecular-replacement calculations indicate that there are three GLO1 dimers in the asymmetric unit, which take up a helical arrangement with their molecular dyads arranged approximately perpendicular to the c axis. Further analysis of these data are under way.

  9. Inhibitory Activity of Eleven Artemisia Species from Iran against Leishmania Major Parasites

    PubMed Central

    Emami, Seyed Ahmad; Zamanai Taghizadeh Rabe, Shahrzad; Ahi, Ali; Mahmoudi, Mahmoud

    2012-01-01

    Objective(s) Annual incidence of cutaneous leishmaniasis is increasingly growing and development of the alternative drugs against it is a major concern. Artemisia genus is a traditional medicinal plant in Iran. The aim of this study was to examine the leishmanicidal activity of various Iranian Artemisia species extracts. Materials and Methods Different extracts were gathered from eleven Iranian Artemisia species. Their leishmanicidal activities against the growth of Leishmania major (L. major) promastigotes were examined as the half maximal inhibitory concentration (IC50) using MTT assay. Results Obtained results showed that ethanol extracts especially those taken from A. ciniformis, A. santolina and A. kulbadica have the strongest effects. Conclusion Looking for the effective leishmanicidal agents from natural resources in Iran, we found that the ethanol extract of collected Artemisia species had significant effect on in vitro leishmanicidal activity and may be suitable candidates in the treatment of leishmaniasis. PMID:23493354

  10. CD8+ T cells Are Preferentially Activated during Primary Low Dose Leishmania major Infection but Are Completely Dispensable during Secondary Anti-Leishmania Immunity

    PubMed Central

    Okwor, Ifeoma B.; Jia, Ping; Mou, Zhirong; Onyilagha, Chukwunonso; Uzonna, Jude E.

    2014-01-01

    We previously showed that CD8+ T cells are required for optimal primary immunity to low dose Leishmania major infection. However, it is not known whether immunity induced by low dose infection is durable and whether CD8+ T cells contribute to secondary immunity following recovery from low dose infection. Here, we compared primary and secondary immunity to low and high dose L. major infections and assessed the influence of infectious dose on the quality and magnitude of secondary anti-Leishmania immunity. In addition, we investigated the contribution of CD8+ T cells in secondary anti-Leishmania immunity following recovery from low and high dose infections. We found that the early immune response to low and high dose infections were strikingly different: while low dose infection preferentially induced proliferation and effector cytokine production by CD8+ T cells, high dose infection predominantly induced proliferation and cytokine production by CD4+ T cells. This differential activation of CD4+ and CD8+ T cells by high and low dose infections respectively, was imprinted during in vitro and in vivo recall responses in healed mice. Both low and high dose-infected mice displayed strong infection-induced immunity and were protected against secondary L. major challenge. While depletion of CD4+ cells in mice that healed low and high dose infections abolished resistance to secondary challenge, depletion of CD8+ cells had no effect. Collectively, our results show that although CD8+ T cells are preferentially activated and may contribute to optimal primary anti-Leishmania immunity following low dose infection, they are completely dispensable during secondary immunity. PMID:25412267

  11. Progressive Perforation of the Nasal Septum Due to Leishmania major: A Case of Mucosal Leishmaniasis in a Traveler.

    PubMed

    Harrison, Nicole; Walochnik, Julia; Ramsebner, Reinhard; Veletzky, Luzia; Lagler, Heimo; Ramharter, Michael

    2017-03-01

    AbstractThis report describes a case of mucosal leishmaniasis caused by Leishmania major with destructive perforation of the nasal septum illustrating the diagnostic challenges of a rare clinical presentation of L. major infection in a traveler. The atypical presentation may have been associated with the use of cortisone as a potential trigger for the progressive destruction of the nasal septum.

  12. Cloning and Constructing a Plasmid Encoding Leishmania Eukaryotic Initiation Factor Gene of Leishmania major Fused with Green Fluorescent Protein Gene as a Vaccine Candidate.

    PubMed

    Maspi, N; Ghaffarifar, F; Sharifi, Z; Dalimi, A

    2015-05-12

    Leishmaniasis is usually treated with chemotherapy; however, toxicity, resistance and high-cost limit use of the chemical drugs. Leishmania eukaryotic initiation factor (LeIF) protein acts the same as interleukin (IL)-12 and reduces the secretion of IL-4 in lymph node cells of mice infected with Leishmania major. The aim of this study was cloning of the gene encoding LeIF antigen into eukaryotic expression plasmid pEGFP-N1. DNA was extracted from Iranian strain of the L major (MRHO/IR/75/ER) promastigotes. The full-length sequence of LeIF was amplified with Pfu DNA polymerase using a specific primer. The amplified LeIF was cloned into a pJET1.2/blunt vector. Then this fragment was digested with HindIII and EcoRI and was subcloned into the pEGFP-N1 vector. Confirmation of the cloning was done by colony polymerase chain reaction (PCR). Leishmania eukaryotic initiation factor gene was successfully cloned and subcloned into pJET1.2 and pEGFP-N1 plasmids, respectively. The results of colony PCR, restriction analysis and sequencing confirmed them. We cloned LeIF gene which could be expressed in eukaryotic cells in vivo and could be used as a vaccine candidate against leishmaniasis in future studies.

  13. Processing of metacaspase into a cytoplasmic catalytic domain mediating cell death in Leishmania major.

    PubMed

    Zalila, Habib; González, Iveth J; El-Fadili, Amal Kuendig; Delgado, Maria Belen; Desponds, Chantal; Schaff, Cédric; Fasel, Nicolas

    2011-01-01

    Metacaspases are cysteine peptidases that could play a role similar to caspases in the cell death programme of plants, fungi and protozoa. The human protozoan parasite Leishmania major expresses a single metacaspase (LmjMCA) harbouring a central domain with the catalytic dyad histidine and cysteine as found in caspases. In this study, we investigated the processing sites important for the maturation of LmjMCA catalytic domain, the cellular localization of LmjMCA polypeptides, and the functional role of the catalytic domain in the cell death pathway of Leishmania parasites. Although LmjMCA polypeptide precursor form harbours a functional mitochondrial localization signal (MLS), we determined that LmjMCA polypeptides are mainly localized in the cytoplasm. In stress conditions, LmjMCA precursor forms were extensively processed into soluble forms containing the catalytic domain. This domain was sufficient to enhance sensitivity of parasites to hydrogen peroxide by impairing the mitochondrion. These data provide experimental evidences of the importance of LmjMCA processing into an active catalytic domain and of its role in disrupting mitochondria, which could be relevant in the design of new drugs to fight leishmaniasis and likely other protozoan parasitic diseases.

  14. Characterization and regulation of Leishmania major 3-hydroxy-3-methylglutaryl-CoA reductase.

    PubMed Central

    Montalvetti, A; Peña-Díaz, J; Hurtado, R; Ruiz-Pérez, L M; González-Pacanowska, D

    2000-01-01

    In eukaryotes the enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyses the synthesis of mevalonic acid, a common precursor to all isoprenoid compounds. Here we report the isolation and overexpression of the gene coding for HMG-CoA reductase from Leishmania major. The protein from Leishmania lacks the membrane domain characteristic of eukaryotic cells but exhibits sequence similarity with eukaryotic reductases. Highly purified protein was achieved by ammonium sulphate precipitation followed by chromatography on hydroxyapatite. Kinetic parameters were determined for the protozoan reductase, obtaining K(m) values for the overall reaction of 40.3+/-5.8 microM for (R,S)-HMG-CoA and 81.4+/-5.3 microM for NADPH; V(max) was 33.55+/-1.8 units x mg(-1). Gel-filtration experiments suggested an apparent molecular mass of 184 kDa with subunits of 46 kDa. Finally, in order to achieve a better understanding of the role of this enzyme in trypanosomatids, the effect of possible regulators of isoprenoid biosynthesis in cultured promastigote cells was studied. Neither mevalonic acid nor serum sterols appear to modulate enzyme activity whereas incubation with lovastatin results in significant increases in the amount of reductase protein. Western- and Northern-blot analyses indicate that this activation is apparently performed via post-transcriptional control. PMID:10861207

  15. Characterization and regulation of Leishmania major 3-hydroxy-3-methylglutaryl-CoA reductase.

    PubMed

    Montalvetti, A; Peña-Díaz, J; Hurtado, R; Ruiz-Pérez, L M; González-Pacanowska, D

    2000-07-01

    In eukaryotes the enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyses the synthesis of mevalonic acid, a common precursor to all isoprenoid compounds. Here we report the isolation and overexpression of the gene coding for HMG-CoA reductase from Leishmania major. The protein from Leishmania lacks the membrane domain characteristic of eukaryotic cells but exhibits sequence similarity with eukaryotic reductases. Highly purified protein was achieved by ammonium sulphate precipitation followed by chromatography on hydroxyapatite. Kinetic parameters were determined for the protozoan reductase, obtaining K(m) values for the overall reaction of 40.3+/-5.8 microM for (R,S)-HMG-CoA and 81.4+/-5.3 microM for NADPH; V(max) was 33.55+/-1.8 units x mg(-1). Gel-filtration experiments suggested an apparent molecular mass of 184 kDa with subunits of 46 kDa. Finally, in order to achieve a better understanding of the role of this enzyme in trypanosomatids, the effect of possible regulators of isoprenoid biosynthesis in cultured promastigote cells was studied. Neither mevalonic acid nor serum sterols appear to modulate enzyme activity whereas incubation with lovastatin results in significant increases in the amount of reductase protein. Western- and Northern-blot analyses indicate that this activation is apparently performed via post-transcriptional control.

  16. Crystallization and preliminary X-ray analysis of Leishmania major glyoxalase I

    PubMed Central

    Ariza, Antonio; Vickers, Tim J.; Greig, Neil; Fairlamb, Alan H.; Bond, Charles S.

    2005-01-01

    Glyoxalase I (GLO1) is a putative drug target for trypanosomatids, which are pathogenic protozoa that include the causative agents of leishmaniasis. Significant sequence and functional differences between Leishmania major and human GLO1 suggest that it may make a suitable template for rational inhibitor design. L. major GLO1 was crystallized in two forms: the first is extremely disordered and does not diffract, while the second, an orthorhombic form, produces diffraction to 2.0 Å. Molecular-replacement calculations indicate that there are three GLO1 dimers in the asymmetric unit, which take up a helical arrangement with their molecular dyads arranged approximately perpendicular to the c axis. Further analysis of these data are under way. PMID:16511153

  17. Monarch-1 Activation in Murine Macrophage Cell Line (J774 A.1) Infected with Iranian Strain of Leishmania major

    PubMed Central

    Fata, A; Mahmoudian, MR; Varasteh, A; Sankian, M

    2013-01-01

    Background Leishmania major is an intracellular parasite transmitted through the bite of the female phlebotomine sand flies. Leishmania major is able to escape the host immune defense and survive within macrophages. Modulation of the NF-κB (Nuclear Factor-Kappa B) activation and suppression of the pro-inflammatory cytokines by L. major are the main evasion mechanisms that remain to be explored. This study aims to examine the expression level of the Monarch-1 in L. major-infected macrophages, as a negative regulator of the NF-κB activation. Methods Murine macrophage cell line (J774 A.1) was infected by metacyclic form of Leishmania promastigotes at macrophage/parasite ratio of 1:10. After harvesting infected cells at different times, total RNA was extracted and converted to cDNA. Semi-quantitative RT-PCR was performed for Monarch-1 by specific primers. Hypoxanthine Phospho-Ribosyl Transferase (HPRT) was used as an internal control to adjust the amount of mRNA in each sample. Results Semiquantitive analysis of Monarch-1 mRNA expression level showed a significant expression increase within 6 to 30 hours after L. major infection of macrophages when compared to the control macrophages. Conclusion Monarch-1 expression level reveals a significant increase in the early phase of macrophage infection with L. major, which in turn may suppress IL-12 production in Leishmania infected macrophages and deeply influence the relationship between host and parasite. PMID:23914232

  18. Comparative analysis of resistant and susceptible macrophage gene expression response to Leishmania major parasite.

    PubMed

    Rabhi, Imen; Rabhi, Sameh; Ben-Othman, Rym; Aniba, Mohamed Radhouane; Trentin, Bernadette; Piquemal, David; Regnault, Béatrice; Guizani-Tabbane, Lamia

    2013-10-22

    Leishmania are obligated intracellular pathogens that replicate almost exclusively in macrophages. The outcome of infection depends largely on parasite pathogenicity and virulence but also on the activation status and genetic background of macrophages. Animal models are essential for a better understanding of pathogenesis of different microbes including Leishmania. Here we compared the transcriptional signatures of resistant (C57BL/6) and susceptible (BALB/c) mouse bone marrow-derived macrophages in response to Leishmania major (L. major) promastigotes infection.Microarray results were first analyzed for significant pathways using the Kyoto Encylopedia of Genes and Genomes (KEGG) database. The analysis revealed that a large set of the shared genes is involved in the immune response and that difference in the expression level of some chemokines and chemokine receptors could partially explain differences in resistance. We next focused on up-regulated genes unique to either BALB/c or C57BL/6 derived macrophages and identified, using KEGG database, signal transduction pathways among the most relevant pathways unique to both susceptible and resistant derived macrophages. Indeed, genes unique to C57BL/6 BMdMs were associated with target of rapamycin (mTOR) signaling pathway while a range of genes unique to BALB/c BMdMs, belong to p53 signaling pathway. We next investigated whether, in a given mice strain derived macrophages, the different up-regulated unique genes could be coordinately regulated. Using GeneMapp Cytoscape, we showed that the induced genes unique to BALB/c or C57BL/6 BMdMs are interconnected. Finally, we examined whether the induced pathways unique to BALB/c derived macrophages interfere with the ones unique to C57BL/6 derived macrophages. Protein-protein interaction analysis using String database highlights the existence of a cross-talk between p53 and mTOR signaling pathways respectively specific to susceptible and resistant BMdMs. Taken together our

  19. Comparative analysis of resistant and susceptible macrophage gene expression response to Leishmania major parasite

    PubMed Central

    2013-01-01

    Background Leishmania are obligated intracellular pathogens that replicate almost exclusively in macrophages. The outcome of infection depends largely on parasite pathogenicity and virulence but also on the activation status and genetic background of macrophages. Animal models are essential for a better understanding of pathogenesis of different microbes including Leishmania. Results Here we compared the transcriptional signatures of resistant (C57BL/6) and susceptible (BALB/c) mouse bone marrow-derived macrophages in response to Leishmania major (L. major) promastigotes infection. Microarray results were first analyzed for significant pathways using the Kyoto Encylopedia of Genes and Genomes (KEGG) database. The analysis revealed that a large set of the shared genes is involved in the immune response and that difference in the expression level of some chemokines and chemokine receptors could partially explain differences in resistance. We next focused on up-regulated genes unique to either BALB/c or C57BL/6 derived macrophages and identified, using KEGG database, signal transduction pathways among the most relevant pathways unique to both susceptible and resistant derived macrophages. Indeed, genes unique to C57BL/6 BMdMs were associated with target of rapamycin (mTOR) signaling pathway while a range of genes unique to BALB/c BMdMs, belong to p53 signaling pathway. We next investigated whether, in a given mice strain derived macrophages, the different up-regulated unique genes could be coordinately regulated. Using GeneMapp Cytoscape, we showed that the induced genes unique to BALB/c or C57BL/6 BMdMs are interconnected. Finally, we examined whether the induced pathways unique to BALB/c derived macrophages interfere with the ones unique to C57BL/6 derived macrophages. Protein-protein interaction analysis using String database highlights the existence of a cross-talk between p53 and mTOR signaling pathways respectively specific to susceptible and resistant BMd

  20. Phototoxic effects of silicon bis (dimetilaminoetanoxi)-phthalocyanine (SiPc) on the viability of Leishmania major and Leishmania braziliensis promastigotes

    NASA Astrophysics Data System (ADS)

    Guerra Pinto, Juliana; Ferreira-Strixino, Juliana; Mittmann, Josane

    2016-06-01

    American cutaneous leishmaniasis (ACL) is an infectious disease caused by protozoans of the genus Leishmania. The treatment may consist of pentavalent antimonials or pentamidine and amphotericin. However, these treatments are extremely aggressive. Photodynamic antimicrobial chemotherapy (PACT) involves the same mechanism of photodynamic therapy which associates a photosensitizer with oxygen and a light source generating a photochemical reaction leading to cell death. The aim of this study was to verify the potential use of silicon bis (dimetilaminoetanoxi)-phthalocyanine (SiPc) compound in photodynamic treatment through evaluation of its phototoxic effect in promastigotes of the genus Leishmania braziliensis and Leishmania major. Treatment with SiPc was able to drastically affect the viability of the parasites as well as affect their growth and morphology, after PACT treatment. The data shown in this study allows us to conclude that SiPc is a promising photosensitizer (PS) since it does not affect parasite growth and viability in the dark. After PACT with this phthalocyanine, over 99% of parasites were killed with the higher concentration and a light dose used. These results suggest that SiPc can be used in future to treat CL, however, further studies are necessary to determine whether the PS are toxic to mononuclear phagocytic cells and epithelial cells which will also be affected by therapy when applied topically.

  1. Murine immune response induced by Leishmania major during the implantation of paraffin tablets.

    PubMed

    Reis, Maria Letícia Costa; Ferreira, Vanessa Martins; Zhang, Xia; Gonçalves, Ricardo; Vieira, Leda Quércia; Tafuri, Washington Luiz; Mosser, David M; Tafuri, Wagner Luiz

    2010-11-01

    We carried out a model of chronic inflammation using a subcutaneous paraffin tablet in mice experimentally infected with Leishmania major. It was previously reported that the parasite load following paraffin implantation occurred at a peak of 21 days in both BALB/c and C57BL/6 mice. At the present study, we have investigated what cytokines and chemokines are directly related to the parasite load in C57BL/6 mice. All mice were divided in four groups: mice implanted with paraffin tablets; mice experimentally infected with L. major; mice implanted with paraffin tablets and experimentally infected with L. major; and mice submitted only to the surgery were used for the Real-Time Polymerase Chain Reaction (RT-PCR) controls. Fragments of skin tissue and the tissue surrounding the paraffin tablets (inflammatory capsule) were collected for histopathology and RT-PCR studies. By 21 days, a diffuse chronic inflammatory reaction was mainly observed in the deep dermis where macrophages parasitized with Leishmania amastigotes were also found. RT-PCR analysis has shown that BALB/c mice showed strong IL-4 and IL-10 mRNA expression than controls with very little expression of IFN-γ. In contrast, both IFN-γ and IL-10 mRNA was found in higher levels in C57BL/6 animals. Moreover, in C57BL/6 mice the expression of chemokines mRNA of CCL3/MIP-1α was more highly expressed than CCL2/MCP-1. We conclude that the Th1 immune response C57BL/6 did not change to a Th2 response, even though C57BL/6 animals presented higher parasitism than BALB/c mice 21 days after infection and paraffin implantation.

  2. Murine immune response induced by Leishmania major during the implantation of paraffin tablets

    PubMed Central

    Reis, Maria Letícia Costa; Ferreira, Vanessa Martins; Zhang, Xia; Gonçalves, Ricardo; Vieira, Leda Quércia; Tafuri, Washington Luiz; Mosser, David M.

    2011-01-01

    We carried out a model of chronic inflammation using a subcutaneous paraffin tablet in mice experimentally infected with Leishmania major. It was previously reported that the parasite load following paraffin implantation occurred at a peak of 21 days in both BALB/c and C57BL/6 mice. At the present study, we have investigated what cytokines and chemokines are directly related to the parasite load in C57BL/6 mice. All mice were divided in four groups: mice implanted with paraffin tablets; mice experimentally infected with L. major; mice implanted with paraffin tablets and experimentally infected with L. major; and mice submitted only to the surgery were used for the Real-Time Polymerase Chain Reaction (RT-PCR) controls. Fragments of skin tissue and the tissue surrounding the paraffin tablets (inflammatory capsule) were collected for histopathology and RT-PCR studies. By 21 days, a diffuse chronic inflammatory reaction was mainly observed in the deep dermis where macrophages parasitized with Leishmania amastigotes were also found. RT-PCR analysis has shown that BALB/c mice showed strong IL-4 and IL-10 mRNA expression than controls with very little expression of IFN-γ. In contrast, both IFN-γ and IL-10 mRNA was found in higher levels in C57BL/6 animals. Moreover, in C57BL/6 mice the expression of chemokines mRNA of CCL3/MIP-1α was more highly expressed than CCL2/MCP-1. We conclude that the Th1 immune response C57BL/6 did not change to a Th2 response, even though C57BL/6 animals presented higher parasitism than BALB/c mice 21 days after infection and paraffin implantation. PMID:20857143

  3. Amplified DNAs in laboratory stocks of Leishmania tarentolae: extrachromosomal circles structurally and functionally similar to the inverted-H-region amplification of methotrexate-resistant Leishmania major

    SciTech Connect

    Petrillo-Peixoto, M.L.; Beverley, S.M. )

    1988-12-01

    We describe the structure of amplified DNA that was discovered in two laboratory stocks of the protozoan parasite Leishmania tarentolae. Restriction mapping and molecular cloning revealed that a region of 42 kilobases was amplified 8- to 30-fold in these lines. Southern blot analyses of digested DNAs or chromosomes separated by pulsed-field electrophoresis showed that the amplified DNA corresponded to the H region, a locus defined originally by its amplification in methotrexate-resistant Leishmania major. Similarities between the amplified DNA of the two species included (i) extensive cross-hybridization; (ii) approximate conservation of sequence order; (iii) extrachromosomal localization; (iv) an overall inverted, head-to-head configuration as a circular 140-kilobase tetrameric molecule; (v) two regions of DNA sequence rearrangement, each of which was closely associated with the two centers of the inverted repeats; (vi) association with methotrexate resistance; and (vii) phenotypically conservative amplification, in which the wild-type chromosomal arrangement was retained without apparent modification. Our data showed that amplified DNA mediating drug resistance arose in unselected L. tarentolae, although the pressures leading to apparently spontaneous amplification and maintenance of the H region are not known. The simple structure and limited extent of DNA amplified in these and other Leishmania lines suggests that the study of gene amplification in Leishmania spp. offers an attractive model system for the study of amplification in cultured mammalian cells and tumors. We also introduced a method for measuring the size of large circular DNAs, using gamma-irradiation to introduce limited double-strand breaks followed by sizing of the linear DNAs by pulsed-field electrophoresis.

  4. Miltefosine-induced apoptotic cell death on Leishmania major and L. tropica strains.

    PubMed

    Khademvatan, Shahram; Gharavi, Mohammad Javad; Rahim, Fakher; Saki, Jasem

    2011-03-01

    The aim of this study was to assess the cytotoxic effects of various concentrations of miltefosine on Leishmania major (MRHO/IR/75/ER) and L. tropica (MHOM/IR/02/Mash10) promastigotes and to observe the programmed cell death features. The colorimetric MTT assay was used to find L. major and L. tropica viability and the obtained results were expressed as 50% inhibitory concentration (IC50). Also, 50% effective doses (ED50) for L. major and L. tropica amastigotes were also determined. Annexin-V FLUOS staining was performed to study the cell death properties of miltefosine using FACS analysis. Qualitative analysis of the total genomic DNA fragmentation was performed by agarose gel electrophoresis. Furthermore, to observe changes in cell morphology, promastigotes were examined using light microscopy. In both strains of L. major and L. tropica, miltefosine induced dose-dependent death with features of apoptosis, including cell shrinkage, DNA laddering, and externalization of phosphatidylserine. The IC50 was achieved at 22 µM and 11 µM for L. major and L. tropica after 48 hr of incubation, respectively. ED50 of L. major and L. tropica amastigotes were 5.7 µM and 4.2 µM, respectively. Our results indicate that miltefosine induces apoptosis of the causative agent of cutaneous leishmaniasis in a dose-dependent manner. Interestingly, L. major did not display any apoptotic changes when it was exposed to miltefosine in concentrations sufficient to kill L. tropica.

  5. Development of topical treatment for cutaneous leishmaniasis caused by Leishmania major in experimental animals.

    PubMed Central

    El-On, J; Jacobs, G P; Witztum, E; Greenblatt, C L

    1984-01-01

    Topical treatment, with drug-containing ointments, of cutaneous leishmaniasis caused by Leishmania major in BALB/c mice was studied. Twenty chemotherapeutic agents having potential or established antileishmanial activity were formulated in different ointment and cream bases. Only 15% paromomycin sulfate with 12% methylbenzethonium chloride, 12% benzethonium chloride, 12% cetalkonium chloride, or 12% dimethyl sulfoxide, all incorporated in white soft paraffin (United Kingdom patent application no. 2117237A), were completely effective. Topical treatment twice daily for 6 or more days caused total elimination of the parasites and healing of the lesion in all treated mice. All the other antileishmanial compounds, including sodium stibogluconate, pentamidine, amphotericin B, emetine hydrochloride, metronidazole, co-trimoxazole, allopurinol, and rifampin, either showed a slight effect on the parasites or were highly toxic to the animal host at the concentrations tested. Images PMID:6517557

  6. Structure of the major carbohydrate fragment of the Leishmania donovani lipophosphoglycan

    SciTech Connect

    Turco, S.J.; Hull, S.R.; Orlandi, P.A. Jr.; Shepherd, S.D.; Homans, S.W.; Dwek, R.A.; Rademacher, T.W.

    1987-09-22

    The major carbohydrate fragment from the lipophosphoglycan of Leishmania donovani was generated by mild acid hydrolysis (0.02 N HCl, 5 min, 100/sup 0/C) and purified by chromatography on DE-52 cellulose and thin layer. By a combination of analyses including gas-liquid chromatography-mass spectrometry and /sup 1/H NMR, the structure of the fragment was elucidated as PO/sub 4/..-->..6Gal(..beta..1-..-->..4)Man. Approximately 16 of these phosphorylated disaccharide units occur in the overall glycoconjugate structure. NMR analysis of an alkaline phosphatase treated phosphorylated tetrasaccharide generated from lipophosphoglycan showed that the phosphorylated disaccharide units are linked together via ..cap alpha..-glycosidic linkages. Complete characterization of the phosphorylated disaccharide units of lipophosphoglycan provides the first example of a defined carbohydrate anchored in membranes by a derivative of phosphatidylinositol.

  7. Molecular Detection of Leishmania major and L. turanica in Phlebotomus papatasi and First Natural Infection of P. salehi to L. major in North-East of Iran

    PubMed Central

    Rafizadeh, Sayena; Saraei, Mehrzad; Abaei, Mohammad Reza; Oshaghi, Mohammad Ali; Mohebali, Mehdi; Peymani, Amir; Naserpour-Farivar, Taghi; Bakhshi, Hassan; Rassi, Yavar

    2016-01-01

    Background: Leishmaniasis is an important public health disease in many developing countries as well in Iran. The main objective of this study was to investigate on leishmania infection of wild caught sand flies in an endemic focus of disease in Esfarayen district, north east of Iran. Methods: Sand flies were collected by sticky papers and mounted in a drop of Puri’s medium for species identification. Polymerase chain reaction techniques of kDNA, ITS1-rDNA, followed by restriction fragment length polymorphism were used for identification of DNA of Leishmania parasites within infected sand flies. Results: Among the collected female sand flies, two species of Phlebotomus papatasi and Phlebotomus salehi were found naturally infected with Leishmania major. Furthermore, mixed infection of Leishmania turanica and L. major was observed in one specimen of P. papatasi. Sequence analysis revealed two parasite ITS1 haplotypes including three L. major with accession numbers: KJ425408, KJ425407, KM056403 and one L. turanica. (KJ425406). The haplotype of L. major was identical (100%) to several L. major sequences deposited in GenBank, including isolates from Iran, (Gen Bank accession nos.AY573187, KC505421, KJ194178) and Uzbekistan (Accession no.FN677357). Conclusion: To our knowledge, this is the first detection of L. major within wild caught P. salehi in northeast of Iran. PMID:27308272

  8. Leishmania major Self-Limited Infection Increases Blood Cholesterol and Promotes Atherosclerosis Development.

    PubMed

    Fernandes, Luciana R; Ribeiro, Ana Cecília C; Segatto, Marcela; Santos, Luís Felipe F F; Amaral, Joana; Portugal, Luciane R; Leite, Jacqueline I A

    2013-01-01

    Leishmania major infection of resistant mice causes a self-limited lesion characterized by macrophage activation and a Th1 proinflammatory response. Atherosclerosis is an inflammatory disease involving hypercholesterolemia and macrophage activation. In this study, we evaluated the influence of L. major infection on the development of atherosclerosis using atherosclerosis-susceptible apolipoprotein E-deficient (apoE KO) mice. After 6 weeks of infection, apoE KO mice exhibited reduced footpad swelling and parasitemia similar to C57BL/6 controls, confirming that both strains are resistant to infection with L. major. L. major-infected mice had increased plasma cholesterol levels and reduced triacylglycerols. With regard to atherosclerosis, noninfected mice developed only fatty streak lesions, while the infected mice presented with advanced lesions containing a necrotic core and an abundant inflammatory infiltrate. CD36 expression was increased in the aortic valve of the infected mice, indicating increased macrophage activation. In conclusion, L. major infection, although localized and self-limited in resistant apoE KO mice, has a detrimental effect on the blood lipid profile, increases the inflammatory cell migration to atherosclerotic lesions, and promotes atherogenesis. These effects are consequences of the stimulation of the immune system by L. major, which promotes the inflammatory components of atherosclerosis, which are primarily the parasite-activated macrophages.

  9. Leishmania major Self-Limited Infection Increases Blood Cholesterol and Promotes Atherosclerosis Development

    PubMed Central

    Fernandes, Luciana R.; Ribeiro, Ana Cecília C.; Segatto, Marcela; Santos, Luís Felipe F. F.; Amaral, Joana; Portugal, Luciane R.; Leite, Jacqueline I. A.

    2013-01-01

    Leishmania major infection of resistant mice causes a self-limited lesion characterized by macrophage activation and a Th1 proinflammatory response. Atherosclerosis is an inflammatory disease involving hypercholesterolemia and macrophage activation. In this study, we evaluated the influence of L. major infection on the development of atherosclerosis using atherosclerosis-susceptible apolipoprotein E-deficient (apoE KO) mice. After 6 weeks of infection, apoE KO mice exhibited reduced footpad swelling and parasitemia similar to C57BL/6 controls, confirming that both strains are resistant to infection with L. major. L. major-infected mice had increased plasma cholesterol levels and reduced triacylglycerols. With regard to atherosclerosis, noninfected mice developed only fatty streak lesions, while the infected mice presented with advanced lesions containing a necrotic core and an abundant inflammatory infiltrate. CD36 expression was increased in the aortic valve of the infected mice, indicating increased macrophage activation. In conclusion, L. major infection, although localized and self-limited in resistant apoE KO mice, has a detrimental effect on the blood lipid profile, increases the inflammatory cell migration to atherosclerotic lesions, and promotes atherogenesis. These effects are consequences of the stimulation of the immune system by L. major, which promotes the inflammatory components of atherosclerosis, which are primarily the parasite-activated macrophages. PMID:23710353

  10. Protein network prediction and topological analysis in Leishmania major as a tool for drug target selection

    PubMed Central

    2010-01-01

    Background Leishmaniasis is a virulent parasitic infection that causes a worldwide disease burden. Most treatments have toxic side-effects and efficacy has decreased due to the emergence of resistant strains. The outlook is worsened by the absence of promising drug targets for this disease. We have taken a computational approach to the detection of new drug targets, which may become an effective strategy for the discovery of new drugs for this tropical disease. Results We have predicted the protein interaction network of Leishmania major by using three validated methods: PSIMAP, PEIMAP, and iPfam. Combining the results from these methods, we calculated a high confidence network (confidence score > 0.70) with 1,366 nodes and 33,861 interactions. We were able to predict the biological process for 263 interacting proteins by doing enrichment analysis of the clusters detected. Analyzing the topology of the network with metrics such as connectivity and betweenness centrality, we detected 142 potential drug targets after homology filtering with the human proteome. Further experiments can be done to validate these targets. Conclusion We have constructed the first protein interaction network of the Leishmania major parasite by using a computational approach. The topological analysis of the protein network enabled us to identify a set of candidate proteins that may be both (1) essential for parasite survival and (2) without human orthologs. These potential targets are promising for further experimental validation. This strategy, if validated, may augment established drug discovery methodologies, for this and possibly other tropical diseases, with a relatively low additional investment of time and resources. PMID:20875130

  11. MicroRNA expression profile in human macrophages in response to Leishmania major infection.

    PubMed

    Lemaire, Julien; Mkannez, Ghada; Guerfali, Fatma Z; Gustin, Cindy; Attia, Hanène; Sghaier, Rabiaa M; Dellagi, Koussay; Laouini, Dhafer; Renard, Patricia

    2013-01-01

    Leishmania (L.) are intracellular protozoan parasites able to survive and replicate in the hostile phagolysosomal environment of infected macrophages. They cause leishmaniasis, a heterogeneous group of worldwide-distributed affections, representing a paradigm of neglected diseases that are mainly embedded in impoverished populations. To establish successful infection and ensure their own survival, Leishmania have developed sophisticated strategies to subvert the host macrophage responses. Despite a wealth of gained crucial information, these strategies still remain poorly understood. MicroRNAs (miRNAs), an evolutionarily conserved class of endogenous 22-nucleotide non-coding RNAs, are described to participate in the regulation of almost every cellular process investigated so far. They regulate the expression of target genes both at the levels of mRNA stability and translation; changes in their expression have a profound effect on their target transcripts. We report in this study a comprehensive analysis of miRNA expression profiles in L. major-infected human primary macrophages of three healthy donors assessed at different time-points post-infection (three to 24 h). We show that expression of 64 out of 365 analyzed miRNAs was consistently deregulated upon infection with the same trends in all donors. Among these, several are known to be induced by TLR-dependent responses. GO enrichment analysis of experimentally validated miRNA-targeted genes revealed that several pathways and molecular functions were disturbed upon parasite infection. Finally, following parasite infection, miR-210 abundance was enhanced in HIF-1α-dependent manner, though it did not contribute to inhibiting anti-apoptotic pathways through pro-apoptotic caspase-3 regulation. Our data suggest that alteration in miRNA levels likely plays an important role in regulating macrophage functions following L. major infection. These results could contribute to better understanding of the dynamics of gene

  12. Metacyclogenesis is a major determinant of Leishmania promastigote virulence and attenuation.

    PubMed

    da Silva, R; Sacks, D L

    1987-11-01

    The in vivo virulence patterns of promastigote populations defined on the basis of agglutination by the lectin peanut agglutinin (PNA) were studied for various cloned lines of Leishmania major. Promastigotes derived from logarithmic-phase cultures, which were routinely 100% agglutinated at 100 micrograms of PNA per ml, were relatively avirulent for BALB/c mice. The relative virulence of stationary-phase promastigotes appeared to be attributable to the proportion of nonagglutinable (PNA-) promastigotes contained within these populations. Purification of PNA- organisms from stationary cultures provided for each clone the most virulent inoculum, supporting the view that this change in lectin binding accurately reflects the development of infective metacyclic stage promastigotes. By studying this marker, we found that there was considerable variation in the degree to which different strains and clones underwent metacyclogenesis during growth. Examination of a reportedly avirulent L. major clone revealed that metacyclogenesis was unusually delayed and inefficient for this clone, but that those PNA- promastigotes which could be recovered from late-stationary-phase cultures were virulent for BALB/c mice. The loss of virulence associated with frequent subculture could also be attributed to a drastic diminution in metacyclogenesis potential over time. A clone which yielded over 90% PNA- promastigotes during growth within passage 1 generated fewer than 10% PNA- promastigotes during growth by passage 94. Subcloning of late-passage attenuated promastigotes yielded a clone for which no PNA- promastigotes could be generated during growth, and an infective population could not be derived from this clone. Thus, metacyclogenesis does not appear to be stable for even cloned lines of Leishmania promastigotes, and virulence comparisons between different strains and clones can be meaningfully made only if the metacyclic populations contained within the respective inocula are determined.

  13. Uptake and antileishmanial activity of meglumine antimoniate-containing liposomes in Leishmania (Leishmania) major-infected macrophages.

    PubMed

    Borborema, Samanta Etel Treiger; Schwendener, Reto Albert; Osso, João Alberto; de Andrade, Heitor Franco; do Nascimento, Nanci

    2011-10-01

    Leishmaniasis is a parasitic disease caused by the intramacrophage protozoa Leishmania spp. and may be fatal if left untreated. Although pentavalent antimonials are toxic and their mechanism of action is unclear, they remain the first-line drugs for treatment of leishmaniasis. An effective therapy could be achieved by delivering antileishmanial drugs to the site of infection. Compared with free drugs, antileishmanial agent-containing liposomes are more effective, less toxic and have fewer adverse side effects. The aim of this study was to develop novel meglumine antimoniate (MA)-containing liposome formulations and to analyse their antileishmanial activity and uptake by macrophages. Determination of the 50% inhibitory concentration (IC(50)) values showed that MA-containing liposomes were ≥10-fold more effective than the free drug, with a 5-fold increase in selectivity index, higher activity and reduced macrophage toxicity. The concentration required to kill 100% of intracellular amastigotes was ≥40-fold lower when MA was encapsulated in liposomes containing phosphatidylserine compared with the free drug. Fluorescence microscopy analysis revealed increased uptake of fluorescent liposomes in infected macrophages after short incubation times compared with non-infected macrophages. In conclusion, these data suggest that MA encapsulated in liposome formulations is more effective against Leishmania-infected macrophages than the non-liposomal drug. Development of liposome formulations is a valuable approach to the treatment of infectious diseases involving the mononuclear phagocyte system. Copyright © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

  14. Intracellular Glycosylphosphatidylinositols Accumulate on Endosomes: Toxicity of Alpha-Toxin to Leishmania major

    PubMed Central

    Zheng, Zhifeng; Tweten, Rodney K.; Mensa-Wilmot, Kojo

    2005-01-01

    Glycosylphosphatidylinositols (GPIs) are ubiquitous glycolipids in eukaryotes. In the protozoan Leishmania major, GPIs occur “free” or covalently linked to proteins (e.g., gp63) and polysaccharides. While some free GPIs are detected on the plasma membrane, specific sites where GPIs accumulate intracellularly are unknown in most cells, although the glycolipids are synthesized within the secretory system. Herein, we describe a protocol for identifying intracellular sites of GPI accumulation by using alpha-toxin (from Clostridium septicum). Alpha-toxin bound to gp63 and GPIs from L. major. Intracellular binding sites for alpha-toxin were determined in immunofluorescence assays after removal of GPI-anchored macromolecules (e.g., gp63) from the plasma membrane of fixed cells by using detergent. Endosomes were a major site for GPI accretion in L. major. GPI-less gp63 was detected at the endoplasmic reticulum. In studies with live parasites, alpha-toxin killed L. major with a 50% lethal concentration of 0.77 nM. PMID:15755918

  15. The Transcriptome of Leishmania major Developmental Stages in Their Natural Sand Fly Vector

    PubMed Central

    Inbar, Ehud; Hughitt, V. Keith; Dillon, Laura A. L.; Ghosh, Kashinath

    2017-01-01

    ABSTRACT The life cycle of the Leishmania parasite in the sand fly vector involves differentiation into several distinctive forms, each thought to represent an adaptation to specific microenvironments in the midgut of the fly. Based on transcriptome sequencing (RNA-Seq) results, we describe the first high-resolution analysis of the transcriptome dynamics of four distinct stages of Leishmania major as they develop in a natural vector, Phlebotomus duboscqi. The early transformation from tissue amastigotes to procyclic promastigotes in the blood-fed midgut was accompanied by the greatest number of differentially expressed genes, including the downregulation of amastins, and upregulation of multiple cell surface proteins, sugar and amino acid transporters, and genes related to glucose metabolism and cell cycle progression. The global changes accompanying post-blood meal differentiation of procyclic promastigotes to the nectomonad and metacyclic stages were less extensive, though each displayed a unique signature. The transcriptome of nectomonads, which has not been studied previously, revealed changes consistent with cell cycle arrest and the upregulation of genes associated with starvation and stress, including autophagic pathways of protein recycling. Maturation to the infective, metacyclic stage was accompanied by changes suggesting preadaptation to the intracellular environment of the mammalian host, demonstrated by the amastigote-like profiles of surface proteins and metabolism-related genes. Finally, a direct comparison between sand fly-derived and culture-derived metacyclics revealed a reassuring similarity between the two forms, with the in vivo forms distinguished mainly by a stronger upregulation of transcripts associated with nutrient stress. PMID:28377524

  16. Aquaglyceroporin1 gene expression in antimony resistance and susceptible Leishmania major isolates.

    PubMed

    Eslami, Gilda; Zarchi, Morteza Vakil; Moradi, Alireza; Hejazi, Seyed Hossein; Sohrevardi, Seyed Mojtaba; Vakili, Mahmoud; Khamesipour, Ali

    2016-01-01

    The mechanism of antimony resistance in Leishmania has been studied extensively, in connection with decreased influx and/or increased eflux of the drug. Aquaporin 1 (AQP1) protein has been shown to mediate the uptake of trivalent antimony. This study was aimed to find the expression level of AQP1 gene in resistant versus non-resistant clinical isolates of Leishmania major in Iranian patients. Clinical isolates were obtained from 16 considered patients referred to Navab Safavi Clinical Center, Isfahan, Iran from October 2014 to December 2015. After diagnosis of cutaneous leishmaniasis using microscopic observation, biopsy was performed from lesion(s) of each patient and stored inside RNAlater solution at -20΀C. Written informed consent was obtained from all the patients to participate in the study before recording their information and sampling based on Helsinki declaration. Each patient was treated with Glucantime and followed for three months. All sensitive and resistance isolates were considered and compared with AQP1 gene expression using real time PCR that was analyzed with delta-delta Ct. Out of 16 clinical isolates, four patients were resistant and 12 were non-resistant. The AQP1 gene expression in resistant isolates was significantly higher than the one in response failure isolates (p = 0.001). The significant over expression (0.5 fold) of AQP1 gene in resistant versus non- resistant isolates suggests different mechanism of drug resistance such as mutations. Mutations may change the physiological function of the Aquaporin 1 protein that might affect its expression level.

  17. Modulation of Leishmania major aquaglyceroporin activity by a mitogen-activated protein kinase

    PubMed Central

    Mandal, Goutam; Sharma, Mansi; Kruse, Martin; Sander-Juelch, Claudia; Munro, Laura Anne; Wang, Yong; Vilg, Jenny Veide; Tamás, Markus J; Bhattacharjee, Hiranmoy; Wiese, Martin; Mukhopadhyay, Rita

    2012-01-01

    Summary Leishmania major aquaglyceroporin (LmjAQP1) adventitiously facilitates the uptake of antimonite [Sb(III)], an active form of Pentostam® or Glucantime®, which are the first line of defense against all forms of leishmaniasis. The present paper shows that LmjAQP1 activity is modulated by the mitogen-activated protein kinase, LmjMPK2. Leishmania parasites co-expressing LmjAQP1 and LmjMPK2 show increased Sb(III) uptake and increased Sb(III) sensitivity. When subjected to a hypo-osmotic stress, these cells show faster volume recovery than cells expressing LmjAQP1 alone. LmjAQP1 is phosphorylated in vivo at Thr197 and this phosphorylation requires LmjMPK2 activity. Lys42 of LmjMPK2 is critical for its kinase activity. Cells expressing altered T197A LmjAQP1 or K42A LmjMPK2 showed decreased Sb(III) influx and a slower volume recovery than cells expressing wild type proteins. Phosphorylation of LmjAQP1 led to a decrease in its turnover rate affecting LmjAQP1 activity. Although LmjAQP1 is localized to the flagellum of promastigotes, upon phosphorylation, it is relocalized to the entire surface of the parasite. L. mexicana promastigotes with an MPK2 deletion showed reduced Sb(III) uptake and slower volume recovery than wild type cells. This is the first report where a parasite aquaglyceroporin activity is post-translationally modulated by a MAP kinase. PMID:22779703

  18. Characterization of Chronic Cutaneous Lesions from TNF-Receptor-1-Deficient Mice Infected by Leishmania major

    PubMed Central

    Oliveira, Carolina Ferreira; Manzoni-de-Almeida, Daniel; Mello, Paula Seixas; Natale, Caio Cotta; Santiago, Helton da Costa; Miranda, Luíza da Silva; Ferraz, Fernanda Oliveira; dos Santos, Liliane Martins; Teixeira, Mauro Martins; Arantes, Rosa Maria Esteves; Vieira, Leda Quercia

    2012-01-01

    Leishmania major-infected TNF receptor 1 deficient (TNFR1 KO) mice resolve parasitism but fail to resolve lesions, while wild-type mice completely heal. We investigated the cell composition, cytokine production, and apoptosis in lesions from L. major-infected TNFR1 KO and wild-type (WT) mice. Chronic lesions from L. major-infected TNFR1 KO mice presented larger number of CD8+ T and Ly6G+ cells. In addition, higher concentrations of mRNA for IFN-γ CCL2 and CCL5, as well as protein, but lower numbers of apoptotic cells, were found in lesions from TNFR1 KO mice than in WT, at late time points of infection. Our studies showed that persistent lesions in L. major-infected TNFR1 KO mice may be mediated by continuous migration of cells to the site of inflammation due to the presence of chemokines and also by lower levels of apoptosis. We suggest that this model has some striking similarities to the mucocutaneous clinical form of leishmaniasis. PMID:22203861

  19. First molecular detection of Leishmania major within naturally infected Phlebotomus salehi from a zoonotic cutaneous leishmaniasis focus in southern Iran.

    PubMed

    Azizi, K; Fakoorziba, M R; Jalali, M; Moemenbellah-Fard, M D

    2012-03-01

    Human cutaneous leishmaniasis (CL) is a major notifiable public health problem in many parts of Iran. It is often caused by the zooflagellate parasite Leishmania major which is mainly transmitted by the bites of female phlebotomine sandflies belonging to the genus Phlebotomus (Diptera: Psychodidae). The annual incidence of CL in Fars province, southern Iran, was about 108-144 in 2007. The leishmanial infections of wild sandflies that may act as vectors were thus investigated at an endemic focus in this province. In all 330 female Phlebotomus sandflies were screened for the detection of Leishmania-specific kinetoplast DNA (kDNA) by polymerase chain reaction (PCR) methods. A two stage nested PCR protocol was used to establish the identity of Leishmania major species in naturally infected sandflies. The L. major kDNA was detected in 18 (5.5%) individual sandflies which belonged to four different Phlebotomus species (Phlebotomus papatasi, Phlebotomus salehi, Phlebotomus sergenti and P. major group). For the first time, one naturally infected P. salehi specimen contained the kDNA of the protozoan parasite, L. major, with a main band of 560 base pairs identified using the nested PCR method. It seems most likely therefore that P. salehi is potentially a rare sylvatic vector of L. major parasites in parts of this province. This is the first combined morphological and molecular studies of P. salehi in Iran.

  20. Leishmania major Promastigotes Evade LC3-Associated Phagocytosis through the Action of GP63

    PubMed Central

    Matte, Christine; Casgrain, Pierre-André; Séguin, Olivier; Moradin, Neda; Hong, Wan Jin; Descoteaux, Albert

    2016-01-01

    The protozoan Leishmania parasitizes macrophages and evades the microbicidal consequences of phagocytosis through the inhibition of phagolysosome biogenesis. In this study, we investigated the impact of this parasite on LC3-associated phagocytosis, a non-canonical autophagic process that enhances phagosome maturation and functions. We show that whereas internalization of L. major promastigotes by macrophages promoted LC3 lipidation, recruitment of LC3 to phagosomes was inhibited through the action of the parasite surface metalloprotease GP63. Reactive oxygen species generated by the NOX2 NADPH oxidase are necessary for LC3-associated phagocytosis. We found that L. major promastigotes prevented, in a GP63-dependent manner, the recruitment of NOX2 to phagosomes through a mechanism that does not involve NOX2 cleavage. Moreover, we found that the SNARE protein VAMP8, which regulates phagosomal assembly of the NADPH oxidase NOX2, was down-modulated by GP63. In the absence of VAMP8, recruitment of LC3 to phagosomes containing GP63-deficient parasites was inhibited, indicating that VAMP8 is involved in the phagosomal recruitment of LC3. These findings reveal a role for VAMP8 in LC3-associated phagocytosis and highlight a novel mechanism exploited by L. major promastigotes to interfere with the host antimicrobial machinery. PMID:27280768

  1. Multilocus microsatellite typing shows three different genetic clusters of Leishmania major in Iran.

    PubMed

    Mahnaz, Tashakori; Al-Jawabreh, Amer; Kuhls, Katrin; Schönian, Gabriele

    2011-10-01

    Ten polymorphic microsatellite markers were used to analyse 25 strains of Leishmania major collected from cutaneous leishmaniasis cases in different endemic areas in Iran. Nine of the markers were polymorphic, revealing 21 different genotypes. The data displayed significant microsatellite polymorphism with rare allelic heterozygosity. Bayesian statistic and distance based analyses identified three genetic clusters among the 25 strains analysed. Cluster I represented mainly strains isolated in the west and south-west of Iran, with the exception of four strains originating from central Iran. Cluster II comprised strains from the central part of Iran, and cluster III included only strains from north Iran. The geographical distribution of L. major in Iran was supported by comparing the microsatellite profiles of the 25 Iranian strains to those of 105 strains collected in 19 Asian and African countries. The Iranian clusters I and II were separated from three previously described populations comprising strains from Africa, the Middle East and Central Asia whereas cluster III grouped together with the Central Asian population. The considerable genetic variability of L. major might be related to the existence of different populations of Phlebotomus papatasi and/or to differences in reservoir host abundance in different parts of Iran. Copyright © 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  2. Immunotherapeutic effects of chitin in comparison with chitosan against Leishmania major infection.

    PubMed

    Hoseini, Mostafa Haji Molla; Moradi, Maryam; Alimohammadian, Mohammad Hossein; Shahgoli, Vahid Khaze; Darabi, Hayedeh; Rostami, Ali

    2016-04-01

    Chitin and chitosan microparticles (MPs) are important immune system stimulators. The aim of this study was to evaluate the protective effects of these compounds in comparison with each other against Leishmania infection in BALB/c mice infected with Leishmania major (L. major). Female BALB/c mice were injected subcutaneously with 2×10(5) promastigotes. Chitin and/or chitosan MPs (<40 μm) were subcutaneously injected in the BALB/c mice with two-day intervals until two weeks. Mice in all groups were sacrificed at 12 weeks post-infection. Enumeration of viable parasites was performed using limiting dilution assay. Furthermore, the animals (5 mice/group) were sacrificed two weeks post-infection. The lymph node cells were isolated and the effects of the chitinous MPs on the proliferation and production of cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10) were determined. The mean sizes of lesions were significantly smaller in chitin (0.6±0.12 mm) and chitosan treated groups (1.2±0.8 mm) than in the control group (6.2±1.7 mm) (P<0.05). The parasite load in the lymph nodes of the treated mice was significantly lower than that in the lymph nodes of controls (1.31×10(6) vs 8.24×10(7) parasite/lymph node [P=0.032] and 7.49×10(6) vs 8.24×10(7) parasite/lymph node [P=0.05] for chitin and chitosan MPs treatment, respectively). We found that chitinous MPs induced cell proliferation and that chitin but not chitosan increased TNF-α and IL-10 production. Chitin appears that it has more effect than chitosan against leishmaniasis. The current study revealed that chitinous MPs had significant activity against L. major and could be considered as new therapeutic modality in leishmaniasis.

  3. Aberrant host defense against Leishmania major in the absence of SLPI

    PubMed Central

    McCartney-Francis, Nancy; Jin, Wenwen; Belkaid, Yasmine; McGrady, George; Wahl, Sharon M.

    2014-01-01

    SLPI, a potent epithelial and myeloid-derived serine protease inhibitor with antimicrobial and anti-inflammatory functions, is induced by the intracellular parasite Leishmania major, and increased SLPI expression is evident within lesions that follow L. major infection. In contrast to self-resolving infection in C57Bl/6 WT mice, Slpi−/− mice launch a strong Th1 response to L. major, yet fail to control infection and develop destructive, nonhealing lesions with systemic spread of parasites. Because SLPI is both produced by murine macrophages and antagonizes their function, we examined the contribution of macrophage polarization to the defective host response in the absence of SLPI. Slpi−/− and Slpi+/+ macrophages were first primed with either IFNγ or IL-4 to generate classically activated M1 or alternatively activated M2 macrophages. After infection with L. major, Slpi−/− M1 macrophages expressed elevated iNOS RNA, whereas arginase was more highly expressed in WT than Slpi−/− M2 macrophages. After in vivo infection, we found that both IFNγ and iNOS were persistently overexpressed in chronic lesions in Slpi−/− mice, but surprisingly, IL-4 and arginase concomitantly remained elevated. Moreover, overexpression of the negative regulators SOCS1 and IL-27 provided insight into the failure of IFNγ to clear L. major from the dermal lesions. Notably, adenoviral delivery of SLPI to L. major-infected Slpi−/− mice significantly limited the progression of infection. These studies suggest that convergence of M1 and M2 macrophage responses may influence the outcome of innate host defense against intracellular parasites and that SLPI is critical for coordinating resistance to chronic leishmaniasis. PMID:25030421

  4. Vaccination with TAT-antigen fusion protein induces protective, CD8(+) T cell-mediated immunity against Leishmania major.

    PubMed

    Kronenberg, Katharina; Brosch, Sven; Butsch, Florian; Tada, Yayoi; Shibagaki, Naotaka; Udey, Mark C; von Stebut, Esther

    2010-11-01

    In murine leishmaniasis, healing is mediated by IFN-γ-producing CD4(+) and CD8(+) T cells. Thus, an efficacious vaccine should induce Th1 and Tc1 cells. Dendritic cells (DCs) pulsed with exogenous proteins primarily induce strong CD4-dependent immunity; induction of CD8 responses has proven to be difficult. We evaluated the immunogenicity of fusion proteins comprising the protein transduction domain of HIV-1 TAT and the Leishmania antigen LACK (Leishmania homolog of receptors for activated C kinase), as TAT-fusion proteins facilitate major histocompatibility complex class I-dependent antigen presentation. In vitro, TAT-LACK-pulsed DCs induced stronger proliferation of Leishmania-specific CD8(+) T cells compared with DCs incubated with LACK alone. Vaccination with TAT-LACK-pulsed DCs or fusion proteins plus adjuvant in vivo significantly improved disease outcome in Leishmania major-infected mice and was superior to vaccination with DCs treated with LACK alone. Vaccination with DC+TAT-LACK resulted in stronger proliferation of CD8(+) T cells when compared with immunization with DC+LACK. Upon depletion of CD4(+) or CD8(+) T cells, TAT-LACK-mediated protection was lost. TAT-LACK-pulsed IL-12p40-deficient DCs did not promote protection in vivo. In summary, these data show that TAT-fusion proteins are superior in activating Leishmania-specific Tc1 cells when compared with antigen alone and suggest that IL-12-dependent preferential induction of antigen-specific CD8(+) cells promotes significant protection against this important human pathogen.

  5. Immunization against Leishmania major Infection Using LACK- and IL-12-Expressing Lactococcus lactis Induces Delay in Footpad Swelling

    PubMed Central

    Hugentobler, Felix; Yam, Karen K.; Gillard, Joshua; Mahbuba, Raya; Olivier, Martin; Cousineau, Benoit

    2012-01-01

    Background Leishmania is a mammalian parasite affecting over 12 million individuals worldwide. Current treatments are expensive, cause severe side effects, and emerging drug resistance has been reported. Vaccination is the most cost-effective means to control infectious disease but currently there is no vaccine available against Leishmaniasis. Lactococcus lactis is a non-pathogenic, non-colonizing Gram-positive lactic acid bacterium commonly used in the dairy industry. Recently, L. lactis was used to express biologically active molecules including vaccine antigens and cytokines. Methodology/Principal findings We report the generation of L. lactis strains expressing the protective Leishmania antigen, LACK, in the cytoplasm, secreted or anchored to the bacterial cell wall. L. lactis was also engineered to secrete biologically active single chain mouse IL-12. Subcutaneous immunization with live L. lactis expressing LACK anchored to the cell wall and L. lactis secreting IL-12 significantly delayed footpad swelling in Leishmania major infected BALB/c mice. The delay in footpad swelling correlated with a significant reduction of parasite burden in immunized animals compared to control groups. Immunization with these two L. lactis strains induced antigen-specific multifunctional TH1 CD4+ and CD8+ T cells and a systemic LACK-specific TH1 immune response. Further, protection in immunized animals correlated with a Leishmania-specific TH1 immune response post-challenge. L. lactis secreting mouse IL-12 was essential for directing immune responses to LACK towards a protective TH1 response. Conclusions/Significance This report demonstrates the use of L. lactis as a live vaccine against L. major infection in BALB/c mice. The strains generated in this study provide the basis for the development of an inexpensive and safe vaccine against the human parasite Leishmania. PMID:22348031

  6. First report of Leishmania tropica from a classical focus of L. major in North-Sinai, Egypt.

    PubMed

    Shehata, Magdi G; Samy, Abdallah M; Doha, Said A; Fahmy, Adel R; Kaldas, Rania M; Furman, Barry D; Villinski, Jeffrey T

    2009-08-01

    Cutaneous leishmaniasis (CL) is prevalent in the Egyptian Sinai Peninsula and previous research has consistently documented the etiologic agent to be Leishmania major. We report the first isolation of Leishmania tropica from human cases of CL in a Northern Sinai community bordering Palestine. Parasite culturing, real-time polymerase chain reaction (PCR), gene sequencing, and restriction fragment length polymorphism (RFLP) analyses indicate CL cases in this community were caused by either L. major or L. tropica (three cases each). Two wild-caught rodents (Gerbillus pyramidum floweri) were infected with L. tropica. Phlebotomus papatasi sand flies were found harboring L. major, however only non-infected individuals of Phlebotomus sergenti, a vector for L. tropica, were caught. Patients with L. tropica had not traveled from the region in over a year, suggesting these cases are autochthonous. This scenario is consistent with an incursion of L. tropica from bordering countries and raises concerns about expansion of this parasite further into Egypt.

  7. Implication of different domains of the Leishmania major metacaspase in cell death and autophagy.

    PubMed

    Casanova, M; Gonzalez, I J; Sprissler, C; Zalila, H; Dacher, M; Basmaciyan, L; Späth, G F; Azas, N; Fasel, N

    2015-10-22

    Metacaspases (MCAs) are cysteine peptidases expressed in plants, fungi and protozoa, with a caspase-like histidine-cysteine catalytic dyad, but differing from caspases, for example, in their substrate specificity. The role of MCAs is subject to debate: roles in cell cycle control, in cell death or even in cell survival have been suggested. In this study, using a Leishmania major MCA-deficient strain, we showed that L. major MCA (LmjMCA) not only had a role similar to caspases in cell death but also in autophagy and this through different domains. Upon cell death induction by miltefosine or H2O2, LmjMCA is processed, releasing the catalytic domain, which activated substrates via its catalytic dyad His/Cys and a proline-rich C-terminal domain. The C-terminal domain interacted with proteins, notably proteins involved in stress regulation, such as the MAP kinase LmaMPK7 or programmed cell death like the calpain-like cysteine peptidase. We also showed a new role of LmjMCA in autophagy, acting on or upstream of ATG8, involving Lmjmca gene overexpression and interaction of the C-terminal domain of LmjMCA with itself and other proteins. These results allowed us to propose two models, showing the role of LmjMCA in the cell death and also in the autophagy pathway, implicating different protein domains.

  8. Ribosomal RNA Genes in the Protozoan Parasite Leishmania major Possess a Nucleosomal Structure.

    PubMed

    Vizuet-de-Rueda, Juan C; Florencio-Martínez, Luis E; Padilla-Mejía, Norma E; Manning-Cela, Rebeca; Hernández-Rivas, Rosaura; Martínez-Calvillo, Santiago

    2016-04-01

    Little is known about nucleosome structure and epigenetic regulation of transcription of rRNA genes in early-branched eukaryotes. Here we analyze the chromatin architecture and distribution of some histone modifications in the rRNA genes in the parasitic protozoon Leishmania major. Southern blots of MNase-partially-digested chromatin with DNA probes spanning the whole rRNA gene repeat showed that the intergenic spacer presents a tight nucleosomal structure, whereas the promoter region is practically devoid of nucleosomes. Intermediate levels of nucleosomes were found in the rRNA coding regions. ChIP assays allowed us to determine that H3K14ac, H3K23ac and H3K27ac, epigenetics marks that are generally associated with activation of transcription, are enriched in the promoter region. In contrast, H4K20me3, which is generally related to transcriptional silencing, was absent from the promoter region and intergenic spacer and enriched in the coding region. Interestingly, the distribution pattern for H3K9me3, generally linked to heterochromatin formation, was very similar to the distribution observed with the euchromatin marks, suggesting that this modification could be involved in transcriptional activation of rRNA genes in L. major. Copyright © 2016 Elsevier GmbH. All rights reserved.

  9. Implication of different domains of the Leishmania major metacaspase in cell death and autophagy

    PubMed Central

    Casanova, M; Gonzalez, I J; Sprissler, C; Zalila, H; Dacher, M; Basmaciyan, L; Späth, G F; Azas, N; Fasel, N

    2015-01-01

    Metacaspases (MCAs) are cysteine peptidases expressed in plants, fungi and protozoa, with a caspase-like histidine–cysteine catalytic dyad, but differing from caspases, for example, in their substrate specificity. The role of MCAs is subject to debate: roles in cell cycle control, in cell death or even in cell survival have been suggested. In this study, using a Leishmania major MCA-deficient strain, we showed that L. major MCA (LmjMCA) not only had a role similar to caspases in cell death but also in autophagy and this through different domains. Upon cell death induction by miltefosine or H2O2, LmjMCA is processed, releasing the catalytic domain, which activated substrates via its catalytic dyad His/Cys and a proline-rich C-terminal domain. The C-terminal domain interacted with proteins, notably proteins involved in stress regulation, such as the MAP kinase LmaMPK7 or programmed cell death like the calpain-like cysteine peptidase. We also showed a new role of LmjMCA in autophagy, acting on or upstream of ATG8, involving Lmjmca gene overexpression and interaction of the C-terminal domain of LmjMCA with itself and other proteins. These results allowed us to propose two models, showing the role of LmjMCA in the cell death and also in the autophagy pathway, implicating different protein domains. PMID:26492367

  10. An essential role for the Leishmania major metacaspase in cell cycle progression.

    PubMed

    Ambit, A; Fasel, N; Coombs, G H; Mottram, J C

    2008-01-01

    Metacaspases (MCAs) are distant orthologues of caspases and have been proposed to play a role in programmed cell death in yeast and plants, but little is known about their function in parasitic protozoa. The MCA gene of Leishmania major (LmjMCA) is expressed in actively replicating amastigotes and procyclic promastigotes, but at a lower level in metacyclic promastigotes. LmjMCA has a punctate distribution throughout the cell in interphase cells, but becomes concentrated in the kinetoplast (mitochondrial DNA) at the time of the organelle's segregation. LmjMCA also translocates to the nucleus during mitosis, where it associates with the mitotic spindle. Overexpression of LmjMCA in promastigotes leads to a severe growth retardation and changes in ploidy, due to defects in kinetoplast segregation and nuclear division and an impairment of cytokinesis. LmjMCA null mutants could not be generated and following genetic manipulation to express LmjMCA from an episome, the only mutants that were viable were those expressing LmjMCA at physiological levels. Together these data suggest that in L. major active LmjMCA is essential for the correct segregation of the nucleus and kinetoplast, functions that could be independent of programmed cell death, and that the amount of LmjMCA is crucial. The absence of MCAs from mammals makes the enzyme a potential drug target against protozoan parasites.

  11. Control of cutaneous leishmaniasis caused by Leishmania major in south-eastern Morocco.

    PubMed

    Bennis, Issam; De Brouwere, Vincent; Ameur, Btissam; El Idrissi Laamrani, Abderrahmane; Chichaoui, Smaine; Hamid, Sahibi; Boelaert, Marleen

    2015-10-01

    The incidence of cutaneous leishmaniasis (CL) caused by Leishmania major has increased in Morocco over the last decade, prompting the Ministry of Health to take intersectoral response measures including vector and reservoir control. The aim of this article was to describe the CL outbreak response measures taken in the province of Errachidia, where the reservoir of L. major, a sand rat (Meriones shawi), was targeted using strychnine-poisoned wheat baits from 2010 to 2012. We analysed routine surveillance data and other information using the data of the CL control programme. We present data on the evolution and the extension of CL in this province as well as the epidemiological profile of the disease. Between 2004 and 2013, 7099 cases of CL were recorded in Errachidia Province, gradually affecting all districts. Our results demonstrate that more women were affected than men and that all age groups were represented. Errachidia Province was the epicentre of the recent CL outbreak in Morocco. A notable decline in incidence rates was observed after 2011. The outbreak control measures may have contributed to this decline, as well as climatic trends or progressing herd immunity. © 2015 John Wiley & Sons Ltd.

  12. Structure of Lmaj006129AAA, a hypothetical protein from Leishmania major

    SciTech Connect

    Arakaki, Tracy; Le Trong, Isolde; Phizicky, Eric; Quartley, Erin; DeTitta, George; Luft, Joseph; Lauricella, Angela; Anderson, Lori; Kalyuzhniy, Oleksandr; Worthey, Elizabeth; Myler, Peter J.; Kim, David; Baker, David; Hol, Wim G. J.; Merritt, Ethan A.

    2006-03-01

    The crystal structure of a conserved hypothetical protein from L. major, Pfam sequence family PF04543, structural genomics target ID Lmaj006129AAA, has been determined at a resolution of 1.6 Å. The gene product of structural genomics target Lmaj006129 from Leishmania major codes for a 164-residue protein of unknown function. When SeMet expression of the full-length gene product failed, several truncation variants were created with the aid of Ginzu, a domain-prediction method. 11 truncations were selected for expression, purification and crystallization based upon secondary-structure elements and disorder. The structure of one of these variants, Lmaj006129AAH, was solved by multiple-wavelength anomalous diffraction (MAD) using ELVES, an automatic protein crystal structure-determination system. This model was then successfully used as a molecular-replacement probe for the parent full-length target, Lmaj006129AAA. The final structure of Lmaj006129AAA was refined to an R value of 0.185 (R{sub free} = 0.229) at 1.60 Å resolution. Structure and sequence comparisons based on Lmaj006129AAA suggest that proteins belonging to Pfam sequence families PF04543 and PF01878 may share a common ligand-binding motif.

  13. FasL and TRAIL Induce Epidermal Apoptosis and Skin Ulceration Upon Exposure to Leishmania major

    PubMed Central

    Eidsmo, Liv; Fluur, Caroline; Rethi, Bence; Eriksson Ygberg, Sofia; Ruffin, Nicolas; De Milito, Angelo; Akuffo, Hannah; Chiodi, Francesca

    2007-01-01

    Receptor-mediated apoptosis is proposed as an important regulator of keratinocyte homeostasis in human epidermis. We have previously reported that Fas/FasL interactions in epidermis are altered during cutaneous leishmaniasis (CL) and that keratinocyte death through apoptosis may play a pathogenic role for skin ulceration. To further investigate the alterations of apoptosis during CL, a keratinocyte cell line (HaCaT) and primary human epidermal keratinocytes were incubated with supernatants from Leishmania major-infected peripheral blood mononuclear cells. An apoptosis-specific microarray was used to assess mRNA expression in HaCaT cells exposed to supernatants derived from L. major-infected cultures. Fas and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA and protein expression were significantly up-regulated, and apoptosis was detected in both HaCaT and human epidermal keratinocyte cells. The keratinocyte apoptosis was partly inhibited through blocking of Fas or FasL and even more efficiently through TRAIL neutralization. Up-regulation of Fas on keratinocytes in epidermis and the presence of FasL-expressing macrophages and T cells in dermis were previously reported by us. In this study, keratinocytes expressing TRAIL, as well as the proapoptotic receptor TRAIL-R2, were detected in skin biopsies from CL cases. We propose that activation of Fas and TRAIL apoptosis pathways, in the presence of inflammatory mediators at the site of infection, leads to tissue destruction and ulceration during CL. PMID:17200196

  14. Characterization of a Novel Endoplasmic Reticulum Protein Involved in Tubercidin Resistance in Leishmania major

    PubMed Central

    Aoki, Juliana Ide; Coelho, Adriano Cappellazzo; Muxel, Sandra Marcia; Zampieri, Ricardo Andrade; Sanchez, Eduardo Milton Ramos; Nerland, Audun Helge; Floeter-Winter, Lucile Maria; Cotrim, Paulo Cesar

    2016-01-01

    Background Tubercidin (TUB) is a toxic adenosine analog with potential antiparasitic activity against Leishmania, with mechanism of action and resistance that are not completely understood. For understanding the mechanisms of action and identifying the potential metabolic pathways affected by this drug, we employed in this study an overexpression/selection approach using TUB for the identification of potential targets, as well as, drug resistance genes in L. major. Although, TUB is toxic to the mammalian host, these findings can provide evidences for a rational drug design based on purine pathway against leishmaniasis. Methodology/Principal findings After transfection of a cosmid genomic library into L. major Friedlin (LmjF) parasites and application of the overexpression/selection method, we identified two cosmids (cosTUB1 and cosTU2) containing two different loci capable of conferring significant levels of TUB resistance. In the cosTUB1 contained a gene encoding NUPM1-like protein, which has been previously described as associated with TUB resistance in L. amazonensis. In the cosTUB2 we identified and characterized a gene encoding a 63 kDa protein that we denoted as tubercidin-resistance protein (TRP). Functional analysis revealed that the transfectants were less susceptible to TUB than LmjF parasites or those transfected with the control vector. In addition, the trp mRNA and protein levels in cosTUB2 transfectants were higher than LmjF. TRP immunolocalization revealed that it was co-localized to the endoplasmic reticulum (ER), a cellular compartment with many functions. In silico predictions indicated that TRP contains only a hypothetical transmembrane domain. Thus, it is likely that TRP is a lumen protein involved in multidrug efflux transport that may be involved in the purine metabolic pathway. Conclusions/Significance This study demonstrated for the first time that TRP is associated with TUB resistance in Leishmania. The next challenge is to determine how

  15. In vitro and in vivo activities of Peganum harmala extract against Leishmania major

    PubMed Central

    Rahimi-Moghaddam, Parvaneh; Ebrahimi, Soltan Ahmed; Ourmazdi, Hourmazd; Selseleh, Monawar; Karjalian, Maryam; Haj-Hassani, Giti; Alimohammadian, Mohammad Hossein; Mahmoudian, Massoud; Shafiei, Massoumeh

    2011-01-01

    BACKGROUND: In vitro and in vivo antileishmanial activities of crude hydroalcoholic extract of peganum harmala seeds were investigated against Leishmania major. METHODS: The extract of aerial parts of P harmala was obtained by maceration. The in vitro experiments were performed on promastigotes to assess antileishmanial activity of the extract using amphotericin B as a reference. The in vivo studies were carried out on cutaneous leishmaniasis in outbred mice to evaluate the effects of topical application of the ointment-based extract. RESULTS: The in vitro experiments showed a concentration-dependent decrease of parasites number caused by the extract with an IC50 value of 59.4 μg/ml. In vivo studies demonstrated a significant post-treatment decrease in the lesion size and parasite count in infected animals, compared to placebo and control groups. High performance liquid chromatography (HPLC) of the crude extract demonstrated the existence of harmaline and harmine as beta-carboline alkaloids. CONCLUSIONS: P harmala seeds extract showed significant in vitro and in vivo antileishmanial activities. Most biological activity of the extract could be attributed to its beta-carboline content. However, another alkaloid of P harmala seeds extract, peganine, has also been reported to have antileishmanial activity. These beneficial effects can be attributed to the cumulative effects of various biologically active components present in it. PMID:22279479

  16. In vitro and in vivo activity of major constituents from Pluchea carolinensis against Leishmania amazonensis.

    PubMed

    Montrieux, Elly; Perera, Wilmer H; García, Marley; Maes, Louis; Cos, Paul; Monzote, Lianet

    2014-08-01

    The search for new therapeutic agents from natural sources has been a constant for the treatment of diseases such as leishmaniasis. Herein, in vitro and in vivo pharmacological activities of pure major phenolic constituents (caffeic acid, chlorogenic acid, ferulic acid, quercetin, and rosmarinic acid) from Pluchea carolinensis against Leishmania amazonensis are presented. Pure compounds showed inhibitory activity against promastigotes (IC50 = 0.2-0.9 μg/mL) and intracellular amastigotes (IC50 = 1.3-2.9 μg/mL). Four of them were selected after testing against macrophages of BALB/c mice: caffeic acid, ferulic acid, quercetin, and rosmarinic acid, with selective indices of 11, 17, 10, and 20, respectively. Ferulic acid, rosmarinic acid, and caffeic acid controlled lesion size development and parasite burden in footpads from BALB/c experimentally infected mice, after five injections of compounds by intralesional route at 30 mg/kg every 4 days. Pure compounds from P. carolinensis demonstrated antileishmanial properties.

  17. Leishmania major-Like Antigen for Specific and Sensitive Serodiagnosis of Human and Canine Visceral Leishmaniasis

    PubMed Central

    Barbosa-de-Deus, RosÂngela; Luíz dos Mares-Guia, Marcos; Zacarias Nunes, Adriane; Morais Costa, Kátia; Gonçalves Junqueira, Roberto; Mayrink, Wilson; Genaro, Odair; Pereira Tavares, Carlos Alberto

    2002-01-01

    An antigen (LMS) prepared from Leishmania major-like promastigotes was used in an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of human and dog visceral leishmaniasis. The results were compared with those from the indirect immunofluorescent antibody test (IFAT). A total of 1,822 canine sera were tested, including sera from dogs with visceral leishmaniasis, transmissible venereal tumors, ehrlichiosis, rickettsiosis, or Chagas' disease and sera from healthy dogs. The antigen was also tested with 227 samples of human sera, including sera from patients with visceral, cutaneous, or diffuse cutaneous leishmaniasis and from noninfected individuals, as well as sera from patients with Chagas' disease, toxoplasmosis, rickettsiosis, hepatitis B, schistosomiasis, ascaridiasis, malaria, rheumatoid factor, leprosy and rheumatoid factor, tuberculosis, or leprosy. All dogs and all human patients had a clinical and/or serological and/or parasitological diagnosis. For detecting antibodies in sera from dogs with leishmaniasis, the antigen showed a sensitivity of 98%, specificity of 95%, and concordance of 93% and when used for detecting antibodies in human sera presented a sensitivity of 92%, specificity of 100%, and concordance of 92%. Comparison between ELISA and IFAT demonstrated that ELISA using the LMS antigen yielded more reliable results than IFAT. The LMS antigen displayed no cross-reactivity with sera from patients or dogs that had any of the other diseases tested. PMID:12414775

  18. B-1 cells modulate the murine macrophage response to Leishmania major infection.

    PubMed

    Arcanjo, Angelica F; Nunes, Marise P; Silva-Junior, Elias B; Leandro, Monique; da Rocha, Juliana Dutra Barbosa; Morrot, Alexandre; Decote-Ricardo, Debora; Freire-de-Lima, Celio Geraldo

    2017-05-26

    To investigate the modulatory effect of B-1 cells on murine peritoneal macrophages infected with Leishmania major (L. major) in vitro. Peritoneal macrophages obtained from BALB/c and BALB/c XID mice were infected with L. major and cultured in the presence or absence of B-1 cells obtained from wild-type BALB/c mice. Intracellular amastigotes were counted, and interleukin-10 (IL-10) production was quantified in the cellular supernatants using an enzyme-linked immunosorbent assay. The levels of the lipid mediator prostaglandin E2 (PGE2) were determined using a PGE2 enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI), and the number of lipid bodies was quantified in the cytoplasm of infected macrophages in the presence and absence of B-1 cells. Culturing the cells with selective PGE2-neutralizing drugs inhibited PGE2 production and confirmed the role of this lipid mediator in IL-10 production. In contrast, we demonstrated that B-1 cells derived from IL-10 KO mice did not favor the intracellular growth of L. major. We report that B-1 cells promote the growth of L. major amastigotes inside peritoneal murine macrophages. We demonstrated that the modulatory effect was independent of physical contact between the cells, suggesting that soluble factor(s) were released into the cultures. We demonstrated in our co-culture system that B-1 cells trigger IL-10 production by L. major-infected macrophages. Furthermore, the increased secretion of IL-10 was attributed to the presence of the lipid mediator PGE2 in supernatants of L. major-infected macrophages. The presence of B-1 cells also favors the production of lipid bodies by infected macrophages. In contrast, we failed to obtain the same effect on parasite replication inside L. major-infected macrophages when the B-1 cells were isolated from IL-10 knockout mice. Our results show that elevated levels of PGE2 and IL-10 produced by B-1 cells increase L. major growth, as indicated by the number of parasites in cell cultures.

  19. B-1 cells modulate the murine macrophage response to Leishmania major infection

    PubMed Central

    Arcanjo, Angelica F; Nunes, Marise P; Silva-Junior, Elias B; Leandro, Monique; da Rocha, Juliana Dutra Barbosa; Morrot, Alexandre; Decote-Ricardo, Debora; Freire-de-Lima, Celio Geraldo

    2017-01-01

    AIM To investigate the modulatory effect of B-1 cells on murine peritoneal macrophages infected with Leishmania major (L. major) in vitro. METHODS Peritoneal macrophages obtained from BALB/c and BALB/c XID mice were infected with L. major and cultured in the presence or absence of B-1 cells obtained from wild-type BALB/c mice. Intracellular amastigotes were counted, and interleukin-10 (IL-10) production was quantified in the cellular supernatants using an enzyme-linked immunosorbent assay. The levels of the lipid mediator prostaglandin E2 (PGE2) were determined using a PGE2 enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI), and the number of lipid bodies was quantified in the cytoplasm of infected macrophages in the presence and absence of B-1 cells. Culturing the cells with selective PGE2-neutralizing drugs inhibited PGE2 production and confirmed the role of this lipid mediator in IL-10 production. In contrast, we demonstrated that B-1 cells derived from IL-10 KO mice did not favor the intracellular growth of L. major. RESULTS We report that B-1 cells promote the growth of L. major amastigotes inside peritoneal murine macrophages. We demonstrated that the modulatory effect was independent of physical contact between the cells, suggesting that soluble factor(s) were released into the cultures. We demonstrated in our co-culture system that B-1 cells trigger IL-10 production by L. major-infected macrophages. Furthermore, the increased secretion of IL-10 was attributed to the presence of the lipid mediator PGE2 in supernatants of L. major-infected macrophages. The presence of B-1 cells also favors the production of lipid bodies by infected macrophages. In contrast, we failed to obtain the same effect on parasite replication inside L. major-infected macrophages when the B-1 cells were isolated from IL-10 knockout mice. CONCLUSION Our results show that elevated levels of PGE2 and IL-10 produced by B-1 cells increase L. major growth, as indicated by the number of

  20. Neutrophils Contribute to the Protection Conferred by ArtinM against Intracellular Pathogens: A Study on Leishmania major

    PubMed Central

    Ricci-Azevedo, Rafael; Oliveira, Aline Ferreira; Conrado, Marina C. A. V.; Carvalho, Fernanda Caroline; Roque-Barreira, Maria Cristina

    2016-01-01

    ArtinM, a D-mannose binding lectin from Artocarpus heterophyllus, has immunomodulatory activities through its interaction with N-glycans of immune cells, culminating with the establishment of T helper type 1 (Th1) immunity. This interaction protects mice against intracellular pathogens, including Leishmania major and Leishmania amazonensis. ArtinM induces neutrophils activation, which is known to account for both resistance to pathogens and host tissue injury. Although exacerbated inflammation was not observed in ArtinM-treated animals, assessment of neutrophil responses to ArtinM is required to envisage its possible application to design a novel immunomodulatory agent based on carbohydrate recognition. Herein, we focus on the mechanisms through which neutrophils contribute to ArtinM-induced protection against Leishmania, without exacerbating inflammation. For this purpose, human neutrophils treated with ArtinM and infected with Leishmania major were analyzed together with untreated and uninfected controls, based on their ability to eliminate the parasite, release cytokines, degranulate, produce reactive oxygen species (ROS), form neutrophil extracellular traps (NETs) and change life span. We demonstrate that ArtinM-stimulated neutrophils enhanced L. major clearance and at least duplicated tumor necrosis factor (TNF) and interleukin-1beta (IL-1β) release; otherwise, transforming growth factor-beta (TGF-β) production was reduced by half. Furthermore, ROS production and cell degranulation were augmented. The life span of ArtinM-stimulated neutrophils decreased and they did not form NETs when infected with L. major. We postulate that the enhanced leishmanicidal ability of ArtinM-stimulated neutrophils is due to augmented release of inflammatory cytokines, ROS production, and cell degranulation, whereas host tissue integrity is favored by their shortened life span and the absence of NET formation. Our results reinforce the idea that ArtinM may be considered an

  1. Neutrophils Contribute to the Protection Conferred by ArtinM against Intracellular Pathogens: A Study on Leishmania major.

    PubMed

    Ricci-Azevedo, Rafael; Oliveira, Aline Ferreira; Conrado, Marina C A V; Carvalho, Fernanda Caroline; Roque-Barreira, Maria Cristina

    2016-04-01

    ArtinM, a D-mannose binding lectin from Artocarpus heterophyllus, has immunomodulatory activities through its interaction with N-glycans of immune cells, culminating with the establishment of T helper type 1 (Th1) immunity. This interaction protects mice against intracellular pathogens, including Leishmania major and Leishmania amazonensis. ArtinM induces neutrophils activation, which is known to account for both resistance to pathogens and host tissue injury. Although exacerbated inflammation was not observed in ArtinM-treated animals, assessment of neutrophil responses to ArtinM is required to envisage its possible application to design a novel immunomodulatory agent based on carbohydrate recognition. Herein, we focus on the mechanisms through which neutrophils contribute to ArtinM-induced protection against Leishmania, without exacerbating inflammation. For this purpose, human neutrophils treated with ArtinM and infected with Leishmania major were analyzed together with untreated and uninfected controls, based on their ability to eliminate the parasite, release cytokines, degranulate, produce reactive oxygen species (ROS), form neutrophil extracellular traps (NETs) and change life span. We demonstrate that ArtinM-stimulated neutrophils enhanced L. major clearance and at least duplicated tumor necrosis factor (TNF) and interleukin-1beta (IL-1β) release; otherwise, transforming growth factor-beta (TGF-β) production was reduced by half. Furthermore, ROS production and cell degranulation were augmented. The life span of ArtinM-stimulated neutrophils decreased and they did not form NETs when infected with L. major. We postulate that the enhanced leishmanicidal ability of ArtinM-stimulated neutrophils is due to augmented release of inflammatory cytokines, ROS production, and cell degranulation, whereas host tissue integrity is favored by their shortened life span and the absence of NET formation. Our results reinforce the idea that ArtinM may be considered an

  2. Study of Genetic Variation of Leishmania major Based on Internal Transcribed Spacer 1 (ITS1) in Chabahar, Iran.

    PubMed

    Dabirzadeh, Mansour; Hashemi, Mohammad; Maroufi, Yahya

    2016-06-01

    Zoonotic cutaneous leishmaniasis (ZCL) is polymorphic disease that may show various clinical manifestations. This study investigates the determination of genetic variation within the species of Leishmania major isolates from new cases in Chabahar, a port city in Southeast Iran (situated at the Iran-Pakistan border). Migration in this region indicates that leishmaniasis is spreading gradually, and a new micro-habitat focus appears each year. A variety of nucleic acid detection methods that target both DNA and RNA have been developed. The restriction fragment length polymorphism analysis of amplified internal transcribed spacer 1 with polymerase chain reaction (ITS1-RFLP PCR) assay is a multipurpose tool for the diagnosis of Leishmania from clinical samples and for enabling the determination of the infecting Leishmania species. The goal of this study was the identification of species based on ITS1-RFLP in the ribosomal operon of L. major from clinically different forms of ZCL amplified by PCR, followed by the digestion of the PCR product with restriction enzymes. The profiles were observed and visualized in agarose gel under UV light. We used direct smears to identify the parasites. While taking the smear, samples were collected for culture or direct PCR. We used the PCR-RFLP assay of the ITS1 genes for direct identification of Leishmania species in 24 out of 33 suspected patients. PCR-ITS1 amplification was done on the 24 samples confirmed by culture via growth and parasitological methods. Of the 24 isolates, 21 had 350 bp bands (87.5%) and three had 450 bp bands (12.5%). After using the restriction enzyme, banding patterns including fragments of 210 and 140 bp for L. major were detected in 19 cases. The L. major species causing ZCL in Chabahar have limited genetic variation. There seems to be little manifestation of diversity between these lesions as a new focus of disease, and new micro-habitats for the disease are appearing in parts of this region.

  3. An in vitro model for infection with Leishmania major that mimics the immune response in mice.

    PubMed Central

    Soares, M B; David, J R; Titus, R G

    1997-01-01

    By using a primary in vitro response specific for Leishmania major, normal T cells from resistant CBA/CaH-T6J and susceptible BALB/c mice commit to a Th1 and a Th2 response, respectively. Since commitment occurred, we measured the production of gamma interferon (IFN-gamma), interleukin-1 (IL-1), IL-2, IL-4, IL-5, IL-10, and IL-12, prostaglandin E2 (PGE2), transforming growth factor beta (TGF-beta), and nitric oxide in the first 7 days of the response to identify factors that are critical for Th1 and Th2 development. While cells from resistant CBA mice produced more IFN-gamma, IL-10, and nitric oxide, cells from susceptible BALB/c mice produced more IL-1alpha, IL-5, PGE2, and TGF-beta. Although substantial amounts of IL-12 were detected, IL-12 did not associate with either Th1 or Th2 development. We did not anticipate that cells from resistant CBA mice would make more IL-10 in vitro. However, this also occurred in vivo since CBA mice produced substantial amounts of IL-10 following infection with L. major. Moreover, adding anti-IL-10 to primary in vitro responses enhanced production of IFN-gamma and nitric oxide by cells from CBA and BALB/c mice. Therefore, IL-10 cannot be regarded as a cytokine that associates with susceptibility to infection with L. major. Finally, the data presented here suggest that a collection of factors that can be produced by accessory cells influence Th commitment (e.g., IL-1, PGE2, and TGF-beta favor Th2 development). PMID:9199457

  4. MHC class II restricted innate-like double negative T cells contribute to optimal primary and secondary immunity to Leishmania major.

    PubMed

    Mou, Zhirong; Liu, Dong; Okwor, Ifeoma; Jia, Ping; Orihara, Kanami; Uzonna, Jude Ezeh

    2014-09-01

    Although it is generally believed that CD4(+) T cells play important roles in anti-Leishmania immunity, some studies suggest that they may be dispensable, and that MHC II-restricted CD3(+)CD4(-)CD8(-) (double negative, DN) T cells may be more important in regulating primary anti-Leishmania immunity. In addition, while there are reports of increased numbers of DN T cells in Leishmania-infected patients, dogs and mice, concrete evidence implicating these cells in secondary anti-Leishmania immunity has not yet been documented. Here, we report that DN T cells extensively proliferate and produce effector cytokines (IFN-γ, TNF and IL-17) and granzyme B (GrzB) in the draining lymph nodes and spleens of mice following primary and secondary L. major infections. DN T cells from healed mice display functional characteristics of protective anti-Leishmania memory-like cells: rapid and extensive proliferation and effector cytokines production following L. major challenge in vitro and in vivo. DN T cells express predominantly (> 95%) alpha-beta T cell receptor (αβ TCR), are Leishmania-specific, restricted mostly by MHC class II molecules and display transcriptional profile of innate-like genes. Using in vivo depletion and adoptive transfer studies, we show that DN T cells contribute to optimal primary and secondary anti-Leishmania immunity in mice. These results directly identify DN T cells as important players in effective and protective primary and secondary anti-L. major immunity in experimental cutaneous leishmaniasis.

  5. Experimental therapeutic studies of Solanum aculeastrum Dunal. on Leishmania major infection in BALB/c mice

    PubMed Central

    Laban, Linet T; Anjili, Christopher O; Mutiso, Joshua M; Ingonga, Johnstone; Kiige, Samuel G; Ngedzo, Mgala M; Gicheru, Michael M

    2015-01-01

    Objective(s): Solanum acueastrum Dunal. has been shown to have some chemotherapeutic value. Leaf and berry water and methanol compounds of S. acueastrum were evaluated for possible antileishmanial activity In vivo on BALB/c mice and in vitro against Leishmania major promastigotes, amastigotes and vero cells. Materials and Methods: Dry S. aculeastrum berry and leaf material were extracted in methanol and water. L. major parasites were exposed to different concentrations of S. aculeastrum fruit and leaf compounds and the IC50 on the promastigotes, percentage of infection rate of macrophages by amastigotes and the toxicological effect on vero cells were determined. BALB/c mice were infected subcutaneously with 1×106 promastigotes and kept for four weeks to allow for disease establishment. Infected mice were treated with fruit and leaf methanolic and water compounds, amphotericin B (AmB), and sterile phosphate buffered saline (PBS). Results: Fruit methanol compound was most effective in inhibiting the growth of promastigotes with IC5078.62 μg/ml. Fruit water compound showed the best activity in inhibiting infection of macrophages by amastigotes. Fruit methanol compound was more toxic at Ld50=8.06 mg/ml to vero cells than amphotericin B. Analysis of variance computation indicated statistically significant difference in lesion sizes between experimental and control mice groups (P=0.0001). Splenic impression smears ANOVA indicated a highly significant difference in parasitic numbers between the experimental and the control groups (P=0.0001). Conclusion: The results demonstrate that compounds from S. aculeastrum have potential anti-leishmanial activities and the medicinal use of the plant poses considerable toxicity against dividing vero cells. PMID:25810878

  6. Leishmania major telomerase TERT protein has a nuclear/mitochondrial eclipsed distribution that is affected by oxidative stress.

    PubMed

    Campelo, Riward; Díaz Lozano, Isabel; Figarella, Katherine; Osuna, Antonio; Ramírez, José Luis

    2015-01-01

    In its canonical role the reverse transcriptase telomerase recovers the telomeric repeats that are lost during DNA replication. Other locations and activities have been recently described for the telomerase protein subunit TERT in mammalian cells. In the present work, using biochemistry, molecular biology, and electron microscopy techniques, we found that in the human parasite Leishmania major, TERT (and telomerase activity) shared locations between the nuclear, mitochondrial, and cytoplasmic compartments. Also, some telomerase activity and TERT protein could be found in ∼ 100-nm nanovesicles. In the mitochondrial compartment, TERT appears to be mainly associated with the kinetoplast DNA. When Leishmania cells were exposed to H2O2, TERT changed its relative abundance and activity between the nuclear and mitochondrial compartments, with the majority of activity residing in the mitochondrion. Finally, overexpression of TERT in Leishmania transfected cells not only increased the parasitic cell growth rate but also increased their resistance to oxidative stress. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  7. Sheep blood-LB agar base medium (SLM) as a simple and suitable medium for the cultivation of Leishmania major promastigotes.

    PubMed

    Nasiri, Vahid

    2013-11-01

    Several methods of cultivation of Leishmania parasites are available, and among them, biphasic media like Nicolle-Novy-MacNeal medium are considered to be superior for the initial isolation of the parasites and their in vivo infectivity. In the present work, the efficacy of sheep blood-LB agar base medium (SLM) was evaluated for the cultivation of Leishmania major promastigotes. The SLM supports the proliferation of the parasites and can be used for routine isolation and cultivation of Leishmania parasites with acceptable in vivo infectivity for research purposes.

  8. Cinnamic Acid Bornyl Ester Derivatives from Valeriana wallichii Exhibit Antileishmanial In Vivo Activity in Leishmania major-Infected BALB/c Mice

    PubMed Central

    Hazra, Sudipta; Glaser, Jan; Holzgrabe, Ulrike; Hazra, Banasri; Schurigt, Uta

    2015-01-01

    Human leishmaniasis covers a broad spectrum of clinical manifestations ranging from self-healing cutaneous leishmaniasis to severe and lethal visceral leishmaniasis caused among other species by Leishmania major or Leishmania donovani, respectively. Some drug candidates are in clinical trials to substitute current therapies, which are facing emerging drug-resistance accompanied with serious side effects. Here, two cinnamic acid bornyl ester derivatives (1 and 2) were assessed for their antileishmanial activity. Good selectivity and antileishmanial activity of bornyl 3-phenylpropanoate (2) in vitro prompted the antileishmanial assessment in vivo. For this purpose, BALB/c mice were infected with Leishmania major promastigotes and treated with three doses of 50 mg/kg/day of compound 2. The treatment prevented the characteristic swelling at the site of infection and correlated with reduced parasite burden. Transmitted light microscopy and transmission electron microscopy of Leishmania major promastigotes revealed that compounds 1 and 2 induce mitochondrial swelling. Subsequent studies on Leishmania major promastigotes showed the loss of mitochondrial transmembrane potential (ΔΨm) as a putative mode of action. As the cinnamic acid bornyl ester derivatives 1 and 2 had exhibited antileishmanial activity in vitro, and compound 2 in Leishmania major-infected BALB/c mice in vivo, they can be regarded as possible lead structures for the development of new antileishmanial therapeutic approaches. PMID:26554591

  9. A New ABC Half-Transporter in Leishmania major Is Involved in Resistance to Antimony

    PubMed Central

    Manzano, J. I.; García-Hernández, R.; Castanys, S.

    2013-01-01

    The characterization of ABCI4, a new intracellular ATP-binding cassette (ABC) half-transporter in Leishmania major, is described. We show that ABCI4 is involved in heavy metal export, thereby conferring resistance to Pentostam, to Sb(III), and to As(III) and Cd(II). Parasites overexpressing ABCI4 showed a lower mitochondrial toxic effect of antimony by decreasing reactive oxygen species production and maintained higher values of both the mitochondrial electrochemical potential and total ATP levels with respect to controls. The ABCI4 half-transporter forms homodimers as determined by a coimmunoprecipitation assay. A combination of subcellular localization studies under a confocal microscope and a surface biotinylation assay using parasites expressing green fluorescent protein- and FLAG-tagged ABCI4 suggests that the transporter presents a dual localization in both mitochondria and the plasma membrane. Parasites overexpressing ABCI4 present an increased replication in mouse peritoneal macrophages. We have determined that porphyrins are substrates for ABCI4. Consequently, the overexpression of ABCI4 confers resistance to some toxic porphyrins, such as zinc-protoporphyrin, due to the lower accumulation resulting from a significant efflux, as determined using the fluorescent zinc-mesoporphyrin, a validated heme analog. In addition, ABCI4 has a significant ability to efflux thiol after Sb(III) incubation, thus meaning that ABCI4 could be considered to be a potential thiol-X-pump that is able to recognize metal-conjugated thiols. In summary, we have shown that this new ABC transporter is involved in drug sensitivity to antimony and other compounds by efflux as conjugated thiol complexes. PMID:23716044

  10. A new ABC half-transporter in Leishmania major is involved in resistance to antimony.

    PubMed

    Manzano, J I; García-Hernández, R; Castanys, S; Gamarro, F

    2013-08-01

    The characterization of ABCI4, a new intracellular ATP-binding cassette (ABC) half-transporter in Leishmania major, is described. We show that ABCI4 is involved in heavy metal export, thereby conferring resistance to Pentostam, to Sb(III), and to As(III) and Cd(II). Parasites overexpressing ABCI4 showed a lower mitochondrial toxic effect of antimony by decreasing reactive oxygen species production and maintained higher values of both the mitochondrial electrochemical potential and total ATP levels with respect to controls. The ABCI4 half-transporter forms homodimers as determined by a coimmunoprecipitation assay. A combination of subcellular localization studies under a confocal microscope and a surface biotinylation assay using parasites expressing green fluorescent protein- and FLAG-tagged ABCI4 suggests that the transporter presents a dual localization in both mitochondria and the plasma membrane. Parasites overexpressing ABCI4 present an increased replication in mouse peritoneal macrophages. We have determined that porphyrins are substrates for ABCI4. Consequently, the overexpression of ABCI4 confers resistance to some toxic porphyrins, such as zinc-protoporphyrin, due to the lower accumulation resulting from a significant efflux, as determined using the fluorescent zinc-mesoporphyrin, a validated heme analog. In addition, ABCI4 has a significant ability to efflux thiol after Sb(III) incubation, thus meaning that ABCI4 could be considered to be a potential thiol-X-pump that is able to recognize metal-conjugated thiols. In summary, we have shown that this new ABC transporter is involved in drug sensitivity to antimony and other compounds by efflux as conjugated thiol complexes.

  11. Diverse modes of binding in structures of Leishmania major N-myristoyltransferase with selective inhibitors

    PubMed Central

    Brannigan, James A.; Roberts, Shirley M.; Bell, Andrew S.; Hutton, Jennie A.; Hodgkinson, Michael R.; Tate, Edward W.; Leatherbarrow, Robin J.; Smith, Deborah F.; Wilkinson, Anthony J.

    2014-01-01

    The leishmaniases are a spectrum of global diseases of poverty associated with immune dysfunction and are the cause of high morbidity. Despite the long history of these diseases, no effective vaccine is available and the currently used drugs are variously compromised by moderate efficacy, complex side effects and the emergence of resistance. It is therefore widely accepted that new therapies are needed. N-Myristoyltransferase (NMT) has been validated pre-clinically as a target for the treatment of fungal and parasitic infections. In a previously reported high-throughput screening program, a number of hit compounds with activity against NMT from Leishmania donovani have been identified. Here, high-resolution crystal structures of representative compounds from four hit series in ternary complexes with myristoyl-CoA and NMT from the closely related L. major are reported. The structures reveal that the inhibitors associate with the peptide-binding groove at a site adjacent to the bound myristoyl-CoA and the catalytic α-carboxylate of Leu421. Each inhibitor makes extensive apolar contacts as well as a small number of polar contacts with the protein. Remarkably, the compounds exploit different features of the peptide-binding groove and collectively occupy a substantial volume of this pocket, suggesting that there is potential for the design of chimaeric inhibitors with significantly enhanced binding. Despite the high conservation of the active sites of the parasite and human NMTs, the inhibitors act selectively over the host enzyme. The role of conformational flexibility in the side chain of Tyr217 in conferring selectivity is discussed. PMID:25075346

  12. Site-Dependent Recruitment of Inflammatory Cells Determines the Effective Dose of Leishmania major

    PubMed Central

    Ribeiro-Gomes, Flavia L.; Roma, Eric Henrique; Carneiro, Matheus B. H.; Doria, Nicole A.; Sacks, David L.

    2014-01-01

    The route of pathogen inoculation by needle has been shown to influence the outcome of infection. Employing needle inoculation of the obligately intracellular parasite Leishmania major, which is transmitted in nature following intradermal (i.d.) deposition of parasites by the bite of an infected sand fly, we identified differences in the preexisting and acute cellular responses in mice following i.d. inoculation of the ear, subcutaneous (s.c.) inoculation of the footpad, or inoculation of the peritoneal cavity (intraperitoneal [i.p.] inoculation). Initiation of infection at different sites was associated with different phagocytic populations. Neutrophils were the dominant infected cells following i.d., but not s.c. or i.p., inoculation. Inoculation of the ear dermis resulted in higher frequencies of total and infected neutrophils than inoculation of the footpad, and these higher frequencies were associated with a 10-fold increase in early parasite loads. Following inoculation of the ear in the absence of neutrophils, parasite phagocytosis by other cell types did not increase, and fewer parasites were able to establish infection. The frequency of infected neutrophils within the total infected CD11b+ population was higher than the frequency of total neutrophils within the total CD11b+ population, demonstrating that neutrophils are overrepresented as a proportion of infected cells. Employing i.d. inoculation to model sand fly transmission of parasites has significant consequences for infection outcome relative to that of s.c. or i.p. inoculation, including the phenotype of infected cells and the number of parasites that establish infection. Vector-borne infections initiated in the dermis likely involve adaptations to this unique microenvironment. Bypassing or altering this initial step has significant consequences for infection. PMID:24733090

  13. The Crystal Structure of the Leishmania major Deoxyuridine Triphosphate Nucleotidohydrolase in Complex with Nucleotide Analogues, dUMP, and Deoxyuridine*

    PubMed Central

    Hemsworth, Glyn R.; Moroz, Olga V.; Fogg, Mark J.; Scott, Benjamin; Bosch-Navarrete, Cristina; González-Pacanowska, Dolores; Wilson, Keith S.

    2011-01-01

    Members of the Leishmania genus are the causative agents of the life-threatening disease leishmaniasis. New drugs are being sought due to increasing resistance and adverse side effects with current treatments. The knowledge that dUTPase is an essential enzyme and that the all α-helical dimeric kinetoplastid dUTPases have completely different structures compared with the trimeric β-sheet type dUTPase possessed by most organisms, including humans, make the dimeric enzymes attractive drug targets. Here, we present crystal structures of the Leishmania major dUTPase in complex with substrate analogues, the product dUMP and a substrate fragment, and of the homologous Campylobacter jejuni dUTPase in complex with a triphosphate substrate analogue. The metal-binding properties of both enzymes are shown to be dependent upon the ligand identity, a previously unseen characteristic of this family. Furthermore, structures of the Leishmania enzyme in the presence of dUMP and deoxyuridine coupled with tryptophan fluorescence quenching indicate that occupation of the phosphate binding region is essential for induction of the closed conformation and hence for substrate binding. These findings will aid in the development of dUTPase inhibitors as potential new lead anti-trypanosomal compounds. PMID:21454646

  14. PTR1-dependent synthesis of tetrahydrobiopterin contributes to oxidant susceptibility in the trypanosomatid protozoan parasite Leishmania major

    PubMed Central

    Nare, Bakela; Garraway, Levi A.; Vickers, Tim J.; Beverley, Stephen M.

    2009-01-01

    Leishmania must survive oxidative stress, but lack many classical antioxidant enzymes and rely heavily on trypanothione-dependent pathways. We used forward genetic screens to recover loci mediating oxidant resistance via overexpression in Leishmania major, which identified pteridine reductase 1 (PTR1). Comparisons of isogenic lines showed ptr1- null mutants were 18-fold more sensitive to H2O2 than PTR1-overproducing lines, and significant 3-5 fold differences were seen with a broad panel of oxidant-inducing agents. The toxicities of simple nitric oxide generators and other drug classes (except antifolates) were unaffected by PTR1 levels. H2O2 susceptibility could be modulated by exogenous biopterin but not folate, in a PTR1-but not dihydrofolate reductase-dependent manner, implicating H4B metabolism specifically. Neither H2O2 consumption, nor the level of intracellular oxidative stress, was affected by PTR1 levels. Coupled with the fact that reduced pteridines are at least 100-fold less abundant than cellular thiols), these data argue strongly that reduced pteridines act through a mechanism other than scavenging. The ability of unconjugated pteridines to counter oxidative stress has implications to infectivity and response to chemotherapy. Since the intracellular pteridine levels of Leishmania can be readily manipulated, these organisms offer a powerful setting for the dissection of pteridine-dependent oxidant susceptibility in higher eukaryotes. PMID:19396443

  15. Anti Leishmanial Effect of Zinc Sulphate on the Viability of Leishmania tropica and L. major Promastigotes

    PubMed Central

    Fattahi Bafghi, Ali; Noorbala, Mohammad; Noorbala, Mohammad Taghi; Aghabagheri, Mahdi

    2014-01-01

    Background: Cutaneous leishmaniasis (CL) is an endemic disease in developing countries. Although pentamidine orantimonite (Glucantime) has been recommended for cutaneous leishmaniasis treatment by the World Health Organization, there are some concerns too such as high cost, side effects, need for frequent injections, and restricted efficacy. Therefore, different methods have been used for CL treatment so far. Objectives: This study assessed the sensitivity of two parasite agents of cutaneous leishmaniasis: Leishmania major and L. tropica to zinc sulphate in vitro. In the present study, the zinc sulphate effect on urban and rural strains of cutaneous leishmaniasis, viability of old world, in vitro is under investigation. Materials and Methods: The design of the present study was experimental (laboratory-trial) based. Iranian endemic species of L. major and L. tropica were appropriately collected, proliferated, and maintained in the standard culture. Afterward, the proper concentrations of zinc sulphate were provided, sterilized, and added to the cultures containing parasites. In different intervals, parasites were counted by two methods: the slide and cell proliferation ELISA. Results: Both parasite species showed sensitivity to zinc sulphate in vitro and in comparison with the control group, their numbers were reduced. Zinc sulphate (in concentrations of 0.5, 1, 2, and 3 percent) was added to the cultures containing parasites, and the total number of the live parasites was counted through the slide method (Neubauer slide) every day up to the fifth day. The results were analyzed and found statistically significant (P < 0.05). In the second phase, the counting process was repeated with the addition of zinc sulphate compound with different concentrations (3, 4, 5, and 6 percent) and live parasite numbers were counted by ELISA method after 24 hours. The findings revealed that all the cultures containing zinc sulphate showed a slower growth in comparison to the control

  16. Anti Leishmanial Effect of Zinc Sulphate on the Viability of Leishmania tropica and L. major Promastigotes.

    PubMed

    Fattahi Bafghi, Ali; Noorbala, Mohammad; Noorbala, Mohammad Taghi; Aghabagheri, Mahdi

    2014-09-01

    Cutaneous leishmaniasis (CL) is an endemic disease in developing countries. Although pentamidine orantimonite (Glucantime) has been recommended for cutaneous leishmaniasis treatment by the World Health Organization, there are some concerns too such as high cost, side effects, need for frequent injections, and restricted efficacy. Therefore, different methods have been used for CL treatment so far. This study assessed the sensitivity of two parasite agents of cutaneous leishmaniasis: Leishmania major and L. tropica to zinc sulphate in vitro. In the present study, the zinc sulphate effect on urban and rural strains of cutaneous leishmaniasis, viability of old world, in vitro is under investigation. The design of the present study was experimental (laboratory-trial) based. Iranian endemic species of L. major and L. tropica were appropriately collected, proliferated, and maintained in the standard culture. Afterward, the proper concentrations of zinc sulphate were provided, sterilized, and added to the cultures containing parasites. In different intervals, parasites were counted by two methods: the slide and cell proliferation ELISA. Both parasite species showed sensitivity to zinc sulphate in vitro and in comparison with the control group, their numbers were reduced. Zinc sulphate (in concentrations of 0.5, 1, 2, and 3 percent) was added to the cultures containing parasites, and the total number of the live parasites was counted through the slide method (Neubauer slide) every day up to the fifth day. The results were analyzed and found statistically significant (P < 0.05). In the second phase, the counting process was repeated with the addition of zinc sulphate compound with different concentrations (3, 4, 5, and 6 percent) and live parasite numbers were counted by ELISA method after 24 hours. The findings revealed that all the cultures containing zinc sulphate showed a slower growth in comparison to the control group. The mentioned difference was statistically

  17. Differential Impact of LPG-and PG-Deficient Leishmania major Mutants on the Immune Response of Human Dendritic Cells

    PubMed Central

    Jayakumar, Asha; Hickerson, Suzanne; Mostrom, Janet; Turco, Salvatore J.; Beverley, Stephen M.; McDowell, Mary Ann

    2015-01-01

    Background Leishmania major infection induces robust interleukin-12 (IL12) production in human dendritic cells (hDC), ultimately resulting in Th1-mediated immunity and clinical resolution. The surface of Leishmania parasites is covered in a dense glycocalyx consisting of primarily lipophosphoglycan (LPG) and other phosphoglycan-containing molecules (PGs), making these glycoconjugates the likely pathogen-associated molecular patterns (PAMPS) responsible for IL12 induction. Methodology/Principal Findings Here we explored the role of parasite glycoconjugates on the hDC IL12 response by generating L. major Friedlin V1 mutants defective in LPG alone, (FV1 lpg1-), or generally deficient for all PGs, (FV1 lpg2-). Infection with metacyclic, infective stage, L. major or purified LPG induced high levels of IL12B subunit gene transcripts in hDCs, which was abrogated with FV1 lpg1- infections. In contrast, hDC infections with FV1 lpg2- displayed increased IL12B expression, suggesting other PG-related/LPG2 dependent molecules may act to dampen the immune response. Global transcriptional profiling comparing WT, FV1 lpg1-, FV1 lpg2- infections revealed that FV1 lpg1- mutants entered hDCs in a silent fashion as indicated by repression of gene expression. Transcription factor binding site analysis suggests that LPG recognition by hDCs induces IL-12 in a signaling cascade resulting in Nuclear Factor κ B (NFκB) and Interferon Regulatory Factor (IRF) mediated transcription. Conclusions/Significance These data suggest that L. major LPG is a major PAMP recognized by hDC to induce IL12-mediated protective immunity and that there is a complex interplay between PG-baring Leishmania surface glycoconjugates that result in modulation of host cellular IL12. PMID:26630499

  18. A Newly Emerged Cutaneous Leishmaniasis Focus in Northern Israel and Two New Reservoir Hosts of Leishmania major

    PubMed Central

    Faiman, Roy; Abbasi, Ibrahim; Jaffe, Charles; Motro, Yoav; Nasereddin, Abdelmagid; Schnur, Lionel F.; Torem, Moshe; Pratlong, Francine; Dedet, Jean-Pierre; Warburg, Alon

    2013-01-01

    In 2006/7, 18 cases of cutaneous leishmaniasis (CL) were reported for the first time from Sde Eliyahu (pop. 650), a village in the Beit She'an valley of Israel. Between 2007–2011, a further 88 CL cases were diagnosed bringing the total to 106 (16.3% of the population of Sde Eliyahu). The majority of cases resided in the south-western part of the village along the perimeter fence. The causative parasite was identified as Leishmania major Yakimoff & Schokhor, 1914 (Kinetoplastida: Trypanosomatidae). Phlebotomus papatasi (Scopoli), 1786 (Diptera: Psychodidae) was found to be the most abundant phlebotomine species comprising 97% of the sand flies trapped inside the village, and an average of 7.9% of the females were positive for Leishmania ITS1 DNA. Parasite isolates from CL cases and a sand fly were characterized using several methods and shown to be L. major. During a comprehensive survey of rodents 164 Levant voles Microtus guentheri Danford & Alston, 1880 (Rodentia: Cricetidae) were captured in alfalfa fields bordering the village. Of these 27 (16.5%) tested positive for Leishmania ITS1 DNA and shown to be L. major by reverse line blotting. A very high percentage (58.3% - 21/36) of Tristram's jirds Meriones tristrami Thomas, 1892 (Rodentia: Muridae), found further away from the village also tested positive for ITS1 by PCR. Isolates of L. major were successfully cultured from the ear of a wild jird found positive by ITS1 PCR. Although none of the wild PCR-positive voles exhibited external pathology, laboratory-reared voles that were infected by intradermal L. major inoculation, developed patent lesions and sand flies became infected by feeding on the ears of these laboratory-infected voles. This is the first report implicating M. guentheri and M. tristrami as reservoirs of Leishmania. The widespread co-distribution of M. guentheri and P. papatasi, suggests a significant threat from the spread of CL caused by L. major in the Middle East, central Asia and southern

  19. Comparative analysis of the kinomes of three pathogenic trypanosomatids: Leishmania major, Trypanosoma brucei and Trypanosoma cruzi

    PubMed Central

    Parsons, Marilyn; Worthey, Elizabeth A; Ward, Pauline N; Mottram, Jeremy C

    2005-01-01

    Background The trypanosomatids Leishmania major, Trypanosoma brucei and Trypanosoma cruzi cause some of the most debilitating diseases of humankind: cutaneous leishmaniasis, African sleeping sickness, and Chagas disease. These protozoa possess complex life cycles that involve development in mammalian and insect hosts, and a tightly coordinated cell cycle ensures propagation of the highly polarized cells. However, the ways in which the parasites respond to their environment and coordinate intracellular processes are poorly understood. As a part of an effort to understand parasite signaling functions, we report the results of a genome-wide analysis of protein kinases (PKs) of these three trypanosomatids. Results Bioinformatic searches of the trypanosomatid genomes for eukaryotic PKs (ePKs) and atypical PKs (aPKs) revealed a total of 176 PKs in T. brucei, 190 in T. cruzi and 199 in L. major, most of which are orthologous across the three species. This is approximately 30% of the number in the human host and double that of the malaria parasite, Plasmodium falciparum. The representation of various groups of ePKs differs significantly as compared to humans: trypanosomatids lack receptor-linked tyrosine and tyrosine kinase-like kinases, although they do possess dual-specificity kinases. A relative expansion of the CMGC, STE and NEK groups has occurred. A large number of unique ePKs show no strong affinity to any known group. The trypanosomatids possess few ePKs with predicted transmembrane domains, suggesting that receptor ePKs are rare. Accessory Pfam domains, which are frequently present in human ePKs, are uncommon in trypanosomatid ePKs. Conclusion Trypanosomatids possess a large set of PKs, comprising approximately 2% of each genome, suggesting a key role for phosphorylation in parasite biology. Whilst it was possible to place most of the trypanosomatid ePKs into the seven established groups using bioinformatic analyses, it has not been possible to ascribe function

  20. Identification of Potent Chemotypes Targeting Leishmania major Using a High-Throughput, Low-Stringency, Computationally Enhanced, Small Molecule Screen

    PubMed Central

    Sharlow, Elizabeth R.; Close, David; Shun, Tongying; Leimgruber, Stephanie; Reed, Robyn; Mustata, Gabriela; Wipf, Peter; Johnson, Jacob; O'Neil, Michael; Grögl, Max; Magill, Alan J.; Lazo, John S.

    2009-01-01

    Patients with clinical manifestations of leishmaniasis, including cutaneous leishmaniasis, have limited treatment options, and existing therapies frequently have significant untoward liabilities. Rapid expansion in the diversity of available cutaneous leishmanicidal chemotypes is the initial step in finding alternative efficacious treatments. To this end, we combined a low-stringency Leishmania major promastigote growth inhibition assay with a structural computational filtering algorithm. After a rigorous assay validation process, we interrogated ∼200,000 unique compounds for L. major promastigote growth inhibition. Using iterative computational filtering of the compounds exhibiting >50% inhibition, we identified 553 structural clusters and 640 compound singletons. Secondary confirmation assays yielded 93 compounds with EC50s ≤ 1 µM, with none of the identified chemotypes being structurally similar to known leishmanicidals and most having favorable in silico predicted bioavailability characteristics. The leishmanicidal activity of a representative subset of 15 chemotypes was confirmed in two independent assay formats, and L. major parasite specificity was demonstrated by assaying against a panel of human cell lines. Thirteen chemotypes inhibited the growth of a L. major axenic amastigote-like population. Murine in vivo efficacy studies using one of the new chemotypes document inhibition of footpad lesion development. These results authenticate that low stringency, large-scale compound screening combined with computational structure filtering can rapidly expand the chemotypes targeting in vitro and in vivo Leishmania growth and viability. PMID:19888337

  1. Enzymatic Mechanism of Leishmania major Peroxidase and the Critical Role of Specific Ionic Interactions

    PubMed Central

    Chreifi, Georges; Hollingsworth, Scott A.; Li, Huiying; Tripathi, Sarvind; Arce, Anton P.; Magaña-Garcia, Hugo I.; Poulos, Thomas L.

    2015-01-01

    Leishmania major peroxidase (LmP) is very similar to the well-known yeast cytochrome c peroxidase (CcP). Both enzymes catalyze the peroxidation of cytochrome c. Like CcP, LmP reacts with H2O2 to form Compound I, which consists of a ferryl heme and a Trp radical, FeIV= O;Trp•+. Cytochrome c (Cytc) reduces the Trp radical to give Compound II, FeIV= O;Trp, which is followed by an intramolecular electron transfer to give FeIII–OH;Trp•+, and in the last step, Cytc reduces the Trp radical. In this study, we have used steady-state and single-turnover kinetics to improve our understanding of the overall mechanism of LmP catalysis. While the activity of CcP greatly increases with ionic strength, the kcat for LmP remains relatively constant at all ionic strengths tested. Therefore, unlike CcP, where dissociation of oxidized Cytc is limiting at low ionic strengths, association/dissociation reactions are not limiting at any ionic strength in LmP. We conclude that in LmP, the intramolecular electron transfer reaction, FeIV= O;Trp to FeIII–OH;Trp•+, is limiting at all ionic strengths. Unlike CcP, LmP depends on key intermolecular ion pairs to form the electron transfer competent complex. Mutating these sites causes the initial rate of association to decrease by 2 orders of magnitude and a substantial decrease in kcat. The drop in kcat is due to a switch in the rate-limiting step of the mutants from intramolecular electron transfer to the rate of association in forming the LmP–LmCytc complex. These studies show that while LmP and CcP form very similar complexes and exhibit similar activities, they substantially differ in how their activity changes as a function of ionic strength. This difference is primarily due to the heavy reliance of LmP on highly specific intermolecular ion pairs, while CcP relies mainly on nonpolar interactions. PMID:25941976

  2. Myeloid-derived suppressor cells help protective immunity to Leishmania major infection despite suppressed T cell responses.

    PubMed

    Pereira, Wânia F; Ribeiro-Gomes, Flávia L; Guillermo, Landi V Costilla; Vellozo, Natália S; Montalvão, Fabrício; Dosreis, George A; Lopes, Marcela F

    2011-12-01

    Th1/Th2 cytokines play a key role in immune responses to Leishmania major by controlling macrophage activation for NO production and parasite killing. MDSCs, including myeloid precursors and immature monocytes, produce NO and suppress T cell responses in tumor immunity. We hypothesized that NO-producing MDSCs could help immunity to L. major infection. Gr1(hi)(Ly6C(hi)) CD11b(hi) MDSCs elicited by L. major infection suppressed polyclonal and antigen-specific T cell proliferation. Moreover, L. major-induced MDSCs killed intracellular parasites in a NO-dependent manner and reduced parasite burden in vivo. By contrast, treatment with ATRA, which induces MDSCs to differentiate into macrophages, increased development of lesions, parasite load, and T cell proliferation in draining LNs. Altogether, these results indicate that NO-producing MDSCs help protective immunity to L. major infection, despite suppressed T cell proliferation.

  3. Study of Genetic Variation of Leishmania major Based on Internal Transcribed Spacer 1 (ITS1) in Chabahar, Iran

    PubMed Central

    Dabirzadeh, Mansour; Hashemi, Mohammad; Maroufi, Yahya

    2016-01-01

    Background Zoonotic cutaneous leishmaniasis (ZCL) is polymorphic disease that may show various clinical manifestations. Objectives This study investigates the determination of genetic variation within the species of Leishmania major isolates from new cases in Chabahar, a port city in Southeast Iran (situated at the Iran-Pakistan border). Migration in this region indicates that leishmaniasis is spreading gradually, and a new micro-habitat focus appears each year. Materials and Methods A variety of nucleic acid detection methods that target both DNA and RNA have been developed. The restriction fragment length polymorphism analysis of amplified internal transcribed spacer 1 with polymerase chain reaction (ITS1-RFLP PCR) assay is a multipurpose tool for the diagnosis of Leishmania from clinical samples and for enabling the determination of the infecting Leishmania species. The goal of this study was the identification of species based on ITS1-RFLP in the ribosomal operon of L. major from clinically different forms of ZCL amplified by PCR, followed by the digestion of the PCR product with restriction enzymes. The profiles were observed and visualized in agarose gel under UV light. We used direct smears to identify the parasites. While taking the smear, samples were collected for culture or direct PCR. We used the PCR-RFLP assay of the ITS1 genes for direct identification of Leishmania species in 24 out of 33 suspected patients. PCR-ITS1 amplification was done on the 24 samples confirmed by culture via growth and parasitological methods. Results Of the 24 isolates, 21 had 350 bp bands (87.5%) and three had 450 bp bands (12.5%). After using the restriction enzyme, banding patterns including fragments of 210 and 140 bp for L. major were detected in 19 cases. Conclusions The L. major species causing ZCL in Chabahar have limited genetic variation. There seems to be little manifestation of diversity between these lesions as a new focus of disease, and new micro-habitats for

  4. Immunization against leishmaniasis by PLGA nanospheres loaded with an experimental autoclaved Leishmania major (ALM) and Quillaja saponins.

    PubMed

    Tafaghodi, M; Eskandari, M; Kharazizadeh, M; Khamesipour, A; Jaafari, M R

    2010-12-01

    Immune responses against the Leishmania antigens are not sufficient to protect against a leishmania challenge. Therefore these antigens need to be potentiated by various adjuvants and delivery systems. In this study, Poly (d,l-lactide-co-glycolide (PLGA) nanospheres as antigen delivery system and Quillaja saponins (QS) as immunoadjuvant have been used to enhance the immune response against autoclaved Leishmania major (ALM). PLGA nanospheres were prepared by a double-emulsion (W/O/W) technique. Particulate characteristics were studied by scanning electron microscopy and particle size analysis. Mean diameter for nanospheres loaded with ALM+QS was 294 ± 106 nm. BALB/c mice were immunized three times in 3-weeks intervals using ALM plus QS loaded nanospheres [(ALM+QS)PLGA], ALM encapsulated with PLGA nanospheres [(ALM)PLGA], (ALM)PLGA + QS, ALM + QS, ALM alone or PBS. The intensity of infection induced by L. major challenge was assessed by measuring size of footpad swelling. The strongest protection, showed by significantly (P < 0.05) smaller footpad, were observed in mice immunized with (ALM)PLGA. The (ALM+QS)PLGA group showed the least protection and highest swelling, while the (ALM)PLGA+QS, ALM+QS and ALM showed an intermediate protection with no significant difference. The mice immunized with ALM and ALM+QS showed the highest IgG2a/IgG1 ratio (P < 0.01), followed by (ALM)PLGA+QS. The highest IFN-γ and lowest IL-4 production was seen in (ALM)PLGA+QS, ALM+QS groups. The highest parasite burden was observed in (ALM)PLGA+QS and (ALM+QS)PLGA groups. It is concluded that PLGA nanospheres as a vaccine delivery system could increase the protective immune responses, but QS adjuvant has a reverse effect on protective immune responses and the least protective responses were seen in the presence of this adjuvant.

  5. Methylene tetrahydrofolate dehydrogenase/cyclohydrolase and the synthesis of 10-CHO-THF are essential in Leishmania major

    PubMed Central

    Murta, Silvane M. F.; Vickers, Tim J.; Scott, David A.; Beverley, Stephen M.

    2009-01-01

    Summary 10-formyl tetrahydrofolate is a key metabolite in C1 carbon metabolism, arising through the action of formate-tetrahydrofolate ligase (FTL) and/or 5,10-methenyltetrahydrofolate cyclohydrolase/5,10-methylene tetrahydrofolate dehydrogenase (DHCH). Leishmania major possesses single DHCH1 and FTL genes encoding exclusively cytosolic proteins, unlike other organisms where isoforms occur in the mitochondrion as well. Recombinant DHCH1 showed typical NADP+-dependent methylene tetrahydrofolate DH and 5,10-methenyltetrahydrofolate CH activities, and the DH activity was potently inhibited by a substrate analog 5,10-CO-THF (Ki 105 nM), as was Leishmania growth (EC50 1.1 μM). Previous studies showed null ftl− mutants were normal, raising the possibility that loss of the purine synthetic pathway had rendered 10-CHO-THF dispensable in evolution. We were unable to generate dhch1− null mutants by gene replacement, despite using a wide spectrum of nutritional supplements expected to bypass DHCH function. We applied an improved method for testing essential genes in Leishmania, based upon segregational loss of episomal complementing genes rather than transfection; analysis of ~1400 events without successful loss of DHCH1 again established its requirement. Lastly, we employed ‘genetic metabolite complementation’ using ectopically expressed FTL as an alternative source of 10-CHO-THF; now dhch1− null parasites were readily obtained. These data establish a requirement for 10-CHO tetrahydrofolate metabolism in L. major, and provide genetic and pharmacological validation of DHCH as a target for chemotherapy, in this and potentially other protozoan parasites. PMID:19183277

  6. Leishmania infantum sterol 24-c-methyltransferase formulated with MPL-SE induces cross-protection against L. major infection

    PubMed Central

    Goto, Yasuyuki; Bhatia, Ajay; Raman, Vanitha S.; Vidal, Silvia E. Z.; Bertholet, Sylvie; Coler, Rhea N.; Howard, Randall F.; Reed, Steven G.

    2009-01-01

    The enzyme sterol 24-c-methyltranferase (SMT) is required for the biosynthesis of ergosterol, the major membrane sterol in Leishmania parasites. SMT and ergosterol are not found in mammals, so this protein may be an attractive target for anti-leishmanial vaccines and drugs. We have previously demonstrated that SMT from L. infantum, which causes visceral leishmaniasis, is a protective antigen against this parasite. Because this protein is highly conserved among Leishmania species, we evaluated the potential of SMT to cross-protect against a different form of leishmaniasis. Here, we show that immunization with L. infantum SMT, formulated with monophosphoryl lipid A in stable emulsion (MPL-SE), protects mice from cutaneous leishmaniasis caused by L. major. In BALB/c mice the vaccine preparation induced antigen-specific multifunctional CD4+ T cells capable of producing IFN-γ, IL-2, and/or TNF-α upon antigen re-exposure, and MPL-SE was indispensable to direct immune responses to SMT towards Th1. Mice immunized with the SMT/MPL-SE vaccine developed significantly smaller lesions following ear challenge with L. major. These results suggest that SMT is a promising vaccine antigen for multiple forms of leishmaniasis. PMID:19428898

  7. Leishmania infantum sterol 24-c-methyltransferase formulated with MPL-SE induces cross-protection against L. major infection.

    PubMed

    Goto, Yasuyuki; Bhatia, Ajay; Raman, Vanitha S; Vidal, Silvia E Z; Bertholet, Sylvie; Coler, Rhea N; Howard, Randall F; Reed, Steven G

    2009-05-11

    The enzyme sterol 24-c-methyltranferase (SMT) is required for the biosynthesis of ergosterol, the major membrane sterol in Leishmania parasites. SMT and ergosterol are not found in mammals, so this protein may be an attractive target for anti-leishmanial vaccines and drugs. We have previously demonstrated that SMT from L. infantum, which causes visceral leishmaniasis, is a protective antigen against this parasite. Because this protein is highly conserved among Leishmania species, we evaluated the potential of SMT to cross-protect against a different form of leishmaniasis. Here, we show that immunization with L. infantum SMT, formulated with monophosphoryl lipid A in stable emulsion (MPL-SE), protects mice from cutaneous leishmaniasis caused by L. major. In BALB/c mice the vaccine preparation induced antigen-specific multi-functional CD4(+) T cells capable of producing IFN-gamma, IL-2, and/or TNF-alpha upon antigen re-exposure, and MPL-SE was indispensable to direct immune responses to SMT towards Th1. Mice immunized with the SMT/MPL-SE vaccine developed significantly smaller lesions following ear challenge with L. major. These results suggest that SMT is a promising vaccine antigen for multiple forms of leishmaniasis.

  8. Cell homeostasis in a Leishmania major mutant overexpressing the spliced leader RNA is maintained by an increased proteolytic activity.

    PubMed

    Toledo, Juliano S; Ferreira, Tiago R; Defina, Tânia P A; Dossin, Fernando de M; Beattie, Kenneth A; Lamont, Douglas J; Cloutier, Serge; Papadopoulou, Barbara; Schenkman, Sergio; Cruz, Angela K

    2010-10-01

    Although several stage-specific genes have been identified in Leishmania, the molecular mechanisms governing developmental gene regulation in this organism are still not well understood. We have previously reported an attenuation of virulence in Leishmania major and L. braziliensis carrying extra-copies of the spliced leader RNA gene. Here, we surveyed the major differences in proteome and transcript expression profiles between the spliced leader RNA overexpressor and control lines using two-dimensional gel electrophoresis and differential display reverse transcription PCR, respectively. Thirty-nine genes related to stress response, cytoskeleton, proteolysis, cell cycle control and proliferation, energy generation, gene transcription, RNA processing and post-transcriptional regulation have abnormal patterns of expression in the spliced leader RNA overexpressor line. The evaluation of proteolytic pathways in the mutant revealed a selective increase of cysteine protease activity and an exacerbated ubiquitin-labeled protein population. Polysome profile analysis and measurement of cellular protein aggregates showed that protein translation in the spliced leader RNA overexpressor line is increased when compared to the control line. We found that L. major promastigotes maintain homeostasis in culture when challenged with a metabolic imbalance generated by spliced leader RNA surplus through modulation of intracellular proteolysis. However, this might interfere with a fine-tuned gene expression control necessary for the amastigote multiplication in the mammalian host.

  9. Leishmaniasis in Turkey: first clinical isolation of Leishmania major from 18 autochthonous cases of cutaneous leishmaniasis in four geographical regions.

    PubMed

    Özbilgin, Ahmet; Çulha, Gülnaz; Uzun, Soner; Harman, Mehmet; Topal, Suhan Günaştı; Okudan, Fulya; Zeyrek, Fadile; Gündüz, Cumhur; Östan, İpek; Karakuş, Mehmet; Töz, Seray; Kurt, Özgür; Akyar, Işın; Erat, Ayşegül; Güngör, Dilek; Kayabaşı, Çağla; Çavuş, İbrahim; Bastien, Patrick; Pratlong, Francine; Kocagöz, Tanıl; Özbel, Yusuf

    2016-06-01

    To report isolation of Leishmania major strains obtained from 18 Turkish autochthonous cutaneous leishmaniasis (CL) patients infected with L. major between 2011 and 2014. Initial diagnosis relied on microscopy and culture in enriched medium, prepared by adding specific amounts of liver extract, protein and lipid sources to NNN medium. Promastigotes were then transferred to RPMI medium including 10% of foetal calf serum for mass culture. Species-specific real-time PCR targeting ITS1 region of Leishmania spp. was performed using both lesion aspiration samples and cultured promastigotes. Two of 18 isolates were identified by isoenzyme analysis in the Leishmaniasis Reference Center in Montpellier, France. Each isolate was inoculated into the footpads of six mice to observe the pathogenicity of L. major. Developing lesions were observed, and the thickening of footpads was measured weekly. Melting curve analyses of 18 isolates showed a peak concordant with L. major, and two of them were confirmed by isoenzyme analyses as L. major zymodeme MON103. In the mouse model, acute lesions seen on day 21 were accepted as an indication of heavy infection. Severe impairments were observed on all mouse footpads over 3 weeks, which even progressed to extremity amputation. Cutaneous leishmaniasis-causing L. major was recently identified in Adana province in southern Turkey, with PCR. Our study shows that such CL cases are not limited to Adana but currently present from western to Southeastern Anatolia, and along the Mediterranean coast. The role of small mammals, the main reservoirs of L. major in Anatolia, needs to be elucidated, as do the underlying factors that cause severe clinical manifestations in L. major infections in Turkey, contrary to the infections in neighbouring countries. © 2016 John Wiley & Sons Ltd.

  10. MHC Class II Restricted Innate-Like Double Negative T Cells Contribute to Optimal Primary and Secondary Immunity to Leishmania major

    PubMed Central

    Mou, Zhirong; Liu, Dong; Okwor, Ifeoma; Jia, Ping; Orihara, Kanami; Uzonna, Jude Ezeh

    2014-01-01

    Although it is generally believed that CD4+ T cells play important roles in anti-Leishmania immunity, some studies suggest that they may be dispensable, and that MHC II-restricted CD3+CD4−CD8− (double negative, DN) T cells may be more important in regulating primary anti-Leishmania immunity. In addition, while there are reports of increased numbers of DN T cells in Leishmania-infected patients, dogs and mice, concrete evidence implicating these cells in secondary anti-Leishmania immunity has not yet been documented. Here, we report that DN T cells extensively proliferate and produce effector cytokines (IFN-γ, TNF and IL-17) and granzyme B (GrzB) in the draining lymph nodes and spleens of mice following primary and secondary L. major infections. DN T cells from healed mice display functional characteristics of protective anti-Leishmania memory-like cells: rapid and extensive proliferation and effector cytokines production following L. major challenge in vitro and in vivo. DN T cells express predominantly (> 95%) alpha-beta T cell receptor (αβ TCR), are Leishmania-specific, restricted mostly by MHC class II molecules and display transcriptional profile of innate-like genes. Using in vivo depletion and adoptive transfer studies, we show that DN T cells contribute to optimal primary and secondary anti-Leishmania immunity in mice. These results directly identify DN T cells as important players in effective and protective primary and secondary anti-L. major immunity in experimental cutaneous leishmaniasis. PMID:25233487

  11. The activity of azithromycin against Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis in the golden hamster model.

    PubMed

    Sinagra, Angel; Luna, Concepción; Abraham, David; Iannella, Maria del Carmen; Riarte, Adelina; Krolewiecki, Alejandro J

    2007-01-01

    New therapeutic alternatives against leishmaniasis remain a priority. The activity of azithromycin against Leishmania (Leishmania) major has been previously demonstrated. Different responses among species of Leishmania make species-specific drug screening necessary. The activity of azithromycin against Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis was evaluated in golden hamsters infected through footpad injections of metacyclic promastigotes, and compared with untreated controls and animals treated with meglumine antimoniate. Footpad thickness, lesion cultures and dissemination sites were analyzed. Treatment of golden hamsters with oral azithromycin at 450mg/kg had no activity against infections with Leishmania (Leishmania) amazonensis. For infections due to Leishmania (Viannia) braziliensis, azithromycin demonstrated significant activity relative to untreated controls, but inferior to meglumine antimoniate, for controlling lesion size. Neither drug was able to totally eliminate parasites from the lesions. It was concluded that azithromycin has activity against Leishmania (Viannia) braziliensis but not against Leishmania (Leishmania) amazonensis in this model.

  12. Cathepsin B-Deficient Mice Resolve Leishmania major Inflammation Faster in a T Cell-Dependent Manner

    PubMed Central

    Mériaux, Véronique; Khan, Erin M.; Borde, Chloé; Ciulean, Ioana S.; Fitting, Catherine; Manoury, Bénédicte; Cavaillon, Jean-Marc; Doyen, Noëlle

    2016-01-01

    A critical role for intracellular TLR9 has been described in recognition and host resistance to Leishmania parasites. As TLR9 requires endolysosomal proteolytic cleavage to achieve signaling functionality, we investigated the contribution of different proteases like asparagine endopeptidase (AEP) or cysteine protease cathepsins B (CatB), L (CatL) and S (CatS) to host resistance during Leishmania major (L. major) infection in C57BL/6 (WT) mice and whether they would impact on TLR9 signaling. Unlike TLR9-/-, which are more susceptible to infection, AEP-/-, CatL-/- and CatS-/- mice are as resistant to L. major infection as WT mice, suggesting that these proteases are not individually involved in TLR9 processing. Interestingly, we observed that CatB-/- mice resolve L. major lesions significantly faster than WT mice, however we did not find evidence for an involvement of CatB on either TLR9-dependent or independent cytokine responses of dendritic cells and macrophages or in the innate immune response to L. major infection. We also found no difference in antigen presenting capacity. We observed a more precocious development of T helper 1 responses accompanied by a faster decline of inflammation, resulting in resolution of footpad inflammation, reduced IFNγ levels and decreased parasite burden. Adoptive transfer experiments into alymphoid RAG2-/-γc-/- mice allowed us to identify CD3+ T cells as responsible for the immune advantage of CatB-/- mice towards L. major. In vitro data confirmed the T cell intrinsic differences between CatB-/- mice and WT. Our study brings forth a yet unappreciated role for CatB in regulating T cell responses during L. major infection. PMID:27182703

  13. Protective response to Leishmania major in BALB/c mice requires antigen processing in the absence of DM.

    PubMed

    Kamala, Tirumalai; Nanda, Navreet K

    2009-04-15

    Protection from the parasite Leishmania major is mediated by CD4 T cells. BALB/c mice are susceptible to L. major and show a nonprotective immunodominant CD4 T cell response to Leishmania homolog of activated receptor for c-kinase (LACK) 158-173. Host genes that underlie BALB/c susceptibility to L. major infections are poorly defined. DM, a nonclassical MHC class II molecule, due to its peptide editing properties has been shown to 1) edit the repertoire of peptides displayed by APC, and 2) focus the display of epitopes by APC to the immunodominant ones. We tested the hypothesis that deficiency of DM, by causing presentation of a different array of epitopes by infected APC than that presented by DM-sufficient APC, may change the course of L. major infection in the susceptible BALB/c mice. We show herein that unlike their susceptible wild-type counterparts, BALB/c mice deficient in DM are protected from infections with L. major. Furthermore, DM-deficient mice fail to display the immunodominant LACK 158-173 on infected APC. In its place, infected DM(-/-) hosts show elicitation of CD4 T cells specific for newer epitopes not presented by wild-type L. major-infected APC. Protection of BALB/c DM(-/-) mice is dependent on IFN-gamma. DM is thus a host susceptibility gene in BALB/c mice, and Ag processing in the absence of DM results in elicitation of a protective T cell response against L. major infections. This report suggests a novel mechanism to trigger host resistance against pathogens.

  14. Effects of Leishmania major clones showing different levels of virulence on infectivity, differentiation and maturation of human dendritic cells.

    PubMed

    Markikou-Ouni, W; Ben Achour-Chenik, Y; Meddeb-Garnaoui, A

    2012-09-01

    Leishmania parasites and dendritic cell interactions (DCs) play an essential role in initiating and directing T cell responses and influence disease evolution. These interactions may vary depending on Leishmania species and strains. To evaluate the correlation between Leishmania major (Lm) virulence and in-vitro human DC response, we compared the ability of high (HV) and low virulent (LV) Lm clones to invade, modulate cytokine production and interfere with differentiation of DCs. Clones derived from HV and LV (HVΔlmpdi and LVΔlmpdi), and deleted for the gene coding for a Lm protein disulphide isomerase (LmPDI), probably involved in parasite natural pathogenicity, were also used. Unlike LV, which fails to invade DCs in half the donors, HV promastigotes were associated with a significant increase of the infected cells percentage and parasite burden. A significant decrease of both parameters was observed in HVΔlmpdi-infected DCs, compared to wild-type cells. Whatever Lm virulence, DC differentiation was accompanied by a significant decrease in CD1a expression. Lm clones decreased interleukin (IL)-12p70 production similarly during lipopolysaccharide (LPS)-induced maturation of DCs. LPS stimulation was associated with a weak increase in tumour necrosis factor (TNF)-α and IL-10 productions in HV-, HVΔlmpdi- and LVΔlmpdi-infected DCs. These results indicate that there is a significant variability in the capacity of Lm clones to infect human DCs which depends upon their virulence, probably involving LmPDI protein. However, independently of their virulence, Lm clones were able to down-regulate CD1a expression during DC differentiation and IL-12p70 production during DC maturation, which may favour their survival.

  15. In vitro and in vivo antileishmanial effects of aloe-emodin on Leishmania major.

    PubMed

    Dalimi, Abdolhossein; Delavari, Mahdi; Ghaffarifar, Fatemeh; Sadraei, Javid

    2015-04-01

    Cutaneous leishmaniasis is a common parasitic disease that is endemic in some parts of Iran. The drugs of choice used for leishmaniasis therapy are associated with a risk of recurrence and serious adverse effects. Therefore, finding a safe and effective treatment is of great importance. In the present study, the effect of aloe-emodin on the growth of Leishmania major amastigotes was evaluated under in vitro conditions. In addition, the efficacy of a topical of aloe-emodin ointment was investigated in BALB/c mice with cutaneous leishmanial ulcers. Different concentrations (40 μg/mL, 80 μg/mL, 120 μg/mL, and 160 μg/mL) of aloe-emodin were tested on Leishmania amastigotes twice: 24 hours and 48 hours. The induced apoptosis and necrotic effects of two concentrations (40 μg/mL and 120 μg/mL) of aloe-emodin on promastigotes were investigated by flow cytometry. Under the in vivo condition, aloe-emodin ointment efficacy was evaluated at two concentrations (i.e., 0.1% and 1%). Serum indicator factors of the test and control groups were tested to evaluate the toxic effects of this compound on the liver and kidney. Results showed that aloe-emodin inhibited the growth of Leishmania amastigotes and induced apoptosis in promastigotes. Topical application of aloe-emodin ointment likewise reduced the ulcer size. No significant differences in biochemical analysis were observed between the control and treated groups. In conclusion, aloe-emodin showed antileishmanial effects under in vitro and in vivo conditions and may be used in clinical trials.

  16. In vitro and in vivo antileishmanial effects of aloe-emodin on Leishmania major

    PubMed Central

    Dalimi, Abdolhossein; Delavari, Mahdi; Ghaffarifar, Fatemeh; Sadraei, Javid

    2015-01-01

    Cutaneous leishmaniasis is a common parasitic disease that is endemic in some parts of Iran. The drugs of choice used for leishmaniasis therapy are associated with a risk of recurrence and serious adverse effects. Therefore, finding a safe and effective treatment is of great importance. In the present study, the effect of aloe-emodin on the growth of Leishmania major amastigotes was evaluated under in vitro conditions. In addition, the efficacy of a topical of aloe-emodin ointment was investigated in BALB/c mice with cutaneous leishmanial ulcers. Different concentrations (40 μg/mL, 80 μg/mL, 120 μg/mL, and 160 μg/mL) of aloe-emodin were tested on Leishmania amastigotes twice: 24 hours and 48 hours. The induced apoptosis and necrotic effects of two concentrations (40 μg/mL and 120 μg/mL) of aloe-emodin on promastigotes were investigated by flow cytometry. Under the in vivo condition, aloe-emodin ointment efficacy was evaluated at two concentrations (i.e., 0.1% and 1%). Serum indicator factors of the test and control groups were tested to evaluate the toxic effects of this compound on the liver and kidney. Results showed that aloe-emodin inhibited the growth of Leishmania amastigotes and induced apoptosis in promastigotes. Topical application of aloe-emodin ointment likewise reduced the ulcer size. No significant differences in biochemical analysis were observed between the control and treated groups. In conclusion, aloe-emodin showed antileishmanial effects under in vitro and in vivo conditions and may be used in clinical trials. PMID:26151018

  17. KSAC, a Defined Leishmania Antigen, plus Adjuvant Protects against the Virulence of L. major Transmitted by Its Natural Vector Phlebotomus duboscqi

    PubMed Central

    Gomes, Regis; Teixeira, Clarissa; Oliveira, Fabiano; Lawyer, Phillip G.; Elnaiem, Dia-Eldin; Meneses, Claudio; Goto, Yasuyuki; Bhatia, Ajay; Howard, Randall F.; Reed, Steven G.; Valenzuela, Jesus G.; Kamhawi, Shaden

    2012-01-01

    Background Recombinant KSAC and L110f are promising Leishmania vaccine candidates. Both antigens formulated in stable emulsions (SE) with the natural TLR4 agonist MPL® and L110f with the synthetic TLR4 agonist GLA in SE protected BALB/c mice against L. major infection following needle challenge. Considering the virulence of vector-transmitted Leishmania infections, we vaccinated BALB/c mice with either KSAC+GLA-SE or L110f+GLA-SE to assess protection against L. major transmitted via its vector Phlebotomus duboscqi. Methods Mice receiving the KSAC or L110f vaccines were challenged by needle or L. major-infected sand flies. Weekly disease progression and terminal parasite loads were determined. Immunological responses to KSAC, L110f, or soluble Leishmania antigen (SLA) were assessed throughout vaccination, three and twelve weeks after immunization, and one week post-challenge. Results Following sand fly challenge, KSAC-vaccinated mice were protected while L110f-vaccinated animals showed partial protection. Protection correlated with the ability of SLA to induce IFN-γ-producing CD4+CD62LlowCCR7low effector memory T cells pre- and post-sand fly challenge. Conclusions This study demonstrates the protective efficacy of KSAC+GLA-SE against sand fly challenge; the importance of vector-transmitted challenge in evaluating vaccine candidates against Leishmania infection; and the necessity of a rapid potent Th1 response against Leishmania to attain true protection. PMID:22509423

  18. Target sites for the design of anti-trypanosomatid drugs based on the structure of dihydroorotate dehydrogenase.

    PubMed

    Pinheiro, Matheus Pinto; Emery, Flávio da Silva; Nonato, M Cristina

    2013-01-01

    Trypanosomatids consist of a large group of flagellated parasitic protozoa, including parasites from the genera Leishmania and Trypanosoma, responsible for causing infections in millions of humans worldwide and for which currently no appropriate therapy is available. The significance of pyrimidines in cellular metabolism makes their de novo and salvage pathways ideal druggable targets for pharmacological intervention and open an opportunity for pharmaceutical innovation. In the current review, we discuss the merits in targeting the enzyme dihydroorotate dehydrogenase (DHODH), a flavin-dependent enzyme that catalyzes the fourth and only redox step in pyrimidine de novo biosynthesis, as a strategy for the development of efficient therapeutic strategies for trypanosomatid-related diseases.We also describe the advances and perspectives from the structural biology point of view in order to unravel the structure-function relationship of trypanosomatid DHODHs, and to identify and validate target sites for drug development.

  19. Three Leishmania/L. species--L. infantum, L. major, L. tropica--as causative agents of mucosal leishmaniasis in Iran.

    PubMed

    Shirian, Sadegh; Oryan, Ahmad; Hatam, Gholam Reza; Daneshbod, Yahya

    2013-07-01

    Cases of human oro-mucosal leishmaniasis are mainly reported in areas where Leishmania (Viannia) braziliensis perpetuates and the damages are mainly located at the cartilaginous nasal septum and frontal portions of the nasal fossa. In Iran, an area free of any L.(V) braziliensis, three Leishmania species are known to perpetuate through distinct (i) blood-feeding sand flies and (ii) rodents or (iii) canidae. Thus while establishing the diagnosis of any human oro-mucosal lesions, three Leishmania species - L. infantum, L. major, and L. tropica - must be considered as potential etiological agents of these damages. With these objectives in mind, features such as localization, extent, severity of oro-mucosal lesions, and duration of symptoms at the time of diagnosis were recorded from 11 patients with respect to the presence or absence of cutaneous lesions in other body parts. The biopsy samples were collected from the oro-mucosal and cutaneous lesions and were processed for further identification of the Leishmania species. The lesions ranged from mucosal nodules without ulceration, nodules with erosion, and shallow to deep ulcerations. Leishmania major was isolated from six (55%) cases showing lesions or scars. The scars were restricted to upper and lower extremities. For the other five patients who did not display any signs of former or active cutaneous leishmaniasis, L. major, L. tropica, and L. infantum were isolated from their lesions. In conclusion L. major, L. infantum, and L. tropica, regardless of common tropism, can be seen in mucosal tissues. However, L. major was the predominant species detected from the lesions in the nasal, gingival, and hard and soft palates, and L. tropica was isolated from the gingival and lower lip lesions. Leishmania infantum was isolated from two severe cases of deep mucosal damage displayed by the epiglottis, cricoarytenoid muscle, and laryngeal mucosa. One important finding was the association of L. major with active or scarred

  20. T Cells That React to the Immunodominant Leishmania major LACK Antigen Prevent Early Dissemination of the Parasite in Susceptible BALB/c Mice

    PubMed Central

    Schilling, Sabrina; Glaichenhaus, Nicolas

    2001-01-01

    Susceptibility of BALB/c mice to Leishmania major depends on the early production of IL-4 by CD4+ T cells which react to the parasite LACK antigen. Here, we show that LACK-specific cells are rapidly recruited to the site of infection and favor the early dissemination of L. major to the internal organs. PMID:11160025

  1. The Leishmania infantum acidic ribosomal protein P0 administered as a DNA vaccine confers protective immunity to Leishmania major infection in BALB/c mice.

    PubMed

    Iborra, Salvador; Soto, Manuel; Carrión, Javier; Nieto, Ana; Fernández, Edgar; Alonso, Carlos; Requena, Jose M

    2003-11-01

    In this study, we examined the immunogenic properties of the Leishmania infantum acidic ribosomal protein P0 (LiP0) in the BALB/c mouse model. The humoral and cellular responses induced by the administration of the LiP0 antigen, either as soluble recombinant LiP0 (rLiP0) or as a plasmid DNA formulation (pcDNA3-LiP0), were determined. Also, the immunological response associated with a prime-boost strategy, consisting of immunization with pcDNA3-LiP0 followed by a boost with rLiP0, was assayed. Immunization with rLiP0 induced a predominant Th2-like humoral response, but no anti-LiP0 antibodies were induced after immunization with pcDNA3-LiP0, whereas a strong humoral response consisting of a mixed immunoglobulin G2a (IgG2a)-IgG1 isotype profile was induced in mice immunized with the prime-boost regime. For all three immunization protocols, rLiP0-stimulated production of gamma interferon (IFN-gamma) in both splenocytes and lymph node cells from immunized mice was observed. However, it was only when mice were immunized with pcDNA3-LiP0 that noticeable protection against L. major infection was achieved, as determined by both lesion development and parasite burden. Immunization of mice with LiP0-DNA primes both CD4(+) and CD8(+) T cells, which, with the L. major challenge, were boosted to produce significant levels of IL-12-dependent, antigen-specific IFN-gamma. Taken together, these data indicate that genetic vaccination with LiP0 induces protective immunological effector mechanisms, yet the immunological response elicited by LiP0 is not sufficient to keep the infection from progressing.

  2. Infection Parameters in the Sand Fly Vector That Predict Transmission of Leishmania major

    PubMed Central

    Stamper, Lisa W.; Patrick, Rachel L.; Fay, Michael P.; Lawyer, Phillip G.; Elnaiem, Dia-Eldin A.; Secundino, Nagila; Debrabant, Alain; Sacks, David L.; Peters, Nathan C.

    2011-01-01

    To identify parameters of Leishmania infection within a population of infected sand flies that reliably predict subsequent transmission to the mammalian host, we sampled groups of infected flies and compared infection intensity and degree of metacyclogenesis with the frequency of transmission. The percentage of parasites within the midgut that were metacyclic promastigotes had the highest correlation with the frequency of transmission. Meta-analysis of multiple transmission experiments allowed us to establish a percent-metacyclic “cutoff” value that predicted transmission competence. Sand fly infections initiated with variable doses of parasites resulted in correspondingly altered percentages of metacyclic promastigotes, resulting in altered transmission frequency and disease severity. Lastly, alteration of sand fly oviposition status and environmental conditions at the time of transmission also influenced transmission frequency. These observations have implications for transmission of Leishmania by the sand fly vector in both the laboratory and in nature, including how the number of organisms acquired by the sand fly from an infection reservoir may influence the clinical outcome of infection following transmission by bite. PMID:21886852

  3. The Leishmania major BBSome subunit BBS1 is essential for parasite virulence in the mammalian host

    PubMed Central

    Price, Helen P; Paape, Daniel; Hodgkinson, Michael R; Farrant, Katie; Doehl, Johannes; Stark, Meg; Smith, Deborah F

    2013-01-01

    Summary Bardet–Biedl syndrome (BBS) is a human genetic disorder with a spectrum of symptoms caused by primary cilium dysfunction. The disease is caused by mutations in one of at least 17 identified genes, of which seven encode subunits of the BBSome, a protein complex required for specific trafficking events to and from the primary cilium. The molecular mechanisms associated with BBSome function remain to be fully elucidated. Here, we generated null and complemented mutants of the BBSome subunit BBS1 in the protozoan parasite, Leishmania. In the absence of BBS1, extracellular parasites have no apparent defects in growth, flagellum assembly, motility or differentiation in vitro but there is accumulation of vacuole-like structures close to the flagellar pocket. Infectivity of these parasites for macrophages in vitro is reduced compared with wild-type controls but the null parasites retain the ability to differentiate to the intracellular amastigote stage. However, infectivity of BBS1 null parasites is severely compromised in a BALB/c mouse footpad model. We hypothesize that the absence of BBS1 in Leishmania leads to defects in specific trafficking events that affect parasite persistence in the host. This is the first report of an association between the BBSome complex and pathogen infectivity. PMID:23998526

  4. Use of two-photon microscopy to study Leishmania major infection of the skin.

    PubMed

    Carneiro, Matheus Batista; Hohman, Leah Shan; Egen, Jackson G; Peters, Nathan C

    2017-08-15

    Intra-vital two-photon microscopy (2P-IVM) allows for in-situ investigation of tissue organization, cell behavior and the dynamic interactions between different cell types in their natural environment. This methodology has also expanded our understanding of the immune response against pathogens. Leishmania are protozoan intracellular parasites that have adapted to successfully establish infection within the context of an inflammatory response in the skin following transmission by the bite of an infected sand fly. The generation of fluorescent transgenic parasites coupled with the increased availability of different types of fluorescent transgenic reporter mice has facilitated the study of the host-parasite interaction in the skin, significantly impacting our understanding of cutaneous leishmaniasis. In this review we will discuss 2P-IVM in the context of Leishmania infection of the mouse ear skin and describe a simple and minimally invasive procedure that allows long-term imaging of this host-pathogen interaction. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Base J represses genes at the end of polycistronic gene clusters in Leishmania major by promoting RNAP II termination

    PubMed Central

    Reynolds, David L.; Hofmeister, Brigitte T.; Cliffe, Laura; Siegel, T. Nicolai; Anderson, Britta A.; Beverley, Stephen M.; Schmitz, Robert J.; Sabatini, Robert

    2016-01-01

    Summary The genomes of kinetoplastids are organized into polycistronic gene clusters that are flanked by the modified DNA base J. Previous work has established a role of base J in promoting RNA polymerase II termination in Leishmania spp. where the loss of J leads to termination defects and transcription into adjacent gene clusters. It remains unclear whether these termination defects affect gene expression and whether read through transcription is detrimental to cell growth, thus explaining the essential nature of J. We now demonstrate that reduction of base J at specific sites within polycistronic gene clusters in L. major leads to read through transcription and increased expression of downstream genes in the cluster. Interestingly, subsequent transcription into the opposing polycistronic gene cluster does not lead to downregulation of sense mRNAs. These findings indicate a conserved role for J regulating transcription termination and expression of genes within polycistronic gene clusters in trypanosomatids. In contrast to the expectations often attributed to opposing transcription, the essential nature of J in Leishmania spp. is related to its role in gene repression rather than preventing transcriptional interference resulting from read through and dual strand transcription. PMID:27125778

  6. Involvement of Different CD4+ T Cell Subsets Producing Granzyme B in the Immune Response to Leishmania major Antigens

    PubMed Central

    Naouar, Ikbel; Boussoffara, Thouraya; Ben Ahmed, Melika; Belhaj Hmida, Nabil; Gharbi, Adel; Gritli, Sami; Ben Salah, Afif; Louzir, Hechmi

    2014-01-01

    The nature of effector cells and the potential immunogenicity of Leishmania major excreted/secreted proteins (LmES) were evaluated using peripheral blood mononuclear cells (PBMCs) from healed zoonotic cutaneous leishmaniasis individuals (HZCL) and healthy controls (HC). First, we found that PBMCs from HZCL individuals proliferate and produce high levels of IFN-γ and granzyme B (GrB), used as a marker of activated cytotoxic T cells, in response to the parasite antigens. IFN-γ is produced by CD4+ T cells, but unexpectedly GrB is also produced by CD4+ T cells in response to stimulation with LmES, which were found to be as effective as soluble Leishmania antigens to induce proliferation and cytokine production by PBMCs from immune individuals. To address the question of regulatory T cell (Tregs) involvement, the frequency of circulating Tregs was assessed and found to be higher in HZCL individuals compared to that of HC. Furthermore, both CD4+CD25+ and CD4+CD25− T cells, purified from HZCL individuals, produced IFN-γ and GrB when stimulated with LmES. Additional experiments showed that CD4+CD25+CD127dim/− Tregs were involved in GrB production. Collectively, our data indicate that LmES are immunogenic in humans and emphasize the involvement of CD4+ T cells including activated and regulatory T cells in the immune response against parasite antigens. PMID:25104882

  7. Structure-based discovery of the first non-covalent inhibitors of Leishmania major tryparedoxin peroxidase by high throughput docking

    PubMed Central

    Brindisi, Margherita; Brogi, Simone; Relitti, Nicola; Vallone, Alessandra; Butini, Stefania; Gemma, Sandra; Novellino, Ettore; Colotti, Gianni; Angiulli, Gabriella; Di Chiaro, Francesco; Fiorillo, Annarita; Ilari, Andrea; Campiani, Giuseppe

    2015-01-01

    Leishmaniasis is a neglected vector-born disease caused by a protozoan of the genus Leishmania and affecting more than 1.300.000 people worldwide. The couple tryparedoxin/tryparedoxin peroxidase is essential for parasite survival in the host since it neutralizes the hydrogen peroxide produced by macrophages during the infection. Herein we report a study aimed at discovering the first class of compounds able to non-covalently inhibit tryparedoxin peroxidase. We have solved the high-resolution structure of Tryparedoxin peroxidase I from Leishmania major (LmTXNPx) in the reduced state and in fully folded conformation. A first series of compounds able to inhibit LmTXNPx was identified by means of the high throughput docking technique. The inhibitory activity of these compounds was validated by a Horseradish peroxidase-based enzymatic assay and their affinity for LmTXNPx calculated by surface plasmon resonance experiments. On the basis of these results, the analysis of the enzyme-inhibitor docked models allowed us to rationally design and synthesize a series of N,N-disubstituted 3-aminomethyl quinolones. These compounds showed an inhibitory potency against LmTXNPx in the micromolar range. Among them, compound 12 represents the first non-covalent LmTXNPx inhibitor reported to date and could pave the way to the discovery of a new class of drugs against leishmaniasis. PMID:25951439

  8. Antileishmanial Activity of Compounds Derived from the Medicines for Malaria Venture Open Access Box against Intracellular Leishmania major Amastigotes

    PubMed Central

    Khraiwesh, Mozna; Leed, Susan; Roncal, Norma; Johnson, Jacob; Sciotti, Richard; Smith, Philip; Read, Lisa; Paris, Robert; Hudson, Thomas; Hickman, Mark; Grogl, Max

    2016-01-01

    Leishmaniasis is a complex tropical disease caused by kinetoplastid parasitic protozoa of the genus Leishmania and is transmitted by the sand fly insect vector. Cutaneous leishmaniasis (CL) is the most common form of this disease, and CL infections often result in serious skin lesions and scars. CL remains a public health problem in many endemic countries worldwide because of the absence of effective, safe, and cost-effective drugs for treatment. One of the strategies we chose to use to find novel chemical entities worthy of further development as antileishmanials involved screening synthetic and natural products libraries. In our study, we developed a Leishmania major intracellular amastigote assay that uses the activity of luciferase as a measure of parasite proliferation and used this assay to screen a collection of 400 compounds obtained from Medicines for Malaria Venture (MMV) for their antileishmanial activity. Our results showed that 14 compounds identified by MMV as antimalarial drugs have antileishmanial activity and can potentially be optimized for CL drug development. PMID:26503273

  9. Antileishmanial Activity of Compounds Derived from the Medicines for Malaria Venture Open Access Box Against Intracellular Leishmania major Amastigotes.

    PubMed

    Khraiwesh, Mozna; Leed, Susan; Roncal, Norma; Johnson, Jacob; Sciotti, Richard; Smith, Philip; Read, Lisa; Paris, Robert; Hudson, Thomas; Hickman, Mark; Grogl, Max

    2016-02-01

    Leishmaniasis is a complex tropical disease caused by kinetoplastid parasitic protozoa of the genus Leishmania and is transmitted by the sand fly insect vector. Cutaneous leishmaniasis (CL) is the most common form of this disease, and CL infections often result in serious skin lesions and scars. CL remains a public health problem in many endemic countries worldwide because of the absence of effective, safe, and cost-effective drugs for treatment. One of the strategies we chose to use to find novel chemical entities worthy of further development as antileishmanials involved screening synthetic and natural products libraries. In our study, we developed a Leishmania major intracellular amastigote assay that uses the activity of luciferase as a measure of parasite proliferation and used this assay to screen a collection of 400 compounds obtained from Medicines for Malaria Venture (MMV) for their antileishmanial activity. Our results showed that 14 compounds identified by MMV as antimalarial drugs have antileishmanial activity and can potentially be optimized for CL drug development. © The American Society of Tropical Medicine and Hygiene.

  10. An integrated approach towards the discovery of novel non-nucleoside Leishmania major pteridine reductase 1 inhibitors.

    PubMed

    Leite, Franco Henrique Andrade; Froes, Thamires Quadros; da Silva, Suellen Gonçalves; de Souza, Evandro Italo Macêdo; Vital-Fujii, Drielli Gomes; Trossini, Gustavo Henrique Goulart; Pita, Samuel Silva da Rocha; Castilho, Marcelo Santos

    2017-05-26

    Despite the fact that Leishmania ssp are pteridine auxotrophs, Dihydrofolate Reductase-Thymidylate Synthase (DHFR-TS) inhibitors are ineffective against Leishmania major. On the other hand Pteridine Reductase 1 (PTR1) inhibitors proved to be lethal to the parasite. Aiming at identifying hits that lie outside the chemical space of known PTR1 inhibitors, pharmacophore models that differentiate true-binders from decoys and explain the structure-activity relationships of known inhibitors were employed to virtually screen the lead-like subset of ZINC database. This approach leads to the identification of Z80393 (IC50 = 32.31 ± 1.18 μM), whose inhibition mechanism was investigated by Thermal Shift Assays. This experimental result supports a competitive mechanism and was crucial to establish the docking search space as well as select the best pose, which was then investigated by molecular dynamics studies that corroborate the hit putative binding profile towards LmPTR1. The information gathered from such studies shall be useful to design more potent non-nucleoside LmPTR1 inhibitors. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  11. In vitro effect of essential oils and isolated mono- and sesquiterpenes on Leishmania major and Trypanosoma brucei.

    PubMed

    Mikus, J; Harkenthal, M; Steverding, D; Reichling, J

    2000-05-01

    The effect of different essential oils as well as of isolated mono- and sesquiterpenes on the viability of bloodstream forms of Trypanosoma brucei, promastigotes of Leishmania major and human HL-60 cells was evaluated using the Almar Blue assay. Of the 12 essential oils and 8 terpenes investigated, only three essential oils, Melissa officinalis (balmmint) oil, Thymus vulgaris (thyme) oil, and Melaleuca alternifolia (tea tree) oil were about 50-fold and 80-fold more toxic to bloodstream forms of T. brucei than to HL-60 cells, respectively. Terpinen-4-ol, the main compound of the Australian tea tree oil, was even 1000-fold more toxic to trypanosomes than to the human cells. On the other hand, none of the essential oils and terpenes tested were more toxic to promastigotes of L. major than to HL-60 cells.

  12. Cathepsin B in Antigen-Presenting Cells Controls Mediators of the Th1 Immune Response during Leishmania major Infection

    PubMed Central

    Gonzalez-Leal, Iris J.; Röger, Bianca; Schwarz, Angela; Schirmeister, Tanja; Reinheckel, Thomas; Lutz, Manfred B.; Moll, Heidrun

    2014-01-01

    Resistance and susceptibility to Leishmania major infection in the murine model is determined by the capacity of the host to mount either a protective Th1 response or a Th2 response associated with disease progression. Previous reports involving the use of cysteine cathepsin inhibitors indicated that cathepsins B (Ctsb) and L (Ctsl) play important roles in Th1/Th2 polarization during L. major infection in both susceptible and resistant mouse strains. Although it was hypothesized that these effects are a consequence of differential patterns of antigen processing, the mechanisms underlying these differences were not further investigated. Given the pivotal roles that dendritic cells and macrophages play during Leishmania infection, we generated bone-marrow derived dendritic cells (BMDC) and macrophages (BMM) from Ctsb−/− and Ctsl−/− mice, and studied the effects of Ctsb and Ctsl deficiency on the survival of L. major in infected cells. Furthermore, the signals used by dendritic cells to instruct Th cell polarization were addressed: the expression of MHC class II and co-stimulatory molecules, and cytokine production. We found that Ctsb−/− BMDC express higher levels of MHC class II molecules than wild-type (WT) and Ctsl−/− BMDC, while there were no significant differences in the expression of co-stimulatory molecules between cathepsin-deficient and WT cells. Moreover, both BMDC and BMM from Ctsb−/− mice significantly up-regulated the levels of interleukin 12 (IL-12) expression, a key Th1-inducing cytokine. These findings indicate that Ctsb−/− BMDC display more pro-Th1 properties than their WT and Ctsl−/− counterparts, and therefore suggest that Ctsb down-regulates the Th1 response to L. major. Moreover, they propose a novel role for Ctsb as a regulator of cytokine expression. PMID:25255101

  13. Effects of combined therapy with silymarin and glucantime on leishmaniasis induced by Leishmania major in BALB/c mice.

    PubMed

    Jabini, R; Jaafari, M R; Vahdati Hasani, F; Ghazizadeh, F; Khamesipour, A; Karimi, G

    2015-03-01

    Leishmania major is resistant to the traditional treatments in many parts of the world. PgpA, a member of (ABC) transporter superfamily, has been identified in Leishmania involved in antimony resistance. Silymarin can inhibit PgpA. The aim of this study was to determine the effect of combined therapy with glucantime and silymarin on Cutaneous Leishmaniasis. The effects of silymarin on response of L. major to glucantime were evaluated with amastigote macrophage and mice model of leishmaniasis. Immediately after injection in mice inoculated into footpads with L. major amastigote, systemic treatment was performed and the size of footpad swelling was measured twice a week. 4 and 8 weeks after the beginning of the treatment, splenic parasite burden was done. Silymarin showed no significant effect on the response of L. major promastigotes to glucantime. 2 formulations (glucantime 25 µm with silymarin 25 µm or 12.5 µm) reduced cell death in amastigote assays. The effect of silymarin on footpad swelling was detected when the combination of low-dose glucantime (20 mg/kg) with 25-50 mg/kg silymarin (especially 50 mg/kg) were used at day 22 of post infection (P<0.05). According to the parasite burden data, use of silymarin in the presence of different doses of glucantime, did not show significant effect compared to glucantime alone. The results of this study suggest that silymarin in conjunction with glucantime may have benefit effects in murine model of cutaneous leishmaniasis. © Georg Thieme Verlag KG Stuttgart · New York.

  14. Therapeutic Effect of Hedera helix Alcoholic Extract Against Cutaneous Leishmaniasis Caused by Leishmania major in Balb/c Mice

    PubMed Central

    Hooshyar, Hossein; Talari, Safarali; Feyzi, Fatemeh

    2014-01-01

    Background: Cutaneous leishmaniasis (CL) is common and endemic in many areas of Iran, caused by species of a protozoan parasite belonging to the genus Leishmania. There is not any effective vaccine against leishmaniasis; so, therapy is important for prevention and separation of disease. Herbal extract for treatment of CL is cost-effective, applicable topically to lesions, and can avoid the development of drug resistance. Objectives: The aim of this study was to evaluate the in vivo activity of an alcoholic extract of Hedera helix (a native Iranian plant) on the experimental ulcer of zoonotic CL in Balb/c mice. Materials and Methods: At least 5x l06 promastigotes of Leishmania major (MHOM/64/IR/ER75) were inoculated subcutaneously in the tail base of Balb/c mice. Fifty six infected mice were distributed in four groups, two groups (16 mice for 20% alcoholic extract of H. helix and 13 for 70% extract) were used as experimental groups, one (15 mice) as placebo control (Control A), and one (12 mice) as negative control. Treatment effects of two concentrations were determined by comparison of placebo and nontreated groups via measuring the size of skin lesions and the number of parasitologically positive and negative mice after the therapy period. Results: This study showed that the main lesion size did not decrease significantly, or the small lesions did not completely disappear after treatment by H. helix alcoholic extract. Amastigotes counts (mean ± SD) of the skin lesions decreased in control A and 20% concentration groups, but in negative control and 70% concentration groups the number of parasites did not reduce. Conclusions: The present study did not support the in vivo antileishmanial effect of H. helix extract. We recommend further studies using major components of H. helix, especially hederasaponin (saponin K10), to investigate the antileishmanial effect of this plant on L. major. PMID:25147703

  15. Structure of Leishmania major methionyl-tRNA synthetase in complex with intermediate products methionyladenylate and pyrophosphate.

    PubMed

    Larson, Eric T; Kim, Jessica E; Zucker, Frank H; Kelley, Angela; Mueller, Natascha; Napuli, Alberto J; Verlinde, Christophe L M J; Fan, Erkang; Buckner, Frederick S; Van Voorhis, Wesley C; Merritt, Ethan A; Hol, Wim G J

    2011-03-01

    Leishmania parasites cause two million new cases of leishmaniasis each year with several hundreds of millions of people at risk. Due to the paucity and shortcomings of available drugs, we have undertaken the crystal structure determination of a key enzyme from Leishmania major in hopes of creating a platform for the rational design of new therapeutics. Crystals of the catalytic core of methionyl-tRNA synthetase from L. major (LmMetRS) were obtained with the substrates MgATP and methionine present in the crystallization medium. These crystals yielded the 2.0 Å resolution structure of LmMetRS in complex with two products, methionyladenylate and pyrophosphate, along with a Mg(2+) ion that bridges them. This is the first class I aminoacyl-tRNA synthetase (aaRS) structure with pyrophosphate bound. The residues of the class I aaRS signature sequence motifs, KISKS and HIGH, make numerous contacts with the pyrophosphate. Substantial differences between the LmMetRS structure and previously reported complexes of Escherichia coli MetRS (EcMetRS) with analogs of the methionyladenylate intermediate product are observed, even though one of these analogs only differs by one atom from the intermediate. The source of these structural differences is attributed to the presence of the product pyrophosphate in LmMetRS. Analysis of the LmMetRS structure in light of the Aquifex aeolicus MetRS-tRNA(Met) complex shows that major rearrangements of multiple structural elements of enzyme and/or tRNA are required to allow the CCA acceptor triplet to reach the methionyladenylate intermediate in the active site. Comparison with sequences of human cytosolic and mitochondrial MetRS reveals interesting differences near the ATP- and methionine-binding regions of LmMetRS, suggesting that it should be possible to obtain compounds that selectively inhibit the parasite enzyme.

  16. Cutaneous Leishmaniasis (Leishmania major Infection) in Dutch Troops Deployed in Northern Afghanistan: Epidemiology, Clinical Aspects, and Treatment

    PubMed Central

    van Thiel, Pieter-Paul; Leenstra, Tjalling; de Vries, Henry J.; van der Sluis, Allard; van Gool, Tom; Krull, Alex C.; van Vugt, Michèle; de Vries, Peter J.; Zeegelaar, Jimmy E.; Bart, Aldert; van der Meide, Wendy F.; Schallig, Henk D. F. H.; Faber, William R.; Kager, Piet A.

    2010-01-01

    Cutaneous leishmaniasis caused by Leishmania major infection affected 172 (18.3%) of 938 Dutch military troops deployed in northern Afghanistan in 2005. The high attack rate was a result of initial insufficient availability of means of prevention and insufficient adherence to preventive measures. At presentation, the lymphatic system was involved in 24.8%. Treatment with intralesional injections of antimony with or without cryotherapy was satisfactory, but 19.5% of patients received secondary treatment with miltefosine. Six months after treatment, 128 (77.1%) of 166 treated patients were cured, 16 (9.6%) were lost to follow-up, and 22 (13.3%) already experienced cure at six weeks but were not seen at six months. Natural evolution played a role in this observational study, which showed cure of all patients seen at six months. In general, management of cutaneous leishmaniasis was feasible under field conditions. PMID:21118937

  17. Immunization against leishmaniasis by PLGA nanospheres encapsulated with autoclaved Leishmania major (ALM) and CpG-ODN.

    PubMed

    Tafaghodi, Mohsen; Khamesipour, Ali; Jaafari, Mahmoud R

    2011-05-01

    Various adjuvants and delivery systems have been evaluated for increasing the protective immune responses against leishmaniasis and mostly have been shown not to be effective enough. In this study, poly(D,L-lactide-co-glycolide) (PLGA) nanospheres as an antigen delivery system and CpG-ODN as an immunoadjuvant have been used for the first time to enhance the immune response against autoclaved Leishmania major (ALM). PLGA nanospheres were prepared by a double-emulsion (W/O/W) technique. Particulate characteristics were studied by scanning electron microscopy and particle size analysis. Mean diameter of ALM + CpG-ODN-loaded nanospheres was 300 ± 128 nm. BALB/c mice were immunized three times in 3-week intervals using ALM plus CpG-ODN-loaded nanospheres [(ALM + CpG-ODN)(PLGA)], ALM encapsulated PLGA nanospheres [(ALM)(PLGA)], (ALM)(PLGA) + CpG, ALM + CpG, ALM alone, or phosphate buffer solution (PBS). The intensity of infection induced by L. major challenge was assessed by measuring size of footpad swelling. The strongest protection, showed by significantly (P<0.05) smaller footpad, was observed in mice immunized with (ALM + CpG-ODN)(PLGA). The (ALM)(PLGA), (ALM)(PLGA) + CpG, and ALM + CpG were also showed a significantly (P<0.05) smaller footpad swelling compared to the groups received either PBS or ALM alone. The mice immunized with (ALM + CpG-ODN)(PLGA), (ALM)(PLGA) + CpG, and ALM + CpG showed the highest IgG2a/IgG1 ratio, interferon-γ production, and lowest interleukin-4 production compared to the other groups. It is concluded that when both PLGA nanospheres and CpG-ODN adjuvants were used simultaneously, it induce stronger immune response and enhance protection rate against Leishmania infection.

  18. Development of an Ex Vivo Lymph Node Explant Model for Identification of Novel Molecules Active against Leishmania major

    PubMed Central

    Peniche, Alex G.; Osorio, Yaneth; Renslo, Adam R.; Frantz, Doug E.; Melby, Peter C.

    2014-01-01

    Leishmaniasis is a vector-borne zoonotic infection affecting people in tropical and subtropical regions of the world. Current treatments for cutaneous leishmaniasis are difficult to administer, toxic, expensive, and limited in effectiveness and availability. Here we describe the development and application of a medium-throughput screening approach to identify new drug candidates for cutaneous leishmaniasis using an ex vivo lymph node explant culture (ELEC) derived from the draining lymph nodes of Leishmania major-infected mice. The ELEC supported intracellular amastigote proliferation and contained lymph node cell populations (and their secreted products) that enabled the testing of compounds within a system that mimicked the immunopathological environment of the infected host, which is known to profoundly influence parasite replication, killing, and drug efficacy. The activity of known antileishmanial drugs in the ELEC system was similar to the activity measured in peritoneal macrophages infected in vitro with L. major. Using the ELEC system, we screened a collection of 334 compounds, some of which we had demonstrated previously to be active against L. donovani, and identified 119 hits, 85% of which were confirmed to be active by determination of the 50% effective concentration (EC50). We found 24 compounds (7%) that had an in vitro therapeutic index (IVTI; 50% cytotoxic/effective concentration [CC50]/EC50) > 100; 19 of the compounds had an EC50 below 1 μM. According to PubChem searchs, 17 of those compounds had not previously been reported to be active against Leishmania. We expect that this novel method will help to accelerate discovery of new drug candidates for treatment of cutaneous leishmaniasis. PMID:24126577

  19. Proteophosphoglycan confers resistance of Leishmania major to midgut digestive enzymes induced by blood feeding in vector sand flies

    PubMed Central

    Secundino, Nagila; Kimblin, Nicola; Peters, Nathan C.; Lawyer, Phillip; Capul, Althea A.; Beverley, Stephen M.; Turco, Salvatore J.; Sacks, David

    2010-01-01

    Summary Leishmania synthesize abundant phosphoglycan-containing molecules made up of [Gal-Man-PO4] repeating units, including the surface lipophosphoglycan (LPG), and the surface and secreted proteophosphoglycan (PPG). The vector competence of Phlebotomus duboscqi and Lutzomyia longipalpis sand flies was tested using L. major knockout mutants deficient in either total phosphoglycans (lpg2− or lpg5A−/5B−) or LPG alone (lpg1−) along with their respective gene add-back controls. Our results confirm that LPG, the major cell surface molecule of Leishmania promastigotes known to mediate attachment to the vector midgut, is necessary to prevent the loss of infection during excretion of the blood meal remnants from a natural vector, P. duboscqi, but not an unnatural vector, L. longipalpis. Midgut digestive enzymes induced by blood feeding pose another potential barrier to parasite survival. Our results show that 36–72 h after the infective feed, all parasites developed well except the lpg2− and lpg5A−/5B− mutants, which showed significantly reduced survival and growth. Protease inhibitors promoted the early survival and growth of lpg2− in the blood meal. PPG was shown to be the key molecule conferring resistance to midgut digestive enzymes, as it prevented killing of lpg2− promastigotes exposed to midgut lysates prepared from blood-fed flies. The protection was not associated with inhibition of enzyme activities, but with cell surface acquisition of the PPG, which appears to function similar to mammalian mucins to protect the surface of developing promastigotes against proteolytic damage. PMID:20088949

  20. Inhibition of caspase-8 activity promotes protective Th1- and Th2-mediated immunity to Leishmania major infection.

    PubMed

    Pereira-Manfro, Wânia F; Ribeiro-Gomes, Flávia L; Filardy, Alessandra Almeida; Vellozo, Natália S; Guillermo, Landi V C; Silva, Elisabeth M; Siegel, Richard M; Dosreis, George A; Lopes, Marcela F

    2014-02-01

    We investigated how apoptosis pathways mediated by death receptors and caspase-8 affect cytokine responses and immunity to Leishmania major parasites. Splenic CD4 T cells undergo activation-induced apoptosis, and blockade of FasL-Fas interaction increased IFN-γ and IL-4 cytokine responses to L. major antigens. To block death receptor-induced death, we used mice expressing a T cell-restricted transgene for vFLIP. Inhibition of caspase-8 activation in vFLIP mice enhanced Th1 and Th2 cytokine responses to L. major infection, even in the Th1-prone B6 background. We also observed increased NO production by splenocytes from vFLIP mice upon T cell activation. Despite an exacerbated Th2 response, vFLIP mice controlled better L. major infection, with reduced lesions and lower parasite loads compared with WT mice. Moreover, injection of anti-IL-4 mAb in infected vFLIP mice disrupted control of parasite infection. Therefore, blockade of caspase-8 activity in T cells improves immunity to L. major infection by promoting increased Th1 and Th2 responses.

  1. A label-free, PCR-free and signal-on electrochemical DNA biosensor for Leishmania major based on gold nanoleaves.

    PubMed

    Moradi, M; Sattarahmady, N; Rahi, A; Hatam, G R; Sorkhabadi, S M Rezayat; Heli, H

    2016-12-01

    Detection of leishmaniasis is important in clinical diagnoses. In the present study, identification of Leishmania parasites was performed by a label-free, PCR-free and signal-on ultrasensitive electrochemical DNA biosensor. Gold nanoleaves were firstly electrodeposited by an electrodeposition method using spermidine as a shape directing agent. The biosensor was fabricated by immobilization of a Leishmania major specific DNA probe onto gold nanoleaves, and methylene blue was employed as a marker. Hybridization of the complementary single stranded DNA sequence with the biosensor under the selected conditions was then investigated. The biosensor could detect a synthetic DNA target in a range of 1.0×10(-10) to 1.0×10(-19)molL(-1) with a limit of detection of 1.8×10(-20)molL(-1), and genomic DNA in a range of 0.5-20ngμL(-1) with a limit of detection of 0.07ngμL(-1). The biosensor could distinguish Leishmania major from a non-complementary-sequence oligonucleotide and the tropica species with a high selectivity. The biosensor was applicable to detect Leishmania major in patient samples.

  2. Monoterpenic aldehydes as potential anti-Leishmania agents: activity of Cymbopogon citratus and citral on L. infantum, L. tropica and L. major.

    PubMed

    Machado, M; Pires, P; Dinis, A M; Santos-Rosa, M; Alves, V; Salgueiro, L; Cavaleiro, C; Sousa, M C

    2012-03-01

    In order to contribute for the search of new drugs for leishmaniasis, we study the susceptibility of Leishmania infantum, Leishmania tropica and Leishmania major to Cymbopogon citratus essential oil and major compounds, mrycene and citral. C. citratus and citral were the most active inhibiting L. infantum, L. tropica and L. major growth at IC(50) concentrations ranging from 25 to 52 μg/ml and from 34 to 42 μg/ml, respectively. L. infantum promastigotes exposed to essential oil and citral underwent considerable ultrastructural alterations, namely mitochondrial and kinetoplast swelling, autophagosomal structures, disruption of nuclear membrane and nuclear chromatin condensation. C. citratus essential oil and citral promoted the leishmanicidal effect by triggering a programmed cell death. In fact, the leishmanicidal activity was mediated via apoptosis as evidenced by externalization of phosphatidylserine, loss of mitochondrial membrane potential, and cell-cycle arrest at the G(0)/G(1) phase. Taken together, ours findings lead us to propose that citral was responsible for anti-Leishmania activity of the C. citratus and both may represent a valuable source for therapeutic control of leishmaniasis. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Crystallization and preliminary crystallographic characterization of LmACR2, an arsenate/antimonate reductase from Leishmania major

    SciTech Connect

    Bisacchi, Davide; Zhou, Yao; Rosen, Barry P.; Mukhopadhyay, Rita; Bordo, Domenico

    2006-10-01

    LmACR2 from L. major is the first rhodanese-like enzyme directly involved in the reduction of arsenate and antimonate to be crystallized. Diffraction data have been collected to 1.99 Å resolution using synchrotron X-rays. Arsenic is present in the biosphere owing either to the presence of pesticides and herbicides used in agricultural and industrial activities or to leaching from geological formations. The health effects of prolonged exposure to arsenic can be devastating and may lead to various forms of cancer. Antimony(V), which is chemically very similar to arsenic, is used instead in the treatment of leishmaniasis, an infection caused by the protozoan parasite Leishmania sp.; the reduction of pentavalent antimony contained in the drug Pentostam to the active trivalent form arises from the presence in the Leishmania genome of a gene, LmACR2, coding for the protein LmACR2 (14.5 kDa, 127 amino acids) that displays weak but significant sequence similarity to the catalytic domain of Cdc25 phosphatase and to rhodanese enzymes. For structural characterization, LmACR2 was overexpressed, purified to homogeneity and crystallized in a trigonal space group (P321 or P3{sub 1}21/P3{sub 2}21). The protein crystallized in two distinct trigonal crystal forms, with unit-cell parameters a = b = 111.0, c = 86.1 Å and a = b = 111.0, c = 175.6 Å, respectively. At a synchrotron beamline, the diffraction pattern extended to a resolution limit of 1.99 Å.

  4. TWO FUNCTIONALLY DIVERGENT UDP-GAL NUCLEOTIDE-SUGAR TRANSPORTERS PARTICIPATE IN PHOSPHOGLYCAN SYNTHESIS IN LEISHMANIA MAJOR

    PubMed Central

    Capul, Althea A.; Barron, Tamara; Dobson, Deborah E.; Turco, Salvatore J.; Beverley, Stephen M.

    2009-01-01

    In the protozoan parasite Leishmania, abundant surface and secreted molecules such as lipophosphoglycan (LPG)1 and proteophosphoglycans (PPGs) contain extensive galactose residues in the form of phosphoglycans (PGs) containing [Gal-Man-PO4] repeating units. PGs are synthesized in the parasite Golgi apparatus and require transport of cytoplasmic nucleotide-sugar precursors to the Golgi lumen by nucleotide sugar transporters (NSTs). GDP-Man transport is mediated by the LPG2 gene product, and here we focused on transporters for UDP-Gal. Database mining revealed twelve candidate NST genes in the L. major genome, including LPG2, as well as a candidate endoplasmic reticulum UDP-glucose transporter (HUT1L), and several pseudogenes. Gene knockout studies established that two genes (LPG5A and LPG5B) encoded UDP-Gal NSTs. While the single lpg5A− and lpg5B− mutants produced PGs, an lpg5A−/5B− double mutant was completely deficient. PG synthesis was restored in the lpg5A−/5B− mutant by heterologous expression of the human UDP-Gal transporter, and heterologous expression of LPG5A and LPG5B rescued the glycosylation defects of the mammalian Lec8 mutant, which is deficient in UDP-Gal uptake. Interestingly, the LPG5A and LPG5B functions overlap but are not equivalent, as the lpg5A− mutant showed a partial defect in LPG but not PPG phosphoglycosylation, while the lpg5B− mutant showed a partial defect in PPG but not LPG phosphoglycosylation. Identification of these key NSTs in Leishmania will facilitate the dissection of glycoconjugate synthesis and its role(s) in the parasite life cycle and further our understanding of NSTs generally. PMID:17347153

  5. The Contribution of the Fas/FasL Apoptotic Pathway in Ulcer Formation during Leishmania major-Induced Cutaneous Leishmaniasis

    PubMed Central

    Eidsmo, Liv; Nylen, Susanne; Khamesipour, Ali; Hedblad, Mari-Anne; Chiodi, Francesca; Akuffo, Hannah

    2005-01-01

    Cutaneous leishmaniasis (CL), caused by the intracellular protozoan Leishmania major, is characterized by lesion formation and ulceration at the site of infection. The mechanism of ulcer formation during CL is not fully understood. The expression of Fas and FasL and the levels of apoptosis in skin biopsies and in restimulated blood mononuclear cells from patients with 1 to 7 months of L. major-induced CL were analyzed using immunohistochemistry and fluorescence-activated cell sorting analysis. The levels of soluble Fas and FasL were also analyzed by enzyme-linked immunosorbent assay. A substantial number of apoptotic keratinocytes were observed mainly in the superficial epidermis of morphologically active and healing CL skin samples. Fas expression was increased on epidermis in active CL, whereas Fas expression was similar in healing and healthy epidermis. FasL-expressing macrophages and T cells were found in subepidermal infiltrate, mainly in active disease. When CL peripheral blood mononuclear cells were restimulated with L. major, Fas was up-regulated on effector T cells, and high levels of sFasL were secreted. Supernatants from restimulated cultures induced apoptosis in human keratinocytes (HaCaT), possibly through Fas/FasL interactions. Our results indicate that FasL-expressing effector T cells and macrophages may act to induce apoptosis and ulcer formation in Fas-expressing keratinocytes during L. major infection. PMID:15793290

  6. Qualitative differences in the early immune response to live and killed Leishmania major: Implications for vaccination strategies against Leishmaniasis.

    PubMed

    Okwor, Ifeoma; Liu, Dong; Uzonna, Jude

    2009-04-28

    Recovery from natural or deliberate infection with Leishmania major leads to the development of lifelong immunity against rechallenge infections. In contrast, vaccination with killed parasites or defined leishmanial antigens generally induces only short-term protection. The reasons for this difference are currently not known but may be related to differences in the quality of the early immune responses to live and killed parasites. Here, we report that live and killed L. major parasites elicit comparable early inflammatory response as evidenced by influx and/or proliferation of cells in the draining lymph nodes (dLNs). In contrast, the early cytokine responses were qualitatively different. Cells from mice inoculated with killed parasites produced significantly more antigen-specific IL-4 and less IFN-gamma than those from mice injected with live parasites. Inclusion of CpG ODN into killed parasite preparations changed the early response to killed parasites from IL-4 to a predominantly IFN-gamma response, resulting in better protection following secondary high dose virulent L. major challenge. Interestingly, CpG-mediated enhancement of killed parasites-induced protection was short-lived and waned after 12 weeks. Taken together, these results suggest that the nature of primary immunity induced by killed and live parasites are qualitatively different and that these differences may account for the differential protection seen in mice following vaccination with live and killed parasites. They further suggest that modulating the early response with an appropriate adjuvant could enhance efficacy of killed parasite vaccines.

  7. Magnesium oxide nanoparticles coated with glucose can silence important genes of Leishmania major at sub-toxic concentrations.

    PubMed

    Bafghi, Ali Fatahi; Daghighi, Mojtaba; Daliri, Karim; Jebali, Ali

    2015-12-01

    The aim of this study was to investigate the effect of magnesium oxide nanoparticles (MgO NPs) and MgO NPs coated with glucose (MONPCG) on Leishmania (L) major. First, the promastigotes of L. major were separately incubated with serial concentrations of MgO NPs and MONPCG for 24, 48, and 72 h at 37 °C. Then, the cell viability of promastigotes was evaluated by MTT assay. On the other hand, the relative expression of Cpb and GP63 genes was detected by quantitative-real time PCR. Based on results, the increase of concentration, both MgO NPs and MONPCG, and incubation time led to decrease of cell viability. Moreover, the expression of Cpb and GP63 genes was decreased with increase of concentration of MgO NPs and MONPCG. Also, the increase of incubation time led to decrease of their expression in MgO NPs treated promastogotes. But, in case of MONPCG treated promastogotes, the increase of incubation time did not change the expression of Cpb and GP63. Interestingly, MONPCG could silence Cpb and GP63 genes better than MgO NPs. Note, the capability was also seen at sub-toxic concentrations of MONPCG.

  8. Inbred Strains Derived from Feral Mice Reveal New Pathogenic Mechanisms of Experimental Leishmaniasis Due to Leishmania major

    PubMed Central

    Babay, Besma E. C.; Louzir, Hechmi; Kebaïer, Chahnaz; Boubaker, Samir; Dellagi, Koussay; Cazenave, Pierre-André

    2004-01-01

    Two inbred mouse strains, derived from feral founders, are susceptible to experimental leishmaniasis due to Leishmania major and support a disease of a severity intermediate between those observed in strains C57BL/6 and BALB/c. Mice of the MAI strain develop a severe, nonhealing, but nonfatal disease with no resistance to a secondary parasite challenge. The immunological responses showed a TH2 dominance characterized by an early peak of interleukin-4 (IL-4) and IL-13. However, neutralization of IL-4, which leads to a resistance phenotype in BALB/c mice, has no effect on disease progression in MAI mice. Mice of strain PWK develop a protracted but self-healing disease, characterized by a mixed TH1-plus-TH2 pattern of immune responses in which IL-10 plays an aggravating role, and acquire resistance to a secondary challenge. These features are close to those observed in human cutaneous leishmaniasis due to L. major and make PWK mice a suitable model for the human disease. PMID:15271920

  9. Inbred strains derived from feral mice reveal new pathogenic mechanisms of experimental leishmaniasis due to Leishmania major.

    PubMed

    Babay, Besma E C; Louzir, Hechmi; Kebaïer, Chahnaz; Boubaker, Samir; Dellagi, Koussay; Cazenave, Pierre-André

    2004-08-01

    Two inbred mouse strains, derived from feral founders, are susceptible to experimental leishmaniasis due to Leishmania major and support a disease of a severity intermediate between those observed in strains C57BL/6 and BALB/c. Mice of the MAI strain develop a severe, nonhealing, but nonfatal disease with no resistance to a secondary parasite challenge. The immunological responses showed a TH2 dominance characterized by an early peak of interleukin-4 (IL-4) and IL-13. However, neutralization of IL-4, which leads to a resistance phenotype in BALB/c mice, has no effect on disease progression in MAI mice. Mice of strain PWK develop a protracted but self-healing disease, characterized by a mixed TH1-plus-TH2 pattern of immune responses in which IL-10 plays an aggravating role, and acquire resistance to a secondary challenge. These features are close to those observed in human cutaneous leishmaniasis due to L. major and make PWK mice a suitable model for the human disease.

  10. Therapeutic Effect of Scrophularia striata Ethanolic Extract against Localized Cutaneous Leishmaniasis Caused by Leishmania major (MRHO/IR/75/ER)

    PubMed Central

    ZAHIRI, Malihe; MOHEBALI, Mehdi; KHANAVI, Mahnaz; SAHEBGHARANI, Mousa; SAGHAFIPOUR, Abedin; ESMAEILI, Jamileh; HAJJARAN, Homa; AKHAVAN, Amir Ahmad; REZAYAT, Seyed Mahdi

    2016-01-01

    Background: We evaluated the effect of the ethanolic extract of Scrophularia striata on the Iranian strain of Leishmania major (MRHO/IR/75/ER) both in vitro and in vivo conditions Methods: The effective dose (ED) of ethanolic extract of S. striata were determined using MTT assay on the growth of promastigote forms of L. major in axenic culture media. Then, the ED50 of S. striata on mice peritoneal macrophages was determined using calculation of amastigote forms on mice peritoneal macrophages. For in vivo experiments, the therapeutic effects of various concentrations of S. striata on infected BALB/c mice was studied. A total of 75 infected mice were randomly divided into five groups: two groups (10% and 50% of S. striata) as experimental and three as control (ethanol 50%, Glucantime® and no treatment). The efficacy were determined by comparing the diameters of lesions and the microscopically examinations. Results: The effect of S. striata extract (0/625%, 1/25%, 2/5%, 5%, 10%, 20% and 50%) on peritoneal macrophages of Balb/c mice infected with L.major in tissue-culture slides was assessed. S. striata extract (10%) removed the L.major amastigotes-infected macrophages significantly after 24 h (P < 0.05). The higher concentrations of S. striata ethanolic extract (20%, and 50%) had highly toxic effects on macrophages, resulted in the disintegration of the cytoplasm of macrophages after 48 and 72 h. In concentration 10% of S. striata, more than 85% of L. major amastigotes-infected macrophages were damaged without cytotoxicity effects on macrophages. The higher concentrations had toxic effects on cultured macrophages. Conclusion: S. striata ethanolic extract 10% had anti leishmanial effects in both in vivo and in vitro. PMID:27957441

  11. Replication Attempt: “Effect of BMAP-28 Antimicrobial Peptides on Leishmania Major Promastigote and Amastigote Growth: Role of Leishmanolysin in Parasite Survival”

    PubMed Central

    Iorns, Elizabeth; Gunn, William; Erath, Jessey; Rodriguez, Ana; Zhou, Jian; Benzinou, Michael

    2014-01-01

    This study describes an attempt to replicate experiments from the paper “Effect of BMAP-28 Antimicrobial Peptides on Leishmania major Promastigote and Amastigote Growth: Role of Leishmanolysin in Parasite Survival,” which was submitted to the Reproducibility Initiative for independent validation. The cathelicidin bovine myeloid antimicrobial peptide 28 (BMAP-28) and its isomers were previously shown to have potent antiparasitic activity against Leishmania major. We tested the effectiveness of L-BMAP-28 and two of its isomers, the D-amino acid form (D-BMAP-28) and the retro-inverso form (RI-BMAP-28), in both unamidated and amidated forms, as anti-leishmanial agents against Leishmania major promastigotes in vitro. We observed that L-BMAP-28, as well as its D and RI isomers, demonstrate anti-leishmanial activity against L. major promastigotes in vitro. The inhibitory effect was lower than what was seen in the original study. At 2 µM of amidated peptides, the viability was 94%, 36%, and 66% with L-, D- and RI-peptides, versus 57%, 6%, and 18% in the original study. PMID:25517992

  12. Skin-resident CD4+ T cells protect against Leishmania major by recruiting and activating inflammatory monocytes.

    PubMed

    Glennie, Nelson D; Volk, Susan W; Scott, Phillip

    2017-04-01

    Tissue-resident memory T cells are required for establishing protective immunity against a variety of different pathogens, although the mechanisms mediating protection by CD4+ resident memory T cells are still being defined. In this study we addressed this issue with a population of protective skin-resident, IFNγ-producing CD4+ memory T cells generated following Leishmania major infection. We previously found that resident memory T cells recruit circulating effector T cells to enhance immunity. Here we show that resident memory CD4+ T cells mediate the delayed-hypersensitivity response observed in immune mice and provide protection without circulating T cells. This protection occurs rapidly after challenge, and requires the recruitment and activation of inflammatory monocytes, which limit parasites by production of both reactive oxygen species and nitric oxide. Overall, these data highlight a novel role for tissue-resident memory cells in recruiting and activating inflammatory monocytes, and underscore the central role that skin-resident T cells play in immunity to cutaneous leishmaniasis.

  13. Optimization of the Timing of Induction for the Assessment of Nitric Oxide Production in Leishmania major Infected Macrophage Cells

    PubMed Central

    SADEGHI, Somaye; SEYED, Negar; RAFATI, Sima; TAHERI, Tahereh

    2016-01-01

    Background: The present study was conducted to investigate the optimized timing for macrophages induction and nitric oxide (NO) production against invading Leishmania parasite. Methods: The present study examined the murine macrophage cell line, B10R, in three different states. In the first state, the cells were first infected with L. major and then treated with IFN-γ and LPS as stimulants. In the second state, the cells were infected after stimulation with IFN-γ and LPS. In the third state, the cells were only exposed to stimulants as controls. In all the three states, cell culture supernatants were collected at three points in time (6, 24 and 48 h) and the amount of NO production was measured using Griess assay. Results: The treatment of macrophages with inducers prior to infection with stationary phase parasite led to the secretion of significant amounts of NO, particularly at early time points quit contrary to the cells infected with parasites prior to induction. The amount of NO produced by cells induced after infection was detected significantly lower. Conclusion: The induction of macrophages prior to infection with parasites leads to the production and secretion of greater amounts of NO, resulting in an increased ability to suppress and inhibit parasite proliferation even in the early stages of infection. PMID:28127337

  14. Immunization with a Polyprotein Vaccine Consisting of the T-Cell Antigens Thiol-Specific Antioxidant, Leishmania major Stress-Inducible Protein 1, and Leishmania Elongation Initiation Factor Protects against Leishmaniasis

    PubMed Central

    Coler, Rhea N.; Skeiky, Yasir A. W.; Bernards, Karen; Greeson, Kay; Carter, Darrick; Cornellison, Charisa D.; Modabber, Farrokh; Campos-Neto, Antonio; Reed, Steven G.

    2002-01-01

    Development of an effective vaccine against Leishmania infection is a priority of tropical disease research. We have recently demonstrated protection against Leishmania major in the murine and nonhuman primate models with individual or combinations of purified leishmanial recombinant antigens delivered as plasmid DNA constructs or formulated with recombinant interleukin-12 (IL-12) as adjuvant. In the present study, we immunized BALB/c mice with a recombinant polyprotein comprising a tandem fusion of the leishmanial antigens thiol-specific antioxidant, L. major stress-inducible protein 1 (LmSTI1), and Leishmania elongation initiation factor (LeIF) delivered with adjuvants suitable for human use. Aspects of the safety, immunogenicity, and vaccine efficacy of formulations with each individual component, as well as the polyprotein referred to as Leish-111f, were assessed by using the L. major challenge model with BALB/c mice. No adverse reactions were observed when three subcutaneous injections of the Leish-111f polyprotein formulated with either MPL-squalene (SE) or Ribi 529-SE were given to BALB/c mice. A predominant Th1 immune response characterized by in vitro lymphocyte proliferation, gamma interferon production, and immunoglobulin G2A antibodies was observed with little, if any, IL-4. Moreover, Leish-111f formulated with MPL-SE conferred immunity to leishmaniasis for at least 3 months. These data demonstrate success at designing and developing a prophylactic leishmaniasis vaccine that proved effective in a preclinical model using multiple leishmanial antigens produced as a single protein delivered with a powerful Th1 adjuvant suitable for human use. PMID:12117930

  15. Immunization with a polyprotein vaccine consisting of the T-Cell antigens thiol-specific antioxidant, Leishmania major stress-inducible protein 1, and Leishmania elongation initiation factor protects against leishmaniasis.

    PubMed

    Coler, Rhea N; Skeiky, Yasir A W; Bernards, Karen; Greeson, Kay; Carter, Darrick; Cornellison, Charisa D; Modabber, Farrokh; Campos-Neto, Antonio; Reed, Steven G

    2002-08-01

    Development of an effective vaccine against Leishmania infection is a priority of tropical disease research. We have recently demonstrated protection against Leishmania major in the murine and nonhuman primate models with individual or combinations of purified leishmanial recombinant antigens delivered as plasmid DNA constructs or formulated with recombinant interleukin-12 (IL-12) as adjuvant. In the present study, we immunized BALB/c mice with a recombinant polyprotein comprising a tandem fusion of the leishmanial antigens thiol-specific antioxidant, L. major stress-inducible protein 1 (LmSTI1), and Leishmania elongation initiation factor (LeIF) delivered with adjuvants suitable for human use. Aspects of the safety, immunogenicity, and vaccine efficacy of formulations with each individual component, as well as the polyprotein referred to as Leish-111f, were assessed by using the L. major challenge model with BALB/c mice. No adverse reactions were observed when three subcutaneous injections of the Leish-111f polyprotein formulated with either MPL-squalene (SE) or Ribi 529-SE were given to BALB/c mice. A predominant Th1 immune response characterized by in vitro lymphocyte proliferation, gamma interferon production, and immunoglobulin G2A antibodies was observed with little, if any, IL-4. Moreover, Leish-111f formulated with MPL-SE conferred immunity to leishmaniasis for at least 3 months. These data demonstrate success at designing and developing a prophylactic leishmaniasis vaccine that proved effective in a preclinical model using multiple leishmanial antigens produced as a single protein delivered with a powerful Th1 adjuvant suitable for human use.

  16. Leishmania major metacaspase can replace yeast metacaspase in programmed cell death and has arginine-specific cysteine peptidase activity.

    PubMed

    González, Iveth J; Desponds, Chantal; Schaff, Cédric; Mottram, Jeremy C; Fasel, Nicolas

    2007-02-01

    The human protozoan parasite Leishmania major has been shown to exhibit several morphological and biochemical features characteristic of a cell death program when differentiating into infectious stages and under a variety of stress conditions. Although some caspase-like peptidase activity has been reported in dying parasites, no caspase gene is present in the genome. However, a single metacaspase gene is present in L. major whose encoded protein harbors the predicted secondary structure and the catalytic dyad histidine/cysteine described for caspases and other metacaspases identified in plants and yeast. The Saccharomyces cerevisiae metacaspase YCA1 has been implicated in the death of aging cells, cells defective in some biological functions, and cells exposed to different environmental stresses. In this study, we describe the functional heterologous complementation of a S. cerevisiae yca1 null mutant with the L. major metacaspase (LmjMCA) in cell death induced by oxidative stress. We show that LmjMCA is involved in yeast cell death, similar to YCA1, and that this function depends on its catalytic activity. LmjMCA was found to be auto-processed as occurs for caspases, however LmjMCA did not exhibit any activity with caspase substrates. In contrast and similarly to Arabidopsis thaliana metacaspases, LmjMCA was active towards substrates with arginine in the P1 position, with the activity being abolished following H147A and C202A catalytic site mutations. These results suggest that metacaspases are members of a family of peptidases with a role in cell death conserved in evolution notwithstanding possible differences in their catalytic activity.

  17. Structural and thermodynamic basis of the inhibition of Leishmania major farnesyl diphosphate synthase by nitrogen-containing bisphosphonates

    SciTech Connect

    Aripirala, Srinivas; Gonzalez-Pacanowska, Dolores; Oldfield, Eric; Kaiser, Marcel; Amzel, L. Mario; Gabelli, Sandra B.

    2014-03-01

    Structural insights into L. major farnesyl diphosphate synthase, a key enzyme in the mevalonate pathway, are described. Farnesyl diphosphate synthase (FPPS) is an essential enzyme involved in the biosynthesis of sterols (cholesterol in humans and ergosterol in yeasts, fungi and trypanosomatid parasites) as well as in protein prenylation. It is inhibited by bisphosphonates, a class of drugs used in humans to treat diverse bone-related diseases. The development of bisphosphonates as antiparasitic compounds targeting ergosterol biosynthesis has become an important route for therapeutic intervention. Here, the X-ray crystallographic structures of complexes of FPPS from Leishmania major (the causative agent of cutaneous leishmaniasis) with three bisphosphonates determined at resolutions of 1.8, 1.9 and 2.3 Å are reported. Two of the inhibitors, 1-(2-hydroxy-2,2-diphosphonoethyl)-3-phenylpyridinium (300B) and 3-butyl-1-(2,2-diphosphonoethyl)pyridinium (476A), co-crystallize with the homoallylic substrate isopentenyl diphosphate (IPP) and three Ca{sup 2+} ions. A third inhibitor, 3-fluoro-1-(2-hydroxy-2,2-diphosphonoethyl)pyridinium (46I), was found to bind two Mg{sup 2+} ions but not IPP. Calorimetric studies showed that binding of the inhibitors is entropically driven. Comparison of the structures of L. major FPPS (LmFPPS) and human FPPS provides new information for the design of bisphosphonates that will be more specific for inhibition of LmFPPS. The asymmetric structure of the LmFPPS–46I homodimer indicates that binding of the allylic substrate to both monomers of the dimer results in an asymmetric dimer with one open and one closed homoallylic site. It is proposed that IPP first binds to the open site, which then closes, opening the site on the other monomer, which closes after binding the second IPP, leading to the symmetric fully occupied FPPS dimer observed in other structures.

  18. Anti-Leishmanial Activity (In Vitro and In Vivo) of Allicin and Allicin Cream Using Leishmania major (Sub-strain Zymowme LON4) and Balb/c Mice

    PubMed Central

    Metwally, Dina M.; Al-Olayan, Ebtesam M.; El-Khadragy, Manal F.; Alkathiri, Badriah

    2016-01-01

    Background Leishmania is a unicellular protozoan parasite that produces several human diseases, ranging from localized self-healing cutaneous lesions to deadly visceral infections. Objective The effect of allicin on the growth of Leishmania major (L. major) promastigotes was evaluated under in vitro conditions. Moreover, the efficacy of a topical allicin cream was examined in BALB/c (Bagg albino, laboratory-bred strain of the House Mouse) mice with cutaneous leishmanial lesions compared to the currently used drug, sodiumstibogluconate (pentostam). Methods Cytotoxiciy and promastigote proliferation were measured. Different concentrations (50, 100, 150, and 200 μM) of liquid allicin were tested on L. major promastigotes twice: after 24 and 48 hours using an MTT colorimetric assay. In the in vivo condition, the efficacies of allicin cream and liquid allicin at two concentrations (0.15 μM/mouse and 0.30 μM/mouse) were evaluated. Serum factors of the control and treated groups were tested to evaluate the toxic effects of allicin on the liver and kidney. Results Allicin at a concentration of 50 μM inhibited the growth of Leishmania promastigotes. Topical application of allicin cream reduced lesion sizes in mice. No significant differences in biochemical analysis were observed between the control and treated groups. Conclusions Allicin has antileishmanial effects under in vitro and in vivo conditions and may be used in clinical applications. PMID:27537199

  19. Detection of Leishmania major DNA within wild caught Phlebotomus papatasi and species composition of sand flies in endemic focus of cutaneous leishmaniasis, in western Iran.

    PubMed

    Vahabi, A; Rassi, Y; Oshaghi, M A; Sayyadi, M; Rafizadeh, S

    2016-03-01

    Cutaneous leishmaniasis is one of the most important public health problem in many developing countries. The present study was conducted to determine the vector(s), the parasite and the species composition of sand flies in the Dehloran County during May-November 2012. Sand flies were collected by sticky traps and mounted in Puri's medium for species identification. Polymerase chain reaction (PCR) techniques of kDNA, ITS1-rDNA, followed by restriction fragment length polymorphism (RFLP) were used for identification of DNA of Leishmania parasites in infected sand flies. A total of 82443 specimens comprising 15 species of sand flies (5 Phlebotomus and 10 Sergentomyia) were collected and identified. The species of Phlebotomus papatasi was dominant in outdoor and indoor resting places. Among the 280 specimens of female P. papatasi tested by PCR of kDNA, ITS1-rDNA genes of the parasite followed by RFLP, only 5 of them (1.8 %) were positive to Leishmania major parasites. This is the first molecular detection of leishmania infection of P. papatasi to L. major in this region. The results indicated that, P. papatasi was only species found infected by L. major and the principal vector of disease agent to human.

  20. ABCI3, a new mitochondrial ABC transporter from Leishmania major involved in susceptibility to antimonials and infectivity.

    PubMed

    Arcari, Talia; Manzano, José Ignacio; Gamarro, Francisco

    2017-10-02

    We have identified and characterized ABCI3 as a new mitochondrial ABC transporter from Leishmania major. Localization studies using confocal microscope, surface biotinylation assay and trypsin digestion after digitonin permeabilization suggested that ABCI3 presents a dual localization in both mitochondria and plasma membrane. Using single-knock out parasites for ABCI3 (ABCI3(+/-) ), we provided evidence that ABCI3 is directly involved in Sb(III) and metal ions susceptibility. Attempts to obtain double knock-out parasites for ABCI3 were unsuccessful, suggesting that ABCI3 could be an essential gene in L. majorABCI3(+/-) promastigotes were 5-fold more resistant to Sb(III), while ABCI3(+/-) amastigotes were approximately 2-fold more resistant to Sb(V) This resistant phenotype was associated with a decreased Sb(III) accumulation due to a decreased Sb(III) uptake. ABCI3(+/-) parasites presented higher ATP levels and generated less mitochondrial superoxide after Sb(III) incubation. Finally, we have observed that ABCI3(+/-) parasites showed a slightly higher infection capacity than wild-type and add-back ABCI3(+/-):3xFABCI3 parasites; however, after 72 h the number of ABC3(+/-) intracellular parasites per macrophage increased significantly. Our results show that ABCI3 is responsible for Sb(III) transport inside mitochondria, where it would contribute to enhance the general toxic effects caused by Sb(III) To our knowledge, ABCI3 is the first ABC transporter which is involved in susceptibility towards antimony, conferring Sb(III) resistance to parasites when partially deleted. Copyright © 2017 American Society for Microbiology.

  1. Evaluation of Apoptotic and Antileishmanial Activities of Artemisinin on Promastigotes and BALB/C Mice Infected with Leishmania major

    PubMed Central

    GHAFFARIFAR, Fatemeh; ESAVAND HEYDARI, Farzad; DALIMI, Abdolhosein; HASSAN, Zuhair M.; DELAVARI, Mahdi; MIKAEILOO, Hajar

    2015-01-01

    Background: In leishmaniasis, some drugs prescribed for treatment have toxic effects and there are reports about drug resistance in some countries. Due to this fact, using herbal drugs such as artemisinin with good efficacy and low toxic effect might be suitable. Methods: We evaluated the apoptotic effect of artemisinin on Leishmania major in vitro and the antileishmanial activities of artemisinin on leishmaniasis in BALB/c mice and at the end INF-γ and IL-4 cytokines levels were detected by ELISA in spleen cell culture supernatants. During treatment the lesion size and survival rate were measured each four and ten days, respectively. Results: Percentage of early and late apoptosis in promastigotes of control group and promastigotes treated with 10, 25, 50 and 100 μg/ml of artemisinin after 48 h were 0.13, 16.04, 41.23, 49.03 and 81.83, respectively. The IFN-γ in ointment treated group were higher than those of other groups (P<0.05). The in vivo results showed that ointment compounds healed the lesions more effectively rather than intraperitoneal injection method (P<0.05). The survival rate of mice 150 days after challenge in treated group with ointment of artemisinin was 66% while all mice in control groups were died. Conclusion: All of in vitro results represented that this drug had antileishmanial effects and these results were confirmed by evaluation effects in vivo condition of leishmaniasis. Interestingly, according to these results it can be concluded that this drug has antileishmanial effects in vitro and in vivo conditions. Artemisinin induces cytotoxic effect on L. major via apoptosis-related mechanism. PMID:26246824

  2. Mesenchymal stem cells alter macrophage immune responses to Leishmania major infection in both susceptible and resistance mice.

    PubMed

    Dameshghi, Safura; Zavaran-Hosseini, Ahmad; Soudi, Sara; Shirazi, Fatemeh Jalali; Nojehdehi, Shahrzad; Hashemi, Seyed Mahmoud

    2016-02-01

    Mesenchymal stem cells (MSCs) are attracted to inflammation site and switch immune system to modulate inflammatory responses. This ability makes MSCs the best candidate cells for stem cell therapy of infection diseases. Therefore, we aimed to evaluate the modulatory effect of adipose-derived MSCs (AD-MSCs) on macrophages in Leishmania (L.) major infection. Macrophages and MSCs were isolated from both susceptible (BALB/c) and resistance (C57BL/6) strains. After co-culture of AD-MSCs with macrophages using a transwell system, we assessed MSCs-educated macrophage responses to L. major infection. Our results indicated suppression in levels of tumor necrosis factor α (TNF-α) and interleukin 10 (IL-10) of MSCs co-cultured macrophages in response to L. major infection. To clarify the effects of this suppression on inflammatory conditions, TNF-α/IL-10 ratio was calculated, indicating an increase in TNF-α/IL-10 ratio in MSCs co-cultured groups. The higher TNF-α/IL-10 ratio was observed in BALB/c macrophages co-cultured with BALB/c MSCs. Nitric oxide (NO) assay presented a significant reduction in the supernatant of all MSCs co-cultured groups compared to control. We observed a significant reduction in phagocytosis of MSCs co-cultured groups in response to L. major infection without any significant differences in the phagocytic index. In conclusion, our results represented a new spectrum of immunomodulation induced by MSCs co-cultured with macrophages in response to L. major infection. The magnitude of immunoregulation was different between BALB/c and C57BL/6 strains. Our findings also showed that MSCs exerted potential effect of M1 polarization due to unequal decrease in levels of TNF-α and IL-10 when we considered TNF-α and IL-10as representatives of M1 and M2 phenotypes, respectively. Induction of inflammatory cytokine milieu and reduction in level of IL-10 provides a new hope for stem cell therapy of leishmaniasis in susceptible models.

  3. Isolation of new monoterpene coumarins from Micromelum minutum leaves and their cytotoxic activity against Leishmania major and cancer cells.

    PubMed

    Sakunpak, A; Matsunami, K; Otsuka, H; Panichayupakaranant, P

    2013-08-15

    On the basis of a leishmanicidal assay-guided isolation, two new monoterpene coumarins, minutin A and minutin B, were purified from Micromelum minutum leaves together with four known coumarins, 8,4″-dihydroxy-3″,4″-dihydrocapnolactone-2',3'-diol, 8-hydroxyisocapnolactone-2',3'-diol, 8-hydroxy-3″,4″-dihydrocapnolactone-2',3'-diol, and clauslactone E. Among these compounds, minutin A, minutin B, 8-hydroxyisocapnolactone-2',3'-diol and clauslactone E showed a significant cytotoxic activity against Leishmania major with IC50 values of 26.2, 20.2, 12.1, and 9.8 μM, respectively, while 8,4″-dihydroxy-3″4″-dihydrocapnolactone-2',3'-diol and 8-hydroxy-3″,4″-dihydrocapnolactone-2',3'-diol were not active. However, all these compounds exhibited some inhibitory activity against one or more lung adenocarcinoma (SBC3 and A549) and leukaemia (K562, and K562/ADM) cell lines. Amongst these, clauslactone E, minutin B and 8-hydroxyisocapnolactone-2',3'-diol possessed the strongest cytotoxic activity against SBC3, A549, K562, and K562/ADM cell lines, with IC50 values of 3.7, 10.4, 12.1, and 10.8 μM for clauslactone E; 9.6, 17.5, 8.7 and 6.7 μM for minutin B; 8.8, 10.1, 16.9, and 10.1 μM for 8-hydroxyisocapnolactone-2',3'-diol, respectively.

  4. Crystal Structure of Leishmania major Oligopeptidase B Gives Insight into the Enzymatic Properties of a Trypanosomatid Virulence Factor*

    PubMed Central

    McLuskey, Karen; Paterson, Neil G.; Bland, Nicholas D.; Isaacs, Neil W.; Mottram, Jeremy C.

    2010-01-01

    Oligopeptidase B (OPB) is a serine peptidase with dibasic substrate specificity. It is found in bacteria, plants, and trypanosomatid pathogens, where it has been identified as a virulence factor and potential drug target. In this study we expressed active recombinant Leishmania major OPB and provide the first structure of an oligopeptidase B at high resolution. The crystallographic study reveals that OPB comprises two domains, a catalytic and a propeller domain, linked together by a hinge region. The structure has been determined in complex with the oligopeptide, protease-inhibitor antipain, giving detailed information on the enzyme active site and extended substrate binding pockets. It shows that Glu-621 plays a critical role in the S1 binding pocket and, along with Phe-603, is largely responsible for the enzyme substrate specificity in P1. In the S2 binding pocket, Tyr-499 was shown to be important for substrate stability. The structure also allowed an investigation into the function of residues highlighted in other studies including Glu-623, which was predicted to be involved in the S1 binding pocket but is found forming an inter-domain hydrogen bond. Additional important salt bridges/hydrogen bonds between the two domains were observed, highlighting the significance of the domain interface in OPB. This work provides a foundation for the study of the role of OPBs as virulence factors in trypanosomatids. It could facilitate the development of specific OPB inhibitors with therapeutic potential by exploiting its unique substrate recognition properties as well as providing a model for OPBs in general. PMID:20926390

  5. Antileishmanial activity of Ferula assa-foetida oleo gum resin against Leishmania major: An in vitro study.

    PubMed

    Bafghi, Ali Fatahi; Bagheri, Seyyed Majid; Hejazian, Seyed Hassan

    2014-01-01

    In Ayurveda, asafetida is introduced as a valuable remedy for flatulence, hysteria, nervous disorders, whooping cough, pneumonia and bronchitis in children and also considered as an aphrodisiac agent. Presently, Leishmaniasis is common in most countries of the world and is a serious health problem in the world. Some plant medicines and natural products have a new candidate for treatment of leishmaniasis. This study was designed to evaluate Ferula assa-foetida oleo gum resin (asafetida) on mortality and morbidity Leishmania major in vitro. Mostigotes were isolated from mice spleens and then transformed to promastigotes in Novy-Nicolle-Mac Neal (NNN medium supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml) and 20% heat-inactivated fetal calf serum (FCS) at 25°C. A fixed initial density of the parasites was transferred to screw-capped vials containing 5 ml of RPMI1640 media to which different concentrations of 2.5, 5, 10 and 20 μg asafetida were added and each concentration was done in triplicates. Each run also included control. The mortality of parasitoids was measured by the slide and the enzyme-linked immunosorbent assay (ELISA) methods. After 72 h, asafetida inhibited growth of parasites in all doses in stationary and logarithmic phases. The ELISA measurement suggested that the viability of parasites significantly decreased after 48h (P < 0.05). The results show that asafetida could prevent from growth and viability of parasites and this oleo gum resin can be useful for treatment of leishmaniasis.

  6. Antileishmanial activity of Ferula assa-foetida oleo gum resin against Leishmania major: An in vitro study

    PubMed Central

    Bafghi, Ali Fatahi; Bagheri, Seyyed Majid; Hejazian, Seyed Hassan

    2014-01-01

    Background: In Ayurveda, asafetida is introduced as a valuable remedy for flatulence, hysteria, nervous disorders, whooping cough, pneumonia and bronchitis in children and also considered as an aphrodisiac agent. Presently, Leishmaniasis is common in most countries of the world and is a serious health problem in the world. Some plant medicines and natural products have a new candidate for treatment of leishmaniasis. Objective: This study was designed to evaluate Ferula assa-foetida oleo gum resin (asafetida) on mortality and morbidity Leishmania major in vitro. Materials and Methods: Mostigotes were isolated from mice spleens and then transformed to promastigotes in Novy-Nicolle-Mac Neal (NNN medium supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml) and 20% heat-inactivated fetal calf serum (FCS) at 25°C. A fixed initial density of the parasites was transferred to screw-capped vials containing 5 ml of RPMI1640 media to which different concentrations of 2.5, 5, 10 and 20 μg asafetida were added and each concentration was done in triplicates. Each run also included control. The mortality of parasitoids was measured by the slide and the enzyme-linked immunosorbent assay (ELISA) methods. Results: After 72 h, asafetida inhibited growth of parasites in all doses in stationary and logarithmic phases. The ELISA measurement suggested that the viability of parasites significantly decreased after 48h (P < 0.05). Conclusion: The results show that asafetida could prevent from growth and viability of parasites and this oleo gum resin can be useful for treatment of leishmaniasis. PMID:25624696

  7. H-11-linked gene has a parallel effect on Leishmania major and L. donovani infections in mice

    SciTech Connect

    Blackwell, J.M.; Hale, C.; Roberts, M.B.; Ulczak, O.M.; Liew, F.Y.; Howard, J.G.

    1985-01-01

    The courses of visceral infection following intravenous injection of Leishmania donovani amastigotes, or lesion growth following subcutaneous injection of L. major promastigotes, were examined in B10.129(10M) (H-2b, H-11b) mice and compared with disease profiles observed in congenic C57BL/10ScSn(= B10) (H-2b, H-11a) and B10.D2/n (H-2d, H-11a) mice, and in BALB/mice. Possession of alternative alleles at H-11 and closely linked loci transformed the normal curing/healing phenotype of B10 mice into a characteristically different noncuring/nonhealing phenotype affecting both visceral and subcutaneous infections in B10.129(10M) mice. In reciprocal radiation bone marrow chimeras made between the congenic B10 and B10.129(10M) strains, both cure and noncure phenotypes were transferable with the donor hematopoietic system. Although it was possible to demonstrate transfer of suppression with T-enriched spleen cells from day 61 L. donovani-infected B10.129(10M) donor mice into 550 rad syngeneic recipients, the pretreatment of mice with sublethal irradiation did not, as in the earlier studies of Scl-controlled L. major nonhealing or H-2-controlled L. donovani noncure phenotypes, have a clear or consistent prophylactic effect. Together with the progressive disease profile observed even for L. donovani at low parasite doses this suggests that, despite their ability to develop initial delayed-type hypersensitivity reactions to parasite antigen early in L. major infection, B10.129(10M) mice possess some inherent defect in ability to mount a cell-mediated response effective at the level of macrophage neishmanial activity in vivo even when suppressor T cells are not generated. Elucidation of this characteristically different noncuring/nonhealing phenotye may provide important insight into common events involved in the development of the cell-mediated immune response to both visceral and subcutaneous forms of leishmaniasis.

  8. Effect of trinitroglycerin therapy on serum zinc and copper levels and liver enzyme activities in BALB/c mice infected with Leishmania major MRHO/IR/75/ER

    PubMed Central

    Najafzade, Mana; Mosapour, Abbas; Nahrevanian, Hossein; Zamani, Zahra; Javadian, Seifoddin; Mirkhani, Fatemeh

    2015-01-01

    Objective(s): To evaluate the effect of trinitroglycerin (TNG) as nitric oxide donor agent on serum copper (Cu) and zinc (Zn) levels and liver enzymes in BALB/c mice infected with Leishmania major (L. major) MRHO/IR/75/ER. Materials and Methods: Inbred female mice were divided into three groups: healthy group (uninfected naive mice), control group (infected with L. major), and test group (L. major infected mice treated with TNG). TNG (200 µg/µl) was inoculated subcutaneously into the mice of the test group. Serum Cu and Zn levels and liver enzymes activities were then evaluated by atomic absorption spectrophometer and colorimetric methods, respectively. Results: Serum Cu levels were significantly higher in the test group than in the control and naive groups (P-value <0.05), while Zn levels were higher in the test group than in the control group with no significant difference. Serum glutamicoxaloacetic transaminase concentrations in the test group were significantly lower than those in other groups (P-value <0.05), while serum glutamate pyruvic transaminase concentrations were significantly higher in test compared with those in other groups (P-value <0.05). Moreover, alkaline phosphatase in the control and test groups were significantly lower than that in the naive group (P-value <0.05). Conclusion: TNG treatment increased Zn and Cu levels and thus increased resistance to Leishmania because of the role of Zn and Cu; therefore, TNG therapy will be useful for treating cutaneous leishmania. In addition, the decrease of serum glutamicoxaloacetic transaminase activity can be an index of therapeutic process of TNG. PMID:25945241

  9. First Report on Isolation and Characterization of Leishmania major from Meriones hurrianae (Rodentia: Gerbillidae) of A Rural Cutaneous leishmaniasis Focus in South-Eastern Iran

    PubMed Central

    Kassiri, Hamid; Naddaf, Saied Reza; Javadian, Ezat–Aldin; Mohebali, Mehdi

    2013-01-01

    Background Zoonotic Cutaneous Leishmaniasis (ZCL) is an endemic health problem in many rural areas of Iran, with doubled number of incidences over the last decade. Different species of rodents serve as natural reservoir host for ZCL. The disease is considered as a major health problem in rural areas of Mirjaveh, Chabahar, and Konarak Counties of Sistan va Baluchistan Province. Objectives This study describes the identity of Leishmania species, isolated from Meriones hurrianae from Chabahar County using RAPD-PCR methodology. Materials and Methods Rodents were entrapped by live traps baited with roasted walnut, tomato, and cucumber during spring and summer. All rodents were identified based on external features including fur color, ears characteristics, tail length, hind feet, body measurements, and internal features of teeth and cranium. Giemsa-stained impressions from rodents’ ears were examined for amastigotes microscopically. The samples from infected rodents were cultured in NNN+LIT medium and then the harvested parasites at the stationary phase were subjected to DNA extraction followed by amplification with RAPD-PCR. Results All the 28 entrapped animals were identified as M. hurrianae. Five animals showed to harbor Leishmania parasite by microscopy. Leishmania DNA isolated from five M. hurrianae produced distinctive bands of L. major with four primers. However, the products that were amplified with primers AB1-07, 327, and 329 were stable and reproducible. This is the first report on the isolation and identification of L. major from M. hurrianae from Iran. Conclusions Regarding infection rate of 17.8%, M. hurrianae seems to play the major role in the maintenance and transmission of disease to humans in this area. PMID:24616787

  10. First Report on Isolation and Characterization of Leishmania major from Meriones hurrianae (Rodentia: Gerbillidae) of A Rural Cutaneous leishmaniasis Focus in South-Eastern Iran.

    PubMed

    Kassiri, Hamid; Naddaf, Saied Reza; Javadian, Ezat-Aldin; Mohebali, Mehdi

    2013-09-01

    Zoonotic Cutaneous Leishmaniasis (ZCL) is an endemic health problem in many rural areas of Iran, with doubled number of incidences over the last decade. Different species of rodents serve as natural reservoir host for ZCL. The disease is considered as a major health problem in rural areas of Mirjaveh, Chabahar, and Konarak Counties of Sistan va Baluchistan Province. This study describes the identity of Leishmania species, isolated from Meriones hurrianae from Chabahar County using RAPD-PCR methodology. Rodents were entrapped by live traps baited with roasted walnut, tomato, and cucumber during spring and summer. All rodents were identified based on external features including fur color, ears characteristics, tail length, hind feet, body measurements, and internal features of teeth and cranium. Giemsa-stained impressions from rodents' ears were examined for amastigotes microscopically. The samples from infected rodents were cultured in NNN+LIT medium and then the harvested parasites at the stationary phase were subjected to DNA extraction followed by amplification with RAPD-PCR. All the 28 entrapped animals were identified as M. hurrianae. Five animals showed to harbor Leishmania parasite by microscopy. Leishmania DNA isolated from five M. hurrianae produced distinctive bands of L. major with four primers. However, the products that were amplified with primers AB1-07, 327, and 329 were stable and reproducible. This is the first report on the isolation and identification of L. major from M. hurrianae from Iran. Regarding infection rate of 17.8%, M. hurrianae seems to play the major role in the maintenance and transmission of disease to humans in this area.

  11. Syzygium cumini (L.) Skeels essential oil and its major constituent α-pinene exhibit anti-Leishmania activity through immunomodulation in vitro.

    PubMed

    Rodrigues, Klinger Antonio da Franca; Amorim, Layane Valéria; Dias, Clarice Noleto; Moraes, Denise Fernandes Coutinho; Carneiro, Sabrina Maria Portela; Carvalho, Fernando Aécio de Amorim

    2015-02-03

    Syzygium cumini (L.) Skeels (Myrtaceae), commonly known as "jambolão" in Brazil is widely used in folk medicine against leishmaniasis, inflammation, chronic diarrhea, and ulcers. It is one of the most commonly used plants for the treatment of diabetes worldwide. In previous studies, Syzygium cumini was shown to possess antihyperlipidemic and anti-allergic properties, and to exhibit good performance as an antimicrobial agent against bacteria, fungi, and protozoa parasites of the genus Leishmania and Trypanosoma. This study was aimed at evaluating the effects of S. cumini essential oil (ScEO) and its major component α-pinene on Leishmania (Leishmania) amazonensis, as well as their cytotoxicity and possible mechanisms of action. To evaluate the anti-proliferative effect on Leishmania, effects on promastigote and axenic amastigote forms were assessed using tetrazolium salt (MTT) assay. The intramacrophagic amastigotes were exposed to ScEO and α-pinene to determine the survival index. To gain insight into the mechanism of action involved in the effect on the samples, we evaluated the modulation of macrophage activation state by observing structural (phagocytic and lysosomal activities) and cellular (nitric oxide increase) changes. To assess the safety profile of ScEO and α-pinene, murine macrophages and human red blood cells were treated with ScEO and α-pinene and the selectivity index was calculated for each treatment. α-Pinene was effective against Leishmania amazonensis promastigote forms, with a half-maximal inhibitory concentration (IC50) value of 19.7µg/mL. α-Pinene was more active (IC50 values of 16.1 and 15.6µg/mL against axenic and intracellular amastigotes, respectively) than ScEO (IC50 values of 43.9 and 38.1µg/mL against axenic and intracellular amastigotes, respectively). Our results showed that the anti-Leishmania effects were mediated by immunomodulatory activity, as evidenced by the observed increases in both phagocytic and lysosomal activity

  12. Characterization of the Early Inflammatory Infiltrate at the Feeding Site of Infected Sand Flies in Mice Protected from Vector-Transmitted Leishmania major by Exposure to Uninfected Bites

    PubMed Central

    Teixeira, Clarissa; Gomes, Regis; Oliveira, Fabiano; Meneses, Claudio; Gilmore, Dana C.; Elnaiem, Dia-Eldin A.; Valenzuela, Jesus G.; Kamhawi, Shaden

    2014-01-01

    Background Mice exposed to sand fly saliva are protected against vector-transmitted Leishmania major. Although protection has been related to IFN- γ producing T cells, the early inflammatory response orchestrating this outcome has not been defined. Methodology/Principal findings Mice exposed to uninfected P. duboscqi bites and naïve mice were challenged with L. major-infected flies to characterize their early immune response at the bite site. Mostly, chemokine and cytokine transcript expression post-infected bites was amplified in exposed compared to naïve mice. In exposed mice, induced chemokines were mostly involved in leukocyte recruitment and T cell and NK cell activation; IL-4 was expressed at 6 h followed by IFN-γ and iNOS2 as well as IL-5 and IL-10 expression. In naïve animals, the transcript expression following Leishmania-infected sand fly bites was suppressed. Expression profiles translated to an earlier and significantly larger recruitment of leukocytes including neutrophils, macrophages, Gr+ monocytes, NK cells and CD4+ T cells to the bite site of exposed compared to naïve mice post-infected bites. Additionally, up to 48 hours post-infected bites the number of IFN-γ-producing CD4+T cells and NK cells arriving at the bite site was significantly higher in exposed compared to naïve mice. Thereafter, NK cells become cytolytic and persist at the bite site up to a week post-bite. Conclusion/Significance The quiet environment induced by a Leishmania-infected sand fly bite in naïve mice was significantly altered in animals previously exposed to saliva of uninfected flies. We propose that the enhanced recruitment of Gr+ monocytes, NK cells and CD4 Th1 cells observed at the bite site of exposed mice creates an inhospitable environment that counters the establishment of L. major infection. PMID:24762408

  13. The killing of Leishmania major by human macrophages is mediated by nitric oxide induced after ligation of the Fc epsilon RII/CD23 surface antigen.

    PubMed Central

    Vouldoukis, I; Riveros-Moreno, V; Dugas, B; Ouaaz, F; Bécherel, P; Debré, P; Moncada, S; Mossalayi, M D

    1995-01-01

    Serum IgE concentrations and the expression of the low-affinity receptor for IgE (Fc epsilon RII/CD23) are increased in cutaneous leishmaniasis or after immune challenge with Leishmania antigens. In vitro, the ligation of CD23 by IgE-anti-IgE immune complexes (IgE-IC) or by anti-CD23 monoclonal antibody (mAb) induces nitric oxide (NO) synthase and the generation of various cytokines by human monocytes/macrophages. The present study shows that IgE-IC, via CD23 binding, induce intracellular killing of Leishmania major in human monocyte-derived macrophages through the induction of the L-arginine:NO pathway. This was demonstrated by increased generation of nitrite (NO2-), the stable oxidation product of NO, and by the ability of NG-monomethyl-L-arginine to block both NO generation and parasite killing. A similar NO-dependent effect was observed with interferon gamma-treated cells. Tumor necrosis factor alpha is involved in this process, since both the induction of NO synthase and the killing of parasites caused by anti-CD23 mAb were inhibited by an anti-tumor necrosis factor alpha mAb. Treatment of noninfected CD23+ macrophages with IgE-IC provided protection against subsequent in vitro infection of these cells by Leishmania major promastigotes. Thus, IgE-IC promote killing of L. major by inducing NO synthase in human macrophages. Images Fig. 1 Fig. 5 PMID:7544003

  14. The structure of tubulin-binding cofactor A from Leishmania major infers a mode of association during the early stages of microtubule assembly

    PubMed Central

    Barrack, Keri L.; Fyfe, Paul K.; Hunter, William N.

    2015-01-01

    Tubulin-binding cofactor A (TBCA) participates in microtubule formation, a key process in eukaryotic biology to create the cytoskeleton. There is little information on how TBCA might interact with β-tubulin en route to microtubule biogenesis. To address this, the protozoan Leishmania major was targeted as a model system. The crystal structure of TBCA and comparisons with three orthologous proteins are presented. The presence of conserved features infers that electrostatic interactions that are likely to involve the C-terminal tail of β-tubulin are key to association. This study provides a reagent and template to support further work in this area. PMID:25945706

  15. Transgenic expression of CXCR3 on T cells enhances susceptibility to cutaneous Leishmania major infection by inhibiting monocyte maturation and promoting a Th2 response.

    PubMed

    Oghumu, Steve; Stock, James C; Varikuti, Sanjay; Dong, Ran; Terrazas, Cesar; Edwards, Jessica A; Rappleye, Chad A; Holovatyk, Ariel; Sharpe, Arlene; Satoskar, Abhay R

    2015-01-01

    Cutaneous leishmaniasis, caused mainly by Leishmania major, an obligate intracellular parasite, is a disfiguring disease characterized by large skin lesions and is transmitted by a sand fly vector. We previously showed that the chemokine receptor CXCR3 plays a critical role in mediating resistance to cutaneous leishmaniasis caused by Leishmania major. Furthermore, T cells from L. major-susceptible BALB/c but not L. major-resistant C57BL/6 mice fail to efficiently upregulate CXCR3 upon activation. We therefore examined whether transgenic expression of CXCR3 on T cells would enhance resistance to L. major infection in susceptible BALB/c mice. We generated BALB/c and C57BL/6 transgenic mice, which constitutively overexpressed CXCR3 under a CD2 promoter, and then examined the outcomes with L. major infection. Contrary to our hypothesis, transgenic expression of CXCR3 (CXCR3(Tg)) on T cells of BALB/c mice resulted in increased lesion sizes and parasite burdens compared to wild-type (WT) littermates after L. major infection. Restimulated lymph node cells from L. major-infected BALB/c-CXCR3(Tg) mice produced more interleukin-4 (IL-4) and IL-10 and less gamma interferon (IFN-γ). Cells in draining lymph nodes from BALB/c-CXCR3(Tg) mice showed enhanced Th2 and reduced Th1 cell accumulation associated with increased neutrophils and inflammatory monocytes. However, monocytes displayed an immature phenotype which correlated with increased parasite burdens. Interestingly, transgenic expression of CXCR3 on T cells did not impact the outcome of L. major infection in C57BL/6 mice, which mounted a predominantly Th1 response and spontaneously resolved their infection similar to WT littermates. Our findings demonstrate that transgenic expression of CXCR3 on T cells increases susceptibility of BALB/c mice to L. major.

  16. Designing and Cloning Molecular Constructs to Knock Out N-Acetylglucosamine Phosphatidylinositol De-N-Acetylase (GPI12) Gene in Leishmania major (MRHO/IR/75/ER)

    PubMed Central

    GHASEMI NEJAD ALMANI, Pooya; SHARIFI, Iraj; KAZEMI, Bahram; BABAEI, Zahra; BANDEHPOUR, Mojgan; SALARI, Samira; SAEDI DEZAKI, Ebrahim

    2016-01-01

    Background: Leishmaniasis represents a major public health concern in tropical and sub-tropical countries. At present, there is no efficacious vaccine against the disease and new control methods are needed. One way to access this important goal is to knock out genes of specific macromolecules to evaluate the effect of deletion on the growth, multiplication, pathogenesis and immunity of the parasite. The aim of this study was to design and clone molecular constructs to knock out N-acetylglucosamine phosphatidylinositol de-N-acetylase (GPI12) gene in Leishmania major. Methods: For designing and making molecular constructs, we used pLEXSY-neo2 and pLEXSY-hyg2 vectors. The molecular constructs were cloned in E. coli strain Top10. The molecular constructs were transfected by electroporation into L. major in two stages. Results: The molecular constructs were confirmed by Colony PCR and sequencing. The recombinant strains were isolated by selective antibiotics, after which they were confirmed by PCR, Southern and Western blots. Conclusion: Recombinant parasites were created and examined for subsequent study. With the use of molecular constructs, it was possible to remove and study gene GPI12 and to achieve a live recombinant Leishmania parasite that maintained the original form of the antigenic parasites. This achievement can be used as an experimental model for vaccine development studies. Further investigations are essential to check this model in a suitable host. PMID:28127356

  17. Antigen-Experienced T cells Limit the Priming of Naïve T cells During Infection with Leishmania major1

    PubMed Central

    Gray, Peter M.; Reiner, Steven L.; Smith, Deborah F.; Kaye, Paul M.; Scott, Phillip

    2009-01-01

    One mechanism to control immune responses following infection is to rapidly down regulate antigen presentation, which has been observed in acute viral and bacterial infections. Here we describe experiments designed to address whether antigen presentation is decreased after an initial response to Leishmania major. Naïve α-β-Leishmania-specific (ABLE) T cell receptor transgenic T cells were adoptively transferred into mice at various times after L. major infection to determine the duration of presentation of parasite-derived antigens. ABLE T cells responded vigorously at the initiation of infection, but the ability to prime these cells quickly diminished, independent of IL-10, regulatory T cells or antigen load. However, antigen-experienced clonal and polyclonal T cell populations could respond, indicating that the diminution in naïve ABLE cell responses was not due to lack of antigen presentation. Since naïve T cell priming could be restored by removal of the endogenous T cell population, or adoptive transfer of antigen pulsed dendritic cells, it appears that T cells that have previously encountered antigen during infection compete with naïve antigen-specific T cells. These results suggest that during L. major infection antigen-experienced T cells, rather than naïve T cells, may be primarily responsible for sustaining the immune response. PMID:16818747

  18. Isolation of a myoinhibitory peptide from Leishmania major (Kinetoplastida: Trypanosomatidae) and its function in the vector sand fly Phlebotomus papatasi (Diptera: Psychodidae).

    PubMed

    Vaidyanathan, Rajeev

    2005-03-01

    Protozoan parasites in the genus Leishmania are ingested by sand flies with blood and multiply in the gut until they are transmitted to a vertebrate host when the sand fly blood feeds again. Infections of the enzootic vector Phlebotomus papatasi Scopoli result in distended midguts with no spontaneous gut contractions. Using a P. papatasi hindgut contraction bioassay, a paralytic factor sensitive to trypsin, chymotrypsin, proteinase-K, and heating at 56 degrees C was detected in crude lysates of Leishmania major promastigotes. Application of parasite lysate to isolated hindguts resulted in reversible, dose-dependent inhibition of spontaneous contractions. Mean volume of isolated midguts and hindguts increased by 50-60% after application of L. major lysate. L. major paralytic factor was purified 10(4)-fold over the total protein preparation and yielded a hydrophobic 12-kDa peptide. Myoinhibitory activity eluted as a single peak in reverse phase-high-pressure liquid chromatography. Tandem mass spectrometry resulted in 15 amino acid sequences, three of them sharing 45-73% homology with short hypothetical gene products of undefined function from Pseudomonas, Halobacterium, and Drosophila. This unique protozoan peptide mimics the function of endogenous insect neuropeptides that control visceral muscle contractions. By this novel mechanism, parasites persist in the expanded, relaxed midgut after blood meal and peritrophic matrix digestion. This allows time for development and migration of infective forms, facilitating sand fly vector competence and parasite transmission.

  19. Colonization of Phlebotomus papatasi changes the effect of pre-immunization with saliva from lack of protection towards protection against experimental challenge with Leishmania major and saliva

    PubMed Central

    2011-01-01

    Background Sand fly saliva has been postulated as a potential vaccine or as a vaccine component within multi component vaccine against leishmaniasis. It is important to note that these studies were performed using long-term colonized Phlebotomus papatasi. The effect of sand flies colonization on the outcome of Leishmania infection is reported. Results While pre-immunization of mice with salivary gland homogenate (SGH) of long-term colonized (F5 and beyond) female Phlebotomus papatasi induced protection against Leishmania major co-inoculated with the same type of SGH, pre-immunization of mice with SGH of recently colonized (F2 and F3) female P. papatasi did not confer protection against L. major co-inoculated with the same type of SGH. Our data showed for the first time that a shift from lack of protection to protection occurs at the fourth generation (F4) during the colonization process of P. papatasi. Conclusion For the development of a sand fly saliva-based vaccine, inferences based on long-term colonized populations of sand flies should be treated with caution as colonization of P. papatasi appears to modulate the outcome of L. major infection from lack of protection to protection. PMID:21726438

  20. A Computational Methodology to Overcome the Challenges Associated With the Search for Specific Enzyme Targets to Develop Drugs Against Leishmania major

    PubMed Central

    Catharina, Larissa; Lima, Carlyle Ribeiro; Franca, Alexander; Guimarães, Ana Carolina Ramos; Alves-Ferreira, Marcelo; Tuffery, Pierre; Derreumaux, Philippe; Carels, Nicolas

    2017-01-01

    We present an approach for detecting enzymes that are specific of Leishmania major compared with Homo sapiens and provide targets that may assist research in drug development. This approach is based on traditional techniques of sequence homology comparison by similarity search and Markov modeling; it integrates the characterization of enzymatic functionality, secondary and tertiary protein structures, protein domain architecture, and metabolic environment. From 67 enzymes represented by 42 enzymatic activities classified by AnEnPi (Analogous Enzymes Pipeline) as specific for L major compared with H sapiens, only 40 (23 Enzyme Commission [EC] numbers) could actually be considered as strictly specific of L major and 27 enzymes (19 EC numbers) were disregarded for having ambiguous homologies or analogies with H sapiens. Among the 40 strictly specific enzymes, we identified sterol 24-C-methyltransferase, pyruvate phosphate dikinase, trypanothione synthetase, and RNA-editing ligase as 4 essential enzymes for L major that may serve as targets for drug development. PMID:28638238

  1. Genomic cartography and proposal of nomenclature for the repeated, interspersed elements of the Leishmania major SIDER2 family and identification of SIDER2-containing transcripts.

    PubMed

    Requena, Jose M; Rastrojo, Alberto; Garde, Esther; López, Manuel C; Thomas, M Carmen; Aguado, Begoña

    2017-03-01

    The genomes of most eukaryotic organisms contain a large number of transposable elements that are able to move from one genomic site to another either by transferring of DNA mobile elements (transposons) or transpose via reverse transcription of an RNA intermediate (retroposons). An exception to this rule is found in protists of the subgenus Leishmania, in which active retroposons degenerated after a flourishing era, leaving only retroposon remains; these have been classified into two families: SIDER1 and SIDER2. In this work, we have re-examined the elements belonging to the family SIDER2 present in the genome of Leishmania major with the aim of providing a nomenclature that will facilitate a future reference to particular elements. According to sequence conservation, the 1100 SIDER2 elements have been grouped into subfamilies, and the inferred taxonomic relationships have also been incorporated into the nomenclature. Additionally, we are providing detailed data regarding the genomic distribution of these elements and their association with specific transcripts, based on the recently established transcriptome for L. major. Thus, the presented data can help to study and better understand the roles played by these degenerated retroposons in both regulation of gene expression and genome plasticity. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Habitats of the sandfly vectors of Leishmania tropica and L. major in a mixed focus of cutaneous leishmaniasis in southeast Tunisia.

    PubMed

    Tabbabi, Ahmed; Ghrab, Jamila; Aoun, Karim; Ready, Paul Donald; Bouratbine, Aïda

    2011-08-01

    From 2009 to 2010, 3129 sandflies were caught in CDC light traps placed in various habitats in Ghomrassen, Tataouine governorate, southeast Tunisia, a mixed focus of human cutaneous leishmaniasis caused by Leishmania tropica and Leishmania major. Species diversity was quantified in anthropogenic, semi-anthropogenic and semi-natural locations. Sandflies were identified according to morphological characters and also by the comparative sequence analysis of a fragment of the mitochondrial cytochrome b gene to distinguish between two putative local vectors of L. tropica, namely Phlebotomus chabaudi and Phlebotomus riouxi. The lowest sandfly diversities were found in L. major sites, where the incriminated vector P. papatasi predominated in the burrows of the rodent reservoir hosts (Meriones) as well as inside and outside houses of human cases. In L. tropica sites, the incriminated peri-domestic vector Phlebotomus sergenti was the most abundant species inside houses, whereas P. riouxi or P. chabaudi was the dominant species in the semi-natural rocky habitats favoured by the putative rodent reservoir, Ctenodactylus gundi. All specimens of P. chabaudi identified molecularly had the diagnostic cytochrome b characters of P. riouxi, indicating either that the latter represents only a geographical variant of P. chabaudi or that these two species may sometimes hybridize.

  3. Molecular detection of Leishmania major kDNA from wild rodents in a new focus of zoonotic cutaneous leishmaniasis in an Oriental region of Iran.

    PubMed

    Azizi, Kourosh; Moemenbellah-Fard, Mohammad Djaefar; Kalantari, Mohsen; Fakoorziba, Mohammad Reza

    2012-10-01

    Human cutaneous leishmaniasis is one of the most challenging public health issues in many tropical and subtropical countries of the world, including Iran. More than half (54%) of the new zoonotic cutaneous leishmaniasis (ZCL) cases among the Eastern Mediterranean countries were reported from Iran in 2008. The detection of Leishmania parasites in rodents is essential to incriminate them as probable reservoir hosts of ZCL infection. As a result of the annual detection of about 200-250 clinical ZCL cases in the Jask district of southern Iran, feral rodents were trapped, identified to species level, and examined for Leishmania presence by preparing routine blood smears on microscopic slides from 2007 to 2008. Overall, 27 Tatera indica, 17 Gerbillus nanus, 29 Meriones persicus, 26 M. hurrianae, and 7 M. libycus were identified. Females of T. indica, M. hurrianae, and G. nanus appeared to be naturally infected with the protozoan parasite, L. major. This is the first report of microscopic and molecular detection of this trypanosomatid parasite infecting these three rodents reported from Hormozgan province in southeast Iran. More than three-quarters (82%) of the parasite-infected rodents came from the eastern plain of this province, but none of the other rodents were found to be smear-positive or kinetoplast DNA-positive by PCR. M. hurrianae, G. nanus, and T. indica are therefore incriminated as three potential reservoir hosts of L. major in Oriental parts of Iran.

  4. Identification, biochemical characterization, and in-vivo expression of the intracellular invertase BfrA from the pathogenic parasite Leishmania major.

    PubMed

    Belaz, Sorya; Rattier, Thibault; Lafite, Pierre; Moreau, Philippe; Routier, Françoise H; Robert-Gangneux, Florence; Gangneux, Jean-Pierre; Daniellou, Richard

    2015-10-13

    The parasitic life cycle of Leishmania includes an extracellular promastigote stage that occurs in the gut of the insect vector. During that period, the sucrose metabolism and more specifically the first glycosidase of this pathway are essential for growth and survival of the parasite. We investigated the expression of the invertase BfrA in the promastigote and amastigote stages of three parasite species representative of the three various clinical forms and of various geographical areas, namely Leishmania major, L. donovani and L. braziliensis. Thereafter, we cloned, overexpressed and biochemically characterized this invertase BfrA from L. major, heterologously expressed in both Escherichia coli and L. tarentolae. For all species, expression levels of BfrA mRNA were correlated to the time of the culture and the parasitic stage (promastigotes > amastigotes). BfrA exhibited no activity when expressed as a glycoprotein in L. tarentolae but proved to be an invertase when not glycosylated, yet owing low sequence homology with other invertases from the same family. Our data suggest that BfrA is an original invertase that is located inside the parasite. It is expressed in both parasitic stages, though to a higher extent in promastigotes. This work provides new insight into the parasite sucrose metabolism.

  5. Leishmania major encodes an unusual peroxidase that is a close homologue of plant ascorbate peroxidase: a novel role of the transmembrane domain

    PubMed Central

    Adak, Subrata; Datta, Alok K.

    2005-01-01

    Haem-containing enzymes (peroxidase and catalase) are widely distributed among prokaryotes and eukaryotes and play a vital role in H2O2 detoxification. But, to date, no haem-containing enzymatic defence against toxic H2O2 has been discovered in Leishmania species. We cloned, expressed and purified an unusual plant-like APX (ascorbate peroxidase) from Leishmania major (LmAPX) and characterized its catalytic parameters under steady-state conditions. Examination of its protein sequence indicated approx. 30–60% identity with other APXs. The N-terminal extension of LmAPX is characterized by a charged region followed by a stretch of 22 amino acids containing a transmembrane domain. To understand how the transmembrane domain influences the structure–function of LmAPX, we generated, purified and extensively characterized a variant that lacked the transmembrane domain. Eliminating the transmembrane domain had no impact on substrate-binding affinity but slowed down ascorbate oxidation and increased resistance to H2O2-dependent inactivation in the absence of electron donor by 480-fold. Spectral studies show that H2O2 can quickly oxidize the native enzyme to compound (II), which subsequently is reduced back to the native enzyme by an electron donor. In contrast, ascorbate-free transmembrane domain-containing enzyme did not react with H2O2, as revealed by the absence of compound (II) formation. Our findings suggest that the single copy LmAPX gene may play an important role in detoxification of H2O2 that is generated by endogenous processes and as a result of external influences such as the oxidative burst of infected host macrophages or during drug metabolism by Leishmania. PMID:15850459

  6. Concomitant Immunity Induced by Persistent Leishmania major Does Not Preclude Secondary Re-Infection: Implications for Genetic Exchange, Diversity and Vaccination

    PubMed Central

    Mandell, Michael A.

    2016-01-01

    Background Many microbes have evolved the ability to co-exist for long periods of time within other species in the absence of overt pathology. Evolutionary biologists have proposed benefits to the microbe from ‘asymptomatic persistent infections’, most commonly invoking increased likelihood of transmission by longer-lived hosts. Typically asymptomatic persistent infections arise from strong containment by the immune system, accompanied by protective immunity; such ‘vaccination’ from overt disease in the presence of a non-sterilizing immune response is termed premunition or concomitant immunity. Here we consider another potential benefit of persistence and concomitant immunity to the parasite: the ‘exclusion’ of competing super-infecting strains, which would favor transmission of the original infecting organism. Methodology / Principle Findings To investigate this in the protozoan parasite Leishmania major, a superb model for the study of asymptomatic persistence, we used isogenic lines of comparable virulence bearing independent selectable markers. One was then used to infect genetically resistant mice, yielding infections which healed and progressed to asymptomatic persistent infection; these mice were then super-infected with the second marked line. As anticipated, super-infection yielded minimal pathology, showing that protective immunity against disease pathology had been established. The relative abundance of the primary and super-infecting secondary parasites was then assessed by plating on selective media. The data show clearly that super-infecting parasites were able to colonize the immune host effectively, achieving numbers comparable to and sometimes greater than that of the primary parasite. Conclusions / Significance We conclude that induction of protective immunity does not guarantee the Leishmania parasite exclusive occupation of the infected host. This finding has important consequences to the maintenance and generation of parasite

  7. Naturally Occurring Culturable Aerobic Gut Flora of Adult Phlebotomus papatasi, Vector of Leishmania major in the Old World

    PubMed Central

    Mukhopadhyay, Jaba; Braig, Henk R.; Rowton, Edgar D.; Ghosh, Kashinath

    2012-01-01

    Background Cutaneous leishmaniasis is a neglected, vector-borne parasitic disease and is responsible for persistent, often disfiguring lesions and other associated complications. Leishmania, causing zoonotic cutaneous leishmaniasis (ZCL) in the Old World are mainly transmitted by the predominant sand fly vector, Phlebotomus papatasi. To date, there is no efficient control measure or vaccine available for this widespread insect-borne infectious disease. Methodology/Principal Findings A survey was carried out to study the abundance of different natural gut flora in P. papatasi, with the long-term goal of generating a paratransgenic sand fly that can potentially block the development of Leishmania in the sand fly gut, thereby preventing transmission of leishmania in endemic disease foci. Sand flies, in particular, P. papatasi were captured from different habitats of various parts of the world. Gut microbes were cultured and identified using 16S ribosomal DNA analysis and a phylogenetic tree was constructed. We found variation in the species and abundance of gut flora in flies collected from different habitats. However, a few Gram-positive, nonpathogenic bacteria including Bacillus flexus and B. pumilus were common in most of the sites examined. Conclusion/Significance Our results indicate that there is a wide range of variation of aerobic gut flora inhabiting sand fly guts, which possibly reflect the ecological condition of the habitat where the fly breeds. Also, some species of bacteria (B. pumilus, and B. flexus) were found from most of the habitats. Important from an applied perspective of dissemination, our results support a link between oviposition induction and adult gut flora. PMID:22629302

  8. LmABCB3, an atypical mitochondrial ABC transporter essential for Leishmania major virulence, acts in heme and cytosolic iron/sulfur clusters biogenesis.

    PubMed

    Martínez-García, Marta; Campos-Salinas, Jenny; Cabello-Donayre, María; Pineda-Molina, Estela; Gálvez, Francisco J; Orrego, Lina M; Sánchez-Cañete, María P; Malagarie-Cazenave, Sophie; Koeller, David M; Pérez-Victoria, José M

    2016-01-05

    Mitochondria play essential biological functions including the synthesis and trafficking of porphyrins and iron/sulfur clusters (ISC), processes that in mammals involve the mitochondrial ATP-Binding Cassette (ABC) transporters ABCB6 and ABCB7, respectively. The mitochondrion of pathogenic protozoan parasites such as Leishmania is a promising goal for new therapeutic approaches. Leishmania infects human macrophages producing the neglected tropical disease known as leishmaniasis. Like most trypanosomatid parasites, Leishmania is auxotrophous for heme and must acquire porphyrins from the host. LmABCB3, a new Leishmania major protein with significant sequence similarity to human ABCB6/ABCB7, was identified and characterized using bioinformatic tools. Fluorescent microscopy was used to determine its cellular localization, and its level of expression was modulated by molecular genetic techniques. Intracellular in vitro assays were used to demonstrate its role in amastigotes replication, and an in vivo mouse model was used to analyze its role in virulence. Functional characterization of LmABCB3 was carried out in Leishmania promastigotes and Saccharomyces cerevisiae. Structural analysis of LmABCB3 was performed using molecular modeling software. LmABCB3 is an atypical ABC half-transporter that has a unique N-terminal extension not found in any other known ABC protein. This extension is required to target LmABCB3 to the mitochondrion and includes a potential metal-binding domain. We have shown that LmABCB3 interacts with porphyrins and is required for the mitochondrial synthesis of heme from a host precursor. We also present data supporting a role for LmABCB3 in the biogenesis of cytosolic ISC, essential cofactors for cell viability in all three kingdoms of life. LmABCB3 fully complemented the severe growth defect shown in yeast lacking ATM1, an orthologue of human ABCB7 involved in exporting from the mitochondria a gluthatione-containing compound required for the

  9. Role of protein kinase R in the killing of Leishmania major by macrophages in response to neutrophil elastase and TLR4 via TNFα and IFNβ

    PubMed Central

    Faria, Marilia S.; Calegari-Silva, Tereza C.; de Carvalho Vivarini, Aislan; Mottram, Jeremy C.; Lopes, Ulisses Gazos; Lima, Ana Paula C. A.

    2014-01-01

    In cutaneous leishmaniasis, Leishmania amazonensis activates macrophage double-stranded, RNA-activated protein kinase R (PKR) to promote parasite growth. In our study, Leishmania major grew normally in RAW cells, RAW-expressing dominant-negative PKR (PKR-DN) cells, and macrophages of PKR-knockout mice, revealing that PKR is dispensable for L. major growth in macrophages. PKR activation in infected macrophages with poly I:C resulted in parasite death. Fifty percent of L. major-knockout lines for the ecotin-like serine peptidase inhibitor (ISP2; Δisp2/isp3), an inhibitor of neutrophil elastase (NE), died in RAW cells or macrophages from 129Sv mice, as a result of PKR activation. Inhibition of PKR or NE or neutralization of Toll-like receptor 4 or 2(TLR4 or TLR2) prevented the death of Δisp2/isp3. Δisp2/isp3 grew normally in RAW-PKR-DN cells or macrophages from 129Sv pkr−/−, tlr2−/−, trif−/−, and myd88−/− mice, associating NE activity, PKR, and TLR responses with parasite death. Δisp2/isp3 increased the expression of mRNA for TNF-α by 2-fold and of interferon β (IFNβ) in a PKR-dependent manner. Antibodies to TNF-α reversed the 95% killing by Δisp2/isp3, whereas they grew normally in macrophages from IFN receptor–knockout mice. We propose that ISP2 prevents the activation of PKR via an NE-TLR4-TLR2 axis to control innate responses that contribute to the killing of L. major.—Faria, M. S., Calegari-Silva, T. C., de Carvalho Vivarini, A., Mottram, J. C., Lopes, U. G., Lima, A. P. C. A. Role of protein kinase R in the killing of Leishmania major by macrophages in response to neutrophil elastase and TLR4 via TNFα and IFNβ. PMID:24732131

  10. Les leishmanioses cutanées à Leishmania major et à Leishmania tropica au Maroc: aspects épidémio-cliniques comparatifs de 268 cas

    PubMed Central

    Chiheb, Soumia; Slaoui, Widad; Mouttaqui, Tarik; Riyad, Meriem; Benchikhi, Hakima

    2014-01-01

    Introduction Depuis 1995, le Maroc a connu une réactivation des foyers de leishmanioses cutanées (LC) à L. major et une nouvelle répartition géographique des foyers à L. tropica. Le but de cette étude est de comparer les aspects épidémio-cliniques associés aux LC potentiellement dûes à L. major et à L. tropica. Méthodes Une étude rétrospective a colligé 268 cas de LC au service de dermatologie du CHU Ibn Rochd de Casablanca entre Janvier 1995 et Septembre 2010. Les données étaient analysées par Epi info version 3.5.1. Le test X2 était appliqué (Différence significative = p< 0,05). Résultats Deux cent soixante-huit cas de LC ont été colligés, dont 160 femmes et 108 hommes. Ils ont été répartis en 123 patients originaires des foyers à L.major et 145 patients originaires des foyers à L. tropica. L'aspect ulcéronodulaire, ulcérovégétant ou végétant était retrouvé dans 58 cas (47,2%) des cas de LC à L. major versus 24 cas (16,7%) dans la L.C à L. tropica. L'aspect papulonodulaire était retrouvé dans 84 cas (58%) de LC à L. tropica contre 41 cas (33,3%) de LC à L. major. Conclusion Dans la LC à L. major, l'atteinte des membres et les aspects cliniques végétant ou ulcéro-végétant restent toujours prédominants. Dans la L.C à L. tropica, l'atteinte papulonodulaire unique du visage reste prédominante mais des formes ulcéronodulaires, végétantes ou ulcérovégétantes existent également dans les foyers récents à L. tropica, prêtant à confusion cliniquement avec des LC à L. major. PMID:25810796

  11. Vaccination with heat-killed leishmania antigen or recombinant leishmanial protein and CpG oligodeoxynucleotides induces long-term memory CD4+ and CD8+ T cell responses and protection against leishmania major infection.

    PubMed

    Rhee, Elizabeth G; Mendez, Susana; Shah, Javeed A; Wu, Chang-you; Kirman, Joanna R; Turon, Tara N; Davey, Dylan F; Davis, Heather; Klinman, Dennis M; Coler, Rhea N; Sacks, David L; Seder, Robert A

    2002-06-17

    CpG oligodeoxynucleotides (ODN) have potent effects on innate and adaptive cellular immune responses. In this report, the ability of CpG ODN to confer long-term immunity and protection when used as a vaccine adjuvant with a clinical grade of leishmanial antigen, autoclaved Leishmania major (ALM), or a recombinant leishmanial protein was studied. In two different mouse models of L. major infection, vaccination with ALM plus CpG ODN was able to control infection and markedly reduce lesion development in susceptible BALB/c and resistant C57BL/6 (B6) mice, respectively, up to 12 wk after immunization. Moreover, B6 mice immunized with ALM plus CpG ODNs were still protected against infectious challenge even 6 mo after vaccination. In terms of immune correlates of protection, ALM plus CpG ODN-vaccinated mice displayed L. major-specific T helper cell 1 and CD8+ responses. In addition, complete protection was markedly abrogated in mice depleted of CD8+ T cells at the time of vaccination. Similarly, mice vaccinated with a recombinant leishmanial protein plus CpG ODN also had long-term protection that was dependent on CD8+ T cells in vivo. Together, these data demonstrate that CpG ODN, when used as a vaccine adjuvant with either a recombinant protein or heat-killed leishmanial antigen, can induce long-term protection against an intracellular infection in a CD8-dependent manner.

  12. Direct Visualization of Peptide/MHC Complexes at the Surface and in the Intracellular Compartments of Cells Infected In Vivo by Leishmania major

    PubMed Central

    Cazareth, Julie; Hoebeke, Johan; Lippuner, Christoph; Davalos-Misslitz, Ana; Aebischer, Toni; Muller, Sylviane; Glaichenhaus, Nicolas; Mougneau, Evelyne

    2010-01-01

    Protozoa and bacteria infect various types of phagocytic cells including macrophages, monocytes, dendritic cells and eosinophils. However, it is not clear which of these cells process and present microbial antigens in vivo and in which cellular compartments parasite peptides are loaded onto Major Histocompatibility Complex molecules. To address these issues, we have infected susceptible BALB/c (H-2d) mice with a recombinant Leishmania major parasite expressing a fluorescent tracer. To directly visualize the antigen presenting cells that present parasite-derived peptides to CD4+ T cells, we have generated a monoclonal antibody that reacts to an antigenic peptide derived from the parasite LACK antigen bound to I-Ad Major Histocompatibility Complex class II molecule. Immunogold electron microscopic analysis of in vivo infected cells showed that intracellular I-Ad/LACK complexes were present in the membrane of amastigote-containing phagosomes in dendritic cells, eosinophils and macrophages/monocytes. In both dendritic cells and macrophages, these complexes were also present in smaller vesicles that did not contain amastigote. The presence of I-Ad/LACK complexes at the surface of dendritic cells, but neither on the plasma membrane of macrophages nor eosinophils was independently confirmed by flow cytometry and by incubating sorted phagocytes with highly sensitive LACK-specific hybridomas. Altogether, our results suggest that peptides derived from Leishmania proteins are loaded onto Major Histocompatibility Complex class II molecules in the phagosomes of infected phagocytes. Although these complexes are transported to the cell surface in dendritic cells, therefore allowing the stimulation of parasite-specific CD4+ T cells, this does not occur in other phagocytic cells. To our knowledge, this is the first study in which Major Histocompatibility Complex class II molecules bound to peptides derived from a parasite protein have been visualized within and at the surface of

  13. An electrochemical genosensor for Leishmania major detection based on dual effect of immobilization and electrocatalysis of cobalt-zinc ferrite quantum dots.

    PubMed

    Heli, H; Sattarahmady, N; Hatam, G R; Reisi, F; Vais, R Dehdari

    2016-08-15

    Identification of Leishmania parasites is important in diagnosis and clinical studies of leishmaniasis. Although epidemiological and clinical methods are available, they are not sufficient for identification of causative agents of leishmaniasis. In the present study, quantum dots of magnetic cobalt-zinc ferrite (Co0.5Zn0.5Fe2O4) were synthesized and characterized by physicochemical methods. The quantum dots were then employed as an electrode modifier to immobilize a 24-mer specific single stranded DNA probe, and fabrication of a label-free, PCR-free and signal-on electrochemical genosensor for the detection of Leishmania major. Hybridization of the complementary single stranded DNA sequence with the probe under the selected conditions was explored using methylene blue as a redox marker, utilizing the electrocatalytic effect of the quantum dots on the methylene blue electroreduction process. The genosensor could detect a synthetic single stranded DNA target in a range of 1.0×10(-11) to 1.0×10(-18)molL(-1) with a limit of detection of 2.0×10(-19)molL(-1), and genomic DNA in a range of 7.31×10(-14) to 7.31×10(-6)ngμL(-1) with a limit of detection of 1.80×10(-14)ngμL(-1) with a high selectivity and sensitivity. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. The structure of tubulin-binding cofactor A from Leishmania major infers a mode of association during the early stages of microtubule assembly

    SciTech Connect

    Barrack, Keri L.; Fyfe, Paul K.; Hunter, William N.

    2015-04-21

    The structure of a tubulin-binding cofactor from L. major is reported and compared with yeast, plant and human orthologues. Tubulin-binding cofactor A (TBCA) participates in microtubule formation, a key process in eukaryotic biology to create the cytoskeleton. There is little information on how TBCA might interact with β-tubulin en route to microtubule biogenesis. To address this, the protozoan Leishmania major was targeted as a model system. The crystal structure of TBCA and comparisons with three orthologous proteins are presented. The presence of conserved features infers that electrostatic interactions that are likely to involve the C-terminal tail of β-tubulin are key to association. This study provides a reagent and template to support further work in this area.

  15. Inhibition of Dihydroorotate Dehydrogenase Overcomes Differentiation Blockade in Acute Myeloid Leukemia.

    PubMed

    Sykes, David B; Kfoury, Youmna S; Mercier, François E; Wawer, Mathias J; Law, Jason M; Haynes, Mark K; Lewis, Timothy A; Schajnovitz, Amir; Jain, Esha; Lee, Dongjun; Meyer, Hanna; Pierce, Kerry A; Tolliday, Nicola J; Waller, Anna; Ferrara, Steven J; Eheim, Ashley L; Stoeckigt, Detlef; Maxcy, Katrina L; Cobert, Julien M; Bachand, Jacqueline; Szekely, Brian A; Mukherjee, Siddhartha; Sklar, Larry A; Kotz, Joanne D; Clish, Clary B; Sadreyev, Ruslan I; Clemons, Paul A; Janzer, Andreas; Schreiber, Stuart L; Scadden, David T

    2016-09-22

    While acute myeloid leukemia (AML) comprises many disparate genetic subtypes, one shared hallmark is the arrest of leukemic myeloblasts at an immature and self-renewing stage of development. Therapies that overcome differentiation arrest represent a powerful treatment strategy. We leveraged the observation that the majority of AML, despite their genetically heterogeneity, share in the expression of HoxA9, a gene normally downregulated during myeloid differentiation. Using a conditional HoxA9 model system, we performed a high-throughput phenotypic screen and defined compounds that overcame differentiation blockade. Target identification led to the unanticipated discovery that inhibition of the enzyme dihydroorotate dehydrogenase (DHODH) enables myeloid differentiation in human and mouse AML models. In vivo, DHODH inhibitors reduced leukemic cell burden, decreased levels of leukemia-initiating cells, and improved survival. These data demonstrate the role of DHODH as a metabolic regulator of differentiation and point to its inhibition as a strategy for overcoming differentiation blockade in AML. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Structural Plasticity of Malaria Dihydroorotate Dehydrogenase Allows Selective Binding of Diverse Chemical Scaffolds

    SciTech Connect

    Deng, Xiaoyi; Gujjar, Ramesh; El Mazouni, Farah; Kaminsky, Werner; Malmquist, Nicholas A.; Goldsmith, Elizabeth J.; Rathod, Pradipsinh K.; Phillips, Margaret A.

    2010-01-20

    Malaria remains a major global health burden and current drug therapies are compromised by resistance. Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) was validated as a new drug target through the identification of potent and selective triazolopyrimidine-based DHODH inhibitors with anti-malarial activity in vivo. Here we report x-ray structure determination of PfDHODH bound to three inhibitors from this series, representing the first of the enzyme bound to malaria specific inhibitors. We demonstrate that conformational flexibility results in an unexpected binding mode identifying a new hydrophobic pocket on the enzyme. Importantly this plasticity allows PfDHODH to bind inhibitors from different chemical classes and to accommodate inhibitor modifications during lead optimization, increasing the value of PfDHODH as a drug target. A second discovery, based on small molecule crystallography, is that the triazolopyrimidines populate a resonance form that promotes charge separation. These intrinsic dipoles allow formation of energetically favorable H-bond interactions with the enzyme. The importance of delocalization to binding affinity was supported by site-directed mutagenesis and the demonstration that triazolopyrimidine analogs that lack this intrinsic dipole are inactive. Finally, the PfDHODH-triazolopyrimidine bound structures provide considerable new insight into species-selective inhibitor binding in this enzyme family. Together, these studies will directly impact efforts to exploit PfDHODH for the development of anti-malarial chemotherapy.

  17. Gluconeogenesis in Leishmania mexicana

    PubMed Central

    Rodriguez-Contreras, Dayana; Hamilton, Nicklas

    2014-01-01

    Gluconeogenesis is an active pathway in Leishmania amastigotes and is essential for their survival within the mammalian cells. However, our knowledge about this pathway in trypanosomatids is very limited. We investigated the role of glycerol kinase (GK), phosphoenolpyruvate carboxykinase (PEPCK), and pyruvate phosphate dikinase (PPDK) in gluconeogenesis by generating the respective Leishmania mexicana Δgk, Δpepck, and Δppdk null mutants. Our results demonstrated that indeed GK, PEPCK, and PPDK are key players in the gluconeogenesis pathway in Leishmania, although stage-specific differences in their contribution to this pathway were found. GK participates in the entry of glycerol in promastigotes and amastigotes; PEPCK participates in the entry of aspartate in promastigotes, and PPDK is involved in the entry of alanine in amastigotes. Furthermore, the majority of alanine enters into the pathway via decarboxylation of pyruvate in promastigotes, whereas pathway redundancy is suggested for the entry of aspartate in amastigotes. Interestingly, we also found that l-lactate, an abundant glucogenic precursor in mammals, was used by Leishmania amastigotes to synthesize mannogen, entering the pathway through PPDK. On the basis of these new results, we propose a revision in the current model of gluconeogenesis in Leishmania, emphasizing the differences between amastigotes and promastigotes. This work underlines the importance of studying the trypanosomatid intracellular life cycle stages to gain a better understanding of the pathologies caused in humans. PMID:25288791

  18. Production of superoxide/H2O2 by dihydroorotate dehydrogenase in rat skeletal muscle mitochondria.

    PubMed

    Hey-Mogensen, Martin; Goncalves, Renata L S; Orr, Adam L; Brand, Martin D

    2014-07-01

    Dehydrogenases that use ubiquinone as an electron acceptor, including complex I of the respiratory chain, complex II, and glycerol-3-phosphate dehydrogenase, are known to be direct generators of superoxide and/or H2O2. Dihydroorotate dehydrogenase oxidizes dihydroorotate to orotate and reduces ubiquinone to ubiquinol during pyrimidine metabolism, but it is unclear whether it produces superoxide and/or H2O2 directly or does so only indirectly from other sites in the electron transport chain. Using mitochondria isolated from rat skeletal muscle we establish that dihydroorotate oxidation leads to superoxide/H2O2 production at a fairly high rate of about 300pmol H2O2·min(-1)·mg protein(-1) when oxidation of ubiquinol is prevented and complex II is uninhibited. This H2O2 production is abolished by brequinar or leflunomide, known inhibitors of dihydroorotate dehydrogenase. Eighty percent of this rate is indirect, originating from site IIF of complex II, because it can be prevented by malonate or atpenin A5, inhibitors of complex II. In the presence of inhibitors of all known sites of superoxide/H2O2 production (rotenone to inhibit sites in complex I (site IQ and, indirectly, site IF), myxothiazol to inhibit site IIIQo in complex III, and malonate plus atpenin A5 to inhibit site IIF in complex II), dihydroorotate dehydrogenase generates superoxide/H2O2, at a small but significant rate (23pmol H2O2·min(-1)·mg protein(-1)), from the ubiquinone-binding site. We conclude that dihydroorotate dehydrogenase can generate superoxide and/or H2O2 directly at low rates and is also capable of indirect production at higher rates from other sites through its ability to reduce the ubiquinone pool.

  19. Structure of Plasmodium falciparum dihydroorotate dehydrogenase with a bound inhibitor.

    PubMed

    Hurt, Darrell E; Widom, Joanne; Clardy, Jon

    2006-03-01

    Membrane-associated dihydroorotate dehydrogenase (DHODH) is an antimalarial therapeutic target without an effective inhibitor. Studies on human DHODH (HsDHODH) led to a structural mechanistic model in which respiratory quinones bind in a tunnel formed by the highly variable N-terminus that leads to the flavin mononucleotide-binding site. The therapeutic agents leflunomide (Arava) and brequinar sodium inhibit HsDHODH by binding in this tunnel. Plasmodium falciparum DHODH (PfDHODH) and HsDHODH have markedly different sensitivities to the two drugs. To understand the structural basis of this differential sensitivity and begin a structure-based drug-design cycle for PfDHODH inhibitors, the three-dimensional structure (2.4 Angstroms, R = 20.1%) of PfDHODH bound to the active metabolite of leflunomide was determined by X-ray crystallography. Comparison of the structures of HsDHODH and PfDHODH reveals a completely different binding mode for the same inhibitor in these two catalytically identical enzymes and explains the previously observed species-specific preferential binding. Because no effective inhibitors have been described for PfDHODH, this structure provides critical insight for the design of potential antimalarials.

  20. Effect of hydroalcoholic extract of Echinacea purpurea in combination with meglumine antimoniate on treatment of Leishmania major-induced cutaneous leishmaniasis in BALB/c mice

    PubMed Central

    Sarkari, Bahador; Mohseni, Mobin; Moein, Mahmoud Reza; Shahriarirad, Reza; Asgari, Qasem

    2017-01-01

    Context: Progressive resistance of Leishmania parasite to available drugs including, meglumine antimoniate, has been reported from various regions of the world, especially Iran. Aims: This study was conducted to evaluate the effect of hydroalcoholic extract of Echinacea purpurea in a combination therapy with glucantime in the treatment of cutaneous leishmaniasis caused by Leishmania major. Materials and Methods: Hydroalcoholic extract of E. purpurea was prepared from the plant. Amastigote form of L. major was inoculated to the tail base of thirty mice. After their tails became wounded, mice were divided into six groups. The first group was used as control and the second group received 100 mg/kg of Echinacea extract (orally). The third group was treated by meglumine antimoniate with dose of 20 mg/kg. Combination therapy was used for group four, five, and six where the mice received a different concentration of extract (100–200 mg/kg) and glucantime (10–20 mg/kg). The size of the cutaneous lesion on tail base was measured regularly. Findings were analyzed by SPSS software and using Kruskal-Wallis test. Results: The sizes of the lesion were increased in all mice of control group by the time. The mean size of lesions in mice receiving the extract and/or receiving the extract along with meglumine antimoniate was lower than those of control mice, but the differences were not statistically significant (P > 0.05). On the other hand, the differences between the group of mice which received meglumine antimoniate alone, and the rest of groups were statistically significant (P < 0.05). Conclusion: E. purpurea extract in doses which have been used in this study and combination with meglumine antimoniate was not much effective against L. major in BALB/C mice. PMID:28251109

  1. Oral treatment with zinc sulfate increases the expression of Th1 cytokines mRNA in BALB/c mice infected with Leishmania major.

    PubMed

    Afshari, Mahsa; Riazi-Rad, Farhad; Khaze, Vahid; Bahrami, Fariborz; Ajdary, Soheila; Alimohammadian, Mohammad Hossein

    2016-05-01

    Leishmaniases consist of a group of diseases caused by protozoan parasites of Leishmania genus. The outcome of the disease depends on the immune responses of the host as well as the pathogenicity of the strain of the parasite. In murine models, the inoculation of Leishmania major into resistant mice results in Th1 responses and recovery from the infection. However in the susceptible mice, the same inoculation leads to a profile of Th2 responses. Zinc (Zn) is an essential trace element which is required for the growth and development of the immune responses. In this study, the influence of Zn sulfate on mRNA expression of main cytokines of the immune response was studied in susceptible BALB/c mice infected with L. major. The inoculated mice were divided into 3 groups, namely the untreated (control), the zinc sulfate treated (weeks 2, 4 and 8), and the Glucantime-treated (weeks 4 and 8) mice. During different time points post-infection, the lesion sizes and the parasite burden were measured in all the groups. Moreover, the expression of Ifng, Il4, Il10 and Il12 mRNA levels in the draining lymph nodes of the treated mice were compared to the control mice using real-time PCR. Our data demonstrated significant decreases in lesion sizes and parasite loads in Zn sulfate treated group compared to the untreated group. Moreover, significant fold increases in expression of Ifng transcript were observed in mice treated with Zn sulfate compared to the control. The ratio of Ifng/Il4 mRNA was also higher in Zn sulfate-treated mice compared to Glucantime-treated animals. These results indicate that Zn Sulfate has the ability to induce strong Th1 responses in susceptible BALB/c mice inoculated with L. major. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Comparison of the Proteome Profiling of Iranian isolates of Leishmania tropica, L. major and L. infantum by Two-Dimensional Electrophoresis (2-DE) and Mass-spectrometry

    PubMed Central

    HAJJARAN, Homa; MOHAMMADI BAZARGANI, Mitra; MOHEBALI, Mehdi; BURCHMORE, Richard; HOSSEINI SALEKDEH, Ghasem; KAZEMI-RAD, Elham; KHORAMIZADEH, Mohammad Reza

    2015-01-01

    Background: The mechanisms of virulence and species differences of Leishmania parasites are under the influence of gene expression regulations at posttranscriptional stages. In Iran, L. major and L. tropica are known as principal agents of cutaneous leishmaniasis, while L. infantum causes visceral leishmaniasis. Methods: As a preliminary study, we compared the proteome mapping of the above three Iranian isolates of Leishmania species through the 2-dimension electrophoresis (2-DE), and identified the prominent proteins by Liquid Chromatography (LC) mass spectrometry. Results: We reproducibly detected about 700 protein spots in each species by using the Melanie software. Totally, 264 proteins exhibited significant changes among 3 species. Forty nine protein spots identified in both L. tropica and L. major were similar in position in the gel, whereas only 35 of L. major proteins and 10 of L. tropica proteins were matched with those of L. infantum. Having identified 24 proteins in the three species, we sought to provide possible explanations for their differential expression patterns and discuss their relevance to cell biology. Conclusion: The comparison of proteome profiling pattern of the 3 species identified limit up and limit down regulated or absent /present proteins. In addition, the LC-MS data analysis showed that most of the protein spots with differential abundance in the 3 species are involved in cell motility and cytoskeleton, cell signaling and vesicular trafficking, intracellular survival / proteolysis, oxidative stress defense, protein synthesis, protein ubiquitination / proteolysis, and stress related proteins. Differentially proteins distributed among the species maybe implicated in host pathogenecity interactions and parasite tropism to cutaneous or visceral tissue macrophages. PMID:26811718

  3. Effect of hydroalcoholic extract of Echinacea purpurea in combination with meglumine antimoniate on treatment of Leishmania major-induced cutaneous leishmaniasis in BALB/c mice.

    PubMed

    Sarkari, Bahador; Mohseni, Mobin; Moein, Mahmoud Reza; Shahriarirad, Reza; Asgari, Qasem

    2017-01-01

    Progressive resistance of Leishmania parasite to available drugs including, meglumine antimoniate, has been reported from various regions of the world, especially Iran. This study was conducted to evaluate the effect of hydroalcoholic extract of Echinacea purpurea in a combination therapy with glucantime in the treatment of cutaneous leishmaniasis caused by Leishmania major. Hydroalcoholic extract of E. purpurea was prepared from the plant. Amastigote form of L. major was inoculated to the tail base of thirty mice. After their tails became wounded, mice were divided into six groups. The first group was used as control and the second group received 100 mg/kg of Echinacea extract (orally). The third group was treated by meglumine antimoniate with dose of 20 mg/kg. Combination therapy was used for group four, five, and six where the mice received a different concentration of extract (100-200 mg/kg) and glucantime (10-20 mg/kg). The size of the cutaneous lesion on tail base was measured regularly. Findings were analyzed by SPSS software and using Kruskal-Wallis test. The sizes of the lesion were increased in all mice of control group by the time. The mean size of lesions in mice receiving the extract and/or receiving the extract along with meglumine antimoniate was lower than those of control mice, but the differences were not statistically significant (P > 0.05). On the other hand, the differences between the group of mice which received meglumine antimoniate alone, and the rest of groups were statistically significant (P < 0.05). E. purpurea extract in doses which have been used in this study and combination with meglumine antimoniate was not much effective against L. major in BALB/C mice.

  4. Gender Is a Major Determinant of the Clinical Evolution and Immune Response in Hamsters Infected with Leishmania spp.

    PubMed Central

    Travi, Bruno L.; Osorio, Yaneth; Melby, Peter C.; Chandrasekar, Bysani; Arteaga, Lourdes; Saravia, Nancy G.

    2002-01-01

    In regions where leishmaniasis is endemic, clinical disease is usually reported more frequently among males than females. This difference could be due to disparate risks of exposure of males and females, but gender-related differences in the host response to infection may also play a role. Experimental studies of the influence of gender on Leishmania infection have not included parasites of the subgenus Viannia, which is the most common cause of cutaneous leishmaniasis in the Americas. Mice are not readily susceptible to infection by Leishmania (Viannia) spp., but cutaneous infection of hamsters with L. (V.) panamensis or L. (V.) guyanensis resulted in chronic lesions typical of the human disease caused by these parasites. Strikingly, infection of male hamsters resulted in significantly greater lesion size and severity, an increased rate of dissemination to distant cutaneous sites, and a greater parasite burden in the draining lymph node than infection in female animals. Two lines of evidence indicated this gender-related difference in disease evolution was determined at least in part by the sex hormone status of the animal. First, prepubertal male animals had smaller and/or less severe cutaneous lesions than adult male animals. Second, infection of testosterone-treated female animals resulted in significantly larger lesions than in untreated female animals. The increased severity of disease in male compared to female animals was associated with significantly greater intralesional expression of interleukin-4 (IL-4) (P = 0.04), IL-10 (P = 0.04), and transforming growth factor β (TGF-β) (P < 0.001), cytokines known to promote disease in experimental leishmaniasis. There was a direct correlation between the expression of TGF-β mRNA and lesion size (Spearman's correlation coefficient = 0.873; P < 0.001). These findings demonstrate an inherent risk of increased disease severity in male animals, which is associated with a more permissive immune response. PMID:11953362

  5. A novel protein coding potential of long intergenic non-coding RNAs (lincRNAs) in the kinetoplastid protozoan parasite Leishmania major.

    PubMed

    Pawar, Harsh; Pai, Kalpana; Patole, Milind S

    2017-03-01

    Cutaneous leishmaniasis (CL) is caused by a kinetoplastid protozoan parasite Leishmania major, as a skin ulcer at the site of the sandfly bite. CL is curable and in most cases ulcers heal spontaneously within three to six months leaving a scar and disfiguration. Complete genome of L. major was reported in 2005 at the very initial phase of kinetoplastid parasite genome sequencing project. Presently, L. major genome is most studied and comprehensively annotated genome and therefore, it is being used as a reference genome for annotating recently sequenced Leishmanial genomes. A recent study reporting global transcriptome of L. major promastigotes, identified 1884 uniquely expressed non-coding RNAs (ncRNA) in L. major. In the current study, an in-depth analysis of the 1884 novel ncRNAs was carried out using a proteogenomic approach to identify their protein coding potential. Our analysis resulted in identification of eight novel protein coding genes based on mass spectrometry data. We have analyzed each of these eight novel CDS and in the process have improved the genome annotation of L. major on the basis of mass spectrometry derived peptide data. Although sequenced a decade ago, the improvement in the L. major genome annotation thus is an ongoing process. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Leishmania major inhibits IL-12 in macrophages by signaling through CR3 (CD11b/CD18) and downregulation of ETS-mediated transcription

    PubMed Central

    Ricardo-Carter, Cristina; Favila, Michelle; Polando, Rachel E.; Cotton, Rachel N.; Horner, Kimberly Bogard; Condon, David; Ballhorn, Wibke; Whitcomb, James P.; Yadav, Mahesh; Geister, Rebecca L.; Schorey, Jeffery S.; McDowell, Mary Ann

    2013-01-01

    SUMMARY Leishmania major is an etiological agent of cutaneous leishmaniasis. The parasite primarily infects immune sentinel cells, specifically macrophages and dendritic cells, in the mammalian host. Infection is receptor mediated and is known to involve parasite binding to cell surface protein complement receptor 3 (CR3, Mac-1, CD11b/CD18). Engagement of CR3 by various ligands inhibits production of interleukin-12 (IL-12), the cytokine that drives anti-leishmanial T helper 1-type immune responses. Likewise, L. major infection inhibits IL-12 production and activation of host macrophages. Our data indicate that in the absence of CR3, L. major-infected bone marrow-derived macrophages produce more IL-12 and nitric oxide compared to WT cells upon LPS stimulation. We therefore investigated multiple signaling pathways by which L. major may inhibit IL-12 transcription through CR3 ligation. We demonstrate that L. major infection does not elicit significant NFκB p65, MAPK, IRF-1, or IRF-8 activation in WT or CD11b deficient macrophages. Furthermore, infection neither inhibits LPS-induced MAPK or NFκB activation, nor blocks IFN-γ-activated IRF-1 and IRF-8. ETS-mediated transcription, however, is inhibited by L. major infection independently of CR3. Our data indicate that L. major mediated inhibition of IL-12 occurs through CR3 engagement, however the mechanism of inhibition is independent of NFκB, MAPK, IRF, and ETS. PMID:23834512

  7. Leishmania major inhibits IL-12 in macrophages by signalling through CR3 (CD11b/CD18) and down-regulation of ETS-mediated transcription.

    PubMed

    Ricardo-Carter, C; Favila, M; Polando, R E; Cotton, R N; Bogard Horner, K; Condon, D; Ballhorn, W; Whitcomb, J P; Yadav, M; Geister, R L; Schorey, J S; McDowell, M A

    2013-12-01

    Leishmania major is an aetiological agent of cutaneous leishmaniasis. The parasite primarily infects immune sentinel cells, specifically macrophages and dendritic cells, in the mammalian host. Infection is receptor mediated and is known to involve parasite binding to cell surface protein complement receptor 3 (CR3, Mac-1, CD11b/CD18). Engagement of CR3 by various ligands inhibits production of interleukin-12 (IL-12), the cytokine that drives antileishmanial T helper 1-type immune responses. Likewise, L. major infection inhibits IL-12 production and activation of host macrophages. Our data indicate that in the absence of CR3, L. major-infected bone marrow-derived macrophages produce more IL-12 and nitric oxide compared with WT cells upon lipopolysaccharide (LPS) stimulation. We therefore investigated multiple signalling pathways by which L. major may inhibit IL-12 transcription through CR3 ligation. We demonstrate that L. major infection does not elicit significant NFκB p65, MAPK, IRF-1 or IRF-8 activation in WT or CD11b-deficient macrophages. Furthermore, infection neither inhibits LPS-induced MAPK or NFκB activation nor blocks IFN-γ-activated IRF-1 and IRF-8. ETS-mediated transcription, however, is inhibited by L. major infection independently of CR3. Our data indicate that L. major-mediated inhibition of IL-12 occurs through CR3 engagement; however, the mechanism of inhibition is independent of NFκB, MAPK, IRF and ETS. © 2013 John Wiley & Sons Ltd.

  8. Crystal structure of an Fe-S cluster-containing fumarate hydratase enzyme from Leishmania major reveals a unique protein fold

    PubMed Central

    Drennan, Catherine L.; Nonato, M. Cristina

    2016-01-01

    Fumarate hydratases (FHs) are essential metabolic enzymes grouped into two classes. Here, we present the crystal structure of a class I FH, the cytosolic FH from Leishmania major, which reveals a previously undiscovered protein fold that coordinates a catalytically essential [4Fe-4S] cluster. Our 2.05 Å resolution data further reveal a dimeric architecture for this FH that resembles a heart, with each lobe comprised of two domains that are arranged around the active site. Besides the active site, where the substrate S-malate is bound bidentate to the unique iron of the [4Fe-4S] cluster, other binding pockets are found near the dimeric enzyme interface, some of which are occupied by malonate, shown here to be a weak inhibitor of this enzyme. Taken together, these data provide a framework both for investigations of the class I FH catalytic mechanism and for drug design aimed at fighting neglected tropical diseases. PMID:27528683

  9. Immune response of BALB/c mice against an experimental vaccine of Alum precipitated autoclaved Leishmania major (Alum-ALM) mixed with BCG or Mycobacterium vaccae.

    PubMed

    Nateghi Rostami, M; Keshavarz, H; Khamesipour, A

    2010-04-01

    Immune response in BALB/c mice immunized 3 times with different doses (50 μg or 200 μg of protein) of Alum precipitated autoclaved Leishmania major (Alum-ALM) mixed with either BCG (1x10(7); CFU) or different doses of killed Mycobacterium vaccae (1x10(6), 1x10(7)) was assessed. Mice immunized with low dose of Alum-ALM mixed with either BCG or low M. vaccae showed a significantly higher IFN-gamma production and a lower IL-4 level and a significantly lower parasite burden compared to the control PBS injected group. It seems that immunization with a low dose of Alum-ALM mixed with an adjuvant induces a Th1 type of immune response in susceptible BALB/c mice.

  10. Crystal structure of an Fe-S cluster-containing fumarate hydratase enzyme from Leishmania major reveals a unique protein fold.

    PubMed

    Feliciano, Patricia R; Drennan, Catherine L; Nonato, M Cristina

    2016-08-30

    Fumarate hydratases (FHs) are essential metabolic enzymes grouped into two classes. Here, we present the crystal structure of a class I FH, the cytosolic FH from Leishmania major, which reveals a previously undiscovered protein fold that coordinates a catalytically essential [4Fe-4S] cluster. Our 2.05 Å resolution data further reveal a dimeric architecture for this FH that resembles a heart, with each lobe comprised of two domains that are arranged around the active site. Besides the active site, where the substrate S-malate is bound bidentate to the unique iron of the [4Fe-4S] cluster, other binding pockets are found near the dimeric enzyme interface, some of which are occupied by malonate, shown here to be a weak inhibitor of this enzyme. Taken together, these data provide a framework both for investigations of the class I FH catalytic mechanism and for drug design aimed at fighting neglected tropical diseases.

  11. Innate Immunity against Leishmania Infections

    PubMed Central

    Gurung, Prajwal; Kanneganti, Thirumala-Devi

    2015-01-01

    Leishmaniasis is a major health problem that affects more than 300 million people throughout the world. The morbidity associated with the disease causes serious economic burden in Leishmania endemic regions. Despite the morbidity and economic burden associated with Leishmaniasis, this disease rarely gets noticed and is still categorized under neglected tropical diseases. The lack of research combined with the ability of Leishmania to evade immune recognition has rendered our efforts to design therapeutic treatments or vaccines challenging. Herein, we review the literature on Leishmania from innate immune perspective and discuss potential problems as well as solutions and future directions that could aid in identifying novel therapeutic targets to eliminate this parasite. PMID:26249747

  12. Combination therapy using Pentostam and Praziquantel improves lesion healing and parasite resolution in BALB/c mice co-infected with Leishmania major and Schistosoma mansoni

    PubMed Central

    2013-01-01

    Background Most natural host populations are exposed to a diversity of parasite communities and co-infection of hosts by multiple parasites is commonplace across a diverse range of systems. Co-infection with Leishmania major and Schistosoma mansoni may have important consequences for disease development, severity and transmission dynamics. Pentavalent antimonials and Praziquantel (PZQ) have been relied upon as a first line of treatment for Leishmania and Schistosoma infections respectively. However, it is not clear how combined therapy with the standard drugs will affect the host and parasite burden in concomitance. The aim of the current study was to determine the efficacy of combined chemotherapy using Pentostam and PZQ in BALB/c mice co-infected with L. major and S. mansoni. Methods The study used BALB/c mice infected with L. major and S. mansoni. A 3 × 4 factorial design with three parasite infection groups (Lm, Sm, Lm + Sm designated as groups infected with L. major, S. mansoni and L. major + S. mansoni, respectively) and four treatment regimens [P, PZQ, P + PZQ and PBS designating Pentostam®(GlaxoSmithKline UK), Praziquantel (Biltricide®, Bayer Ag. Leverkusen, Germany), Pentostam + Praziquantel and Phosphate buffered saline] as factors was applied. In each treatment group, there were 10 mice. Lesion development was monitored for 10 weeks. The parasite load, body weight, weight of the spleen and liver were determined between week 8 and week 10. Results Chemotherapy using the first line of treatment for L. major and S. mansoni reduced the lesion size and parasite loads but did not affect the growth response, spleen and liver. In the co-infected BALB/c mice, the use of Pentostam or PZQ did not result in any appreciable disease management. However, treatment with P + PZQ resulted in significantly (p < 0.05) larger reduction of lesions, net increase in the body weight, no changes in the spleen and liver weight and reduced Leishman

  13. Metal-drug synergy: new ruthenium(II) complexes of ketoconazole are highly active against Leishmania major and Trypanosoma cruzi and nontoxic to human or murine normal cells.

    PubMed

    Iniguez, Eva; Sánchez, Antonio; Vasquez, Miguel A; Martínez, Alberto; Olivas, Joanna; Sattler, Aaron; Sánchez-Delgado, Roberto A; Maldonado, Rosa A

    2013-10-01

    In our ongoing search for new metal-based chemotherapeutic agents against leishmaniasis and Chagas disease, six new ruthenium-ketoconazole (KTZ) complexes have been synthesized and characterized, including two octahedral coordination complexes-cis,fac-[Ru(II)Cl2(DMSO)3(KTZ)] (1) and cis-[Ru(II)Cl2(bipy)(DMSO)(KTZ)] (2) (where DMSO is dimethyl sulfoxide and bipy is 2,2'-bipyridine)-and four organometallic compounds-[Ru(II)(η(6)-p-cymene)Cl2(KTZ)] (3), [Ru(II)(η(6)-p-cymene)(en)(KTZ)][BF4]2 (4), [Ru(II)(η(6)-p-cymene)(bipy)(KTZ)][BF4]2 (5), and [Ru(II)(η(6)-p-cymene)(acac)(KTZ)][BF4] (6) (where en is ethylenediamine and acac is acetylacetonate); the crystal structure of 3 is described. The central hypothesis of our work is that combining a bioactive compound such as KTZ and a metal in a single molecule results in a synergy that can translate into improved activity and/or selectivity against parasites. In agreement with this hypothesis, complexation of KTZ with Ru(II) in compounds 3-5 produces a marked enhancement of the activity toward promastigotes and intracellular amastigotes of Leishmania major, when compared with uncomplexed KTZ, or with similar ruthenium compounds not containing KTZ. Importantly, the selective toxicity of compounds 3-5 toward the leishmania parasites, in relation to human fibroblasts and osteoblasts or murine macrophages, is also superior to the selective toxicities of the individual constituents of the drug. When tested against Trypanosoma cruzi epimastigotes, some of the organometallic complexes displayed activity and selectivity comparable to those of free KTZ. A dual-target mechanism is suggested to account for the antiparasitic properties of these complexes.

  14. In vitro activity of the essential oil of Cymbopogon citratus and its major component (citral) on Leishmania amazonensis.

    PubMed

    Santin, Marta Regina; dos Santos, Adriana Oliveira; Nakamura, Celso Vataru; Dias Filho, Benedito Prado; Ferreira, Izabel Cristina Piloto; Ueda-Nakamura, Tânia

    2009-11-01

    Leishmaniasis causes considerable mortality throughout the world, affecting more than 12 million people. Cymbopogon citratus (DC) Stapf, Family Poaceae, is a widely used herb in tropical countries and is also known as a source of ethnomedicines. In this study, the inhibitory effect and the morphological and ultrastructural alterations on Leishmania amazonensis by the essential oil (EO) of C. citratus and its main constituent, citral, were evaluated. The results showed that the antiproliferative activity of EO on promastigotes and axenic amastigotes, and intracellular amastigote forms of L. amazonensis was significantly better than citral, and indicated a dose-dependent effect. Neither compound showed a cytotoxic effect on macrophage strain J774G8. The promastigote forms of L. amazonensis underwent remarkable morphological and ultrastructural alterations compared with untreated cultures. These alterations were visible by light, scanning, and transmission electron microscopy of promastigotes treated with EO and citral at concentrations corresponding to the IC(50) (1.7 and 8.0 microg/ml) and IC(90) (3.2 and 25 microg/ml), respectively, after 72 h of incubation. This study revealed that citral-rich essential oil from C. citratus has promising antileishmanial properties, and is a good candidate for further research to develop a new anti-protozoan drug.

  15. Experimental Transmission of Leishmania infantum by Two Major Vectors: A Comparison between a Viscerotropic and a Dermotropic Strain

    PubMed Central

    Maia, Carla; Seblova, Veronika; Sadlova, Jovana; Votypka, Jan; Volf, Petr

    2011-01-01

    We quantified Leishmania infantum parasites transmitted by natural vectors for the first time. Both L. infantum strains studied, dermotropic CUK3 and viscerotropic IMT373, developed well in Phlebotomus perniciosus and Lutzomyia longipalpis. They produced heavy late-stage infection and colonized the stomodeal valve, which is a prerequisite for successful transmission. Infected sand fly females, and especially those that transmit parasites, feed significantly longer on the host (1.5–1.8 times) than non-transmitting females. Quantitative PCR revealed that P. perniciosus harboured more CUK3 strain parasites, while in L. longipalpis the intensity of infection was higher for the IMT373 strain. However, in both sand fly species the parasite load transmitted was higher for the strain with dermal tropism (CUK3). All but one sand fly female infected by the IMT373 strain transmitted less than 600 promastigotes; in contrast, 29% of L. longipalpis and 14% of P. perniciosus infected with the CUK3 strain transmitted more than 1000 parasites. The parasite number transmitted by individual sand flies ranged from 4 up to 4.19×104 promastigotes; thus, the maximal natural dose found was still about 250 times lower than the experimental challenge dose used in previous studies. This finding emphasizes the importance of determining the natural infective dose for the development of an accurate experimental model useful for the evaluation of new drugs and vaccines. PMID:21695108

  16. PTEN Regulates Glutamine Flux to Pyrimidine Synthesis and Sensitivity to Dihydroorotate Dehydrogenase Inhibition.

    PubMed

    Mathur, Deepti; Stratikopoulos, Elias; Ozturk, Sait; Steinbach, Nicole; Pegno, Sarah; Schoenfeld, Sarah; Yong, Raymund; Murty, Vundavalli V; Asara, John M; Cantley, Lewis C; Parsons, Ramon

    2017-04-01

    Metabolic changes induced by oncogenic drivers of cancer contribute to tumor growth and are attractive targets for cancer treatment. Here, we found that increased growth of PTEN-mutant cells was dependent on glutamine flux through the de novo pyrimidine synthesis pathway, which created sensitivity to the inhibition of dihydroorotate dehydrogenase, a rate-limiting enzyme for pyrimidine ring synthesis. S-phase PTEN-mutant cells showed increased numbers of replication forks, and inhibitors of dihydroorotate dehydrogenase led to chromosome breaks and cell death due to inadequate ATR activation and DNA damage at replication forks. Our findings indicate that enhanced glutamine flux generates vulnerability to dihydroorotate dehydrogenase inhibition, which then causes synthetic lethality in PTEN-deficient cells due to inherent defects in ATR activation. Inhibition of dihydroorotate dehydrogenase could thus be a promising therapy for patients with PTEN-mutant cancers.Significance: We have found a prospective targeted therapy for PTEN-deficient tumors, with efficacy in vitro and in vivo in tumors derived from different tissues. This is based upon the changes in glutamine metabolism, DNA replication, and DNA damage response which are consequences of inactivation of PTENCancer Discov; 7(4); 380-90. ©2017 AACR.See related article by Brown et al., p. 391This article is highlighted in the In This Issue feature, p. 339.

  17. Probing the structure of Leishmania major DHFR TS and structure based virtual screening of peptide library for the identification of anti-leishmanial leads.

    PubMed

    Rajasekaran, Rajalakshmi; Chen, Yi-Ping Phoebe

    2012-09-01

    Leishmaniasis, a multi-faceted ethereal disease is considered to be one of the World's major communicable diseases that demands exhaustive research and control measures. The substantial data on these protozoan parasites has not been utilized completely to develop potential therapeutic strategies against Leishmaniasis. Dihydrofolate reductase thymidylate synthase (DHFR-TS) plays a major role in the infective state of the parasite and hence the DHFR-TS based drugs remains of much interest to researchers working on Leishmaniasis. Although, crystal structures of DHFR-TS from different species including Plasmodium falciparum and Trypanosoma cruzi are available, the experimentally determined structure of the Leishmania major DHFR-TS has not yet been reported in the Protein Data Bank. A high quality three dimensional structure of L.major DHFR-TS has been modeled through the homology modeling approach. Carefully refined and the energy minimized structure of the modeled protein was validated using a number of structure validation programs to confirm its structure quality. The modeled protein structure was used in the process of structure based virtual screening to figure out a potential lead structure against DHFR TS. The lead molecule identified has a binding affinity of 0.51 nM and clearly follows drug like properties.

  18. Diagnostic Antigens of Leishmania.

    DTIC Science & Technology

    1994-01-31

    L. major (LTM p-2), L. major (Friedlander), and Trypanosoma cruzi (MHOM/CH/00/Tulahuen C2) were used. Leishmania promastigotes and T. cruzi ...some weak hybridization was observed with L. amazonensis, but none was seen with L. braziliensis, L. guyanensis, or T cruzi . A similar, overlapping... cruzi (8) have been previously isolated by us. To address this possibility in rLt-1, a portion of the repeat was expressed separately as rLt-lr. The

  19. What is a chromosome like in Leishmania?

    PubMed

    Galindo, I; Arguello, C; Ramirez, J L

    1993-01-01

    Hereby, we present evidences of the presence of centromeric proteins in Leishmania mexicana. These proteins were partially purified and used to bind Leishmania DNA. The protein-DNA complex was immunoprecipitated and the DNA extracted, like in the human sample. Two major fragments of 1.2 and 2.2 Kbp, respectively, were resolved in agarose gel electrophoresis. A model of chromosomal structure and chromosomal segregation in Leishmania is presented.

  20. Validity and reliability of enzyme immunoassays using Leishmania major or L. infantum antigens for the diagnosis of canine visceral leishmaniasis in Brazil.

    PubMed

    de Arruda, Mauro Maciel; Figueiredo, Fabiano Borges; Cardoso, Fernanda Alvarenga; Hiamamoto, Roberto Mitsuyoshi; Brazuna, Júlia Cristina Macksoud; de Oliveira, Maria Regina Fernandes; Noronha, Elza Ferreira; Romero, Gustavo Adolfo Sierra

    2013-01-01

    American visceral leishmaniasis is caused by the protozoan Leishmania infantum. Dogs are the main reservoirs in the domestic transmission cycle. The limited accuracy of diagnostic tests for canine leishmaniasis may contribute to the lack of impact of control measures recommended by the Brazilian Ministry of Health. The objective of this study was to estimate the accuracy of two enzyme-linked immunosorbent assays employing L. major or L. infantum antigens and their reliability between three laboratories of different levels of complexity. A validation study of ELISA techniques using L. major or L. infantum antigens was conducted. Direct visualization of the parasite in hematoxylin/eosin-stained histopathological sections, immunohistochemistry, and isolation of the parasite in culture.were used as gold standard. An animal that was positive in at least one of the tests was defined as infected with L. infantum. Serum samples collected from 1,425 dogs were analyzed. Samples were separated in three aliquots and tested in three different laboratories. Sensitivity, specificity and the area under de ROC curve were calculated and the reliability was evaluated between the participant laboratories. The sensitivity was 91.8% and 89.8% for the L. major and L. infantum assays, respectively. The specificity was 83.75% and 82.7% for the L. major and L. infantum assays, respectively. The area under de ROC curve was 0.920 and 0.898 for L. major and L. infantum, respectively. The mean intraclass correlation coefficients between laboratories ranged from 0.890 to 0.948 when L. major was used as antigen, and from 0.818 to 0.879 when L. infantum was used. ELISA tests using L. major or L. infantum antigens have similar accuracy and reliability. Our results do not support the substitution of the L. major antigen of the ELISA test currently used for the diagnosis of canine visceral leishmaniasis in Brazil.

  1. Sphingomyelin Liposomes Containing Soluble Leishmania major antigens Induced Strong Th2 Immune Response in BALB/c Mice

    PubMed Central

    Chavoshian, Omid; Biari, Nazanin; Badiee, Ali; Khamesipour, Ali; Abbasi, Azam; Saberi, Zahra; Jalali, Seyed Amir; Jaafari, Mahmoud Reza

    2013-01-01

    Objective(s): Soluble Leishmania antigens (SLA) provide suitable protection against leishmaniasis in murine model when delivered by an appropriate delivery system. Liposomes have been shown to be suitable vaccine delivery systems against leishmaniasis, however, the phospholipase-A (PLA) activity of SLA is a drawback to prepare a stable liposomal SLA. One strategy to overcome this problem might be using a lipid which is resistant to PLA activity of SLA such as sphingomyelin (SM). The aim of this study was to evaluate the effect of stable SM liposomes containing SLA on the immune response induced against leishmaniasis in BALB/c mice . Materials and Methods: BALB/c mice were immunized subcutaneously, three times with 2-week intervals, with SLA, SM-liposome-SLA, empty liposome or buffer. As criteria for protection, footpads swelling at the site of challenge and foot parasite loads were assessed. The immune responses were also evaluated by determination of IgG subtypes and the level of IFN-γ and IL-4 in cultured splenocytes. Results: The group of mice receiving SM-liposome-SLA, showed a significant large footpad swelling, higher parasite burden in foot and higher IL-4 level compared to the group immunized with buffer. In terms of IgG and IgG isotypes, there was no significant difference between the mice receiving SM-liposome-SLA and the mice that received buffer. Moreover, the immune response induced by SM-liposome-SLA showed no significant difference compared with the one caused by SLA alone. Conclusion: It is concluded that SM-liposome-SLA is not an appropriate strategy to induce Th1 immune response and protect the mice against Leishmaniasis; however, SM-liposomes could be suitable vaccine delivery systems when a Th2 response is needed. PMID:24171074

  2. Increased myelopoiesis during Leishmania major infection in mice: generation of 'safe targets', a possible way to evade the effector immune mechanism.

    PubMed Central

    Mirkovich, A M; Galelli, A; Allison, A C; Modabber, F Z

    1986-01-01

    BALB/c mice are highly susceptible to Leishmania major infection and develop a disseminated lethal disease. Previous experiments indicate that during infection the spleen is heavily populated with large mononuclear cells containing amastigotes. Morphologically these cells resemble undifferentiated monocytes and granulocytes. In this study we examined myelopoiesis in BALB/c and C57BL/6 (resistant) mice during infection with L. major. The number of macrophage-granulocyte precursors in the spleen of infected BALB/c mice, determined by colony forming units in soft-agar cultures (cfu-c), increased steadily to a level of about 60 times that of normal sex- and age-matched controls. In C57BL/6 mice, spleen cfu-c peaked at about 1 month post-infection (four times that of normal controls) and declined thereafter to about two times normal levels. The number of cfu-c in the bone marrow did not change significantly in either strain during the infection. Colony stimulating activity (CSA) was found in supernates of cultures of adherent cells from the spleen of infected BALB/c mice. Under the same conditions, CSA was non-detectable in supernates of nonadherent spleen cells of infected mice, and those of adherent or nonadherent spleen cells of control animals. A possible role of undifferentiated macrophage-granulocytes in the exquisite susceptibility of BALB/c mice to L. major infection is discussed. PMID:3488146

  3. Leishmania major: genetic heterogeneity of Iranian isolates by single-strand conformation polymorphism and sequence analysis of ribosomal DNA internal transcribed spacer.

    PubMed

    Tashakori, Mahnaz; Mahnaz, Tashakori; Kuhls, Katrin; Katrin, Kuhls; Al-Jawabreh, Amer; Amer, Al-Jawabreh; Mauricio, Isabel L; Isabel, Mauricio; Schönian, Gabriele; Gabriele, Schönian; Farajnia, Safar; Safar, Farajnia; Alimohammadian, Mohammad Hossein; Hossein, Alimohammadian Mohammad

    2006-04-01

    Protozoan parasites of Leishmania major are the causative agents of cutaneous leishmaniasis in different parts of Iran. We applied PCR-based methods to analyze L. major parasites isolated from patients with active lesions from different geographic areas in Iran in order to understand DNA polymorphisms within L. major species. Twenty-four isolates were identified as L. major by RFLP analysis of the ribosomal internal transcribed spacer 1 (ITS1) amplicons. These isolates were further studied by single-strand conformation polymorphism (SSCP) analysis and sequencing of ITS1 and ITS2. Data obtained from SSCP analysis of the ITS1 and ITS2 loci revealed three and four different patterns among all studied samples, respectively. Sequencing of ITS1 and ITS2 confirmed the results of SSCP analysis and showed the potential of the PCR-SSCP method for assessing genetic heterogeneity within L. major. Different patterns in ITS1 were due to substitution of one nucleotide, whereas in ITS2 the changes were defined by variation in the number of repeats in two polymorphic microsatellites. In total five genotypic groups LmA, LmB, LmC, LmD and LmE were identified among L. major isolates. The most frequent genotype, LmA, was detected in isolates collected from different endemic areas of cutaneous leishmaniasis in Iran. Genotypes LmC, LmD and LmE were found only in the new focus of CL in Damghan (Semnan province) and LmB was identified exclusively among isolates of Kashan focus (Isfahan province). The distribution of genetic polymorphisms suggests the existence of distinct endemic regions of L. major in Iran.

  4. Identification of potential inhibitors for oncogenic target of dihydroorotate dehydrogenase using in silico approaches

    NASA Astrophysics Data System (ADS)

    Surekha, Kanagarajan; Nachiappan, Mutharasappan; Prabhu, Dhamodharan; Choubey, Sanjay Kumar; Biswal, Jayashree; Jeyakanthan, Jeyaraman

    2017-01-01

    Dihydroorotate dehydrogenase (DHODH) plays a major role in the rate limiting step of de novo pyrimidine biosynthesis pathway and it is pronounced as a novel target for drug development of cancer. The currently available drugs against DHODH are ineffective and bear various side effects. Three-dimensional structure of the targeted protein was constructed using molecular modeling approach followed by 100 ns molecular dynamics simulations. In this study, High Throughput Virtual Screening (HTVS) was performed using various compound libraries to identify pharmacologically potential molecules. The top four identified lead molecules includes NCI_47074, HitFinder_7630, Binding_66981 and Specs_108872 with high docking score of -9.45, -8.29, -8.04 and -8.03 kcal/mol and the corresponding binding free energy were -16.25, -56.37, -26.93 and -48.04 kcal/mol respectively. Arg122, Arg185, Glu255 and Gly257 are the key residues found to be interacting with the ligands. Molecular dynamics simulations of DHODH-inhibitors complexes were performed to assess the stability of various conformations from complex structures of TtDHODH. Furthermore, stereoelectronic features of the ligands were explored to facilitate charge transfer during the protein-ligand interactions using Density Functional Theoretical approach. Based on in silico analysis, the ligand NCI_47074 ((2Z)-3-({6-[(2Z)-3-carboxylatoprop-2-enamido]pyridin-2-yl}carbamoyl)prop-2-enoate) was found to be the most potent lead molecule which was validated using energetic and electronic parameters and it could serve as a template for designing effective anticancerous drug molecule.

  5. Zoonotic disease in a peripheral population: persistence and transmission of Leishmania major in a putative sink-source system in the Negev Highlands, Israel.

    PubMed

    Berger, Ruti; Wasserberg, Gideon; Warburg, Alon; Orshan, Laor; Kotler, Burt P

    2014-08-01

    Populations at the edge of their geographic distributions are referred to as peripheral populations. Very little attention has been given to this topic in the context of persistence of infectious disease in natural populations. In this study, we examined this question using zoonotic cutaneous leishmaniasis (ZCL) caused by Leishmania major in the Negev Desert of Israel as a model system. Here, we suggest that the regional persistence of Phlebotomus papatasi populations and L. major transmission in the Sede Boqer region could be explained through processes akin to sink-source and/or mainland-island metapopulation dynamics. Given its potentially enzootically superior ecological conditions, we hypothesize that the Zin Valley ecotope constitutes the "mainland" or the "source" patch for the Sede Boqer area where L. major transmission is persistent and resistant to local extinctions (die-outs) whereas the local sand fly populations on the Zin Plateau ("island patch" or "sink patch") are more prone to local extinctions. Between 2007 and 2008, we trapped sand flies and sand rats in the two areas and compared sand fly abundance and L. major infection prevalence in both. In both 2007 and 2008, sand fly abundance was high and continuous in the Zin Wadi but low and discontinuous in the Zin Plateau. Infection prevalence of sand rats was significantly higher in the Wadi (13%) compared with the Zin Plateau (3%). Minimum infection rate in sand flies did not differ significantly between the two areas. Overall, our results are consistent with the premise that the Zin Valley population is relatively robust in terms of L. major transmission, whereas transmission is potentially more tenuous in the plateau. Understanding the biotic and abiotic processes enabling the persistence of L. major and other vector-borne diseases in peripheral disease foci is important for predicting the effect of anthropogenic land use and climate change.

  6. Crystal Structure of the Leishmania Major Phosphodiesterase LmjPDEB1 and Insight into the Design of hte Parasite-Selective Inhibitors

    SciTech Connect

    Wang,H.; Yan, Z.; Geng, J.; Kunz, S.; Seebeck, T.; Ke, H.

    2007-01-01

    Human leishmaniasis is a major public health problem in many countries, but chemotherapy is in an unsatisfactory state. Leishmania major phosphodiesterases (LmjPDEs) have been shown to play important roles in cell proliferation and apoptosis of the parasite. Thus LmjPDE inhibitors may potentially represent a novel class of drugs for the treatment of leishmaniasis. Reported here are the kinetic characterization of the LmjPDEB1 catalytic domain and its crystal structure as a complex with 3-isobutyl-1-methylxanthine (IBMX) at 1.55 Angstroms resolution. The structure of LmjPDEB1 is similar to that of human PDEs. IBMX stacks against the conserved phenylalanine and forms a hydrogen bond with the invariant glutamine, in a pattern common to most inhibitors bound to human PDEs. However, an extensive structural comparison reveals subtle, but significant differences between the active sites of LmjPDEB1 and human PDEs. In addition, a pocket next to the inhibitor binding site is found to be unique to LmjPDEB1. This pocket is isolated by two gating residues in human PDE families, but constitutes a natural expansion of the inhibitor binding pocket in LmjPDEB1. The structure particularity might be useful for the development of parasite-selective inhibitors for the treatment of leishmaniasis.

  7. LB broth-lyophilized Rabbit serum (LLR) as a new and suitable culture medium for cultivation of promastigotes of Leishmania major.

    PubMed

    Nasiri, Vahid; Dalimi, Abdolhossein; Ghaffarifar, Fatemeh

    2017-03-01

    Fetal calf serum is the major part and the most expensive ingredient of the Leishmania culture media. Here, the efficacy of the LB broth-lyophilized Rabbit serum medium (LLR) was evaluated in cultivation of Leishmaniamajor. Conventional Luria-Bertani (LB) broth medium was prepared and autoclaved for 15 min at 121 °C and then lyophilized Rabbit serum was added at the 1, 2.5, 5 and 10 % final concentrations. The efficacy of medium was evaluated by assessing the growth ability and replication pattern of the promastigotes of L. major. According to our finding, the LLR medium with 5-10 % lyophilized Rabbit serum supported the growth of the parasites and can be used for cultivation of Leishmanian parasites with acceptable In vivo infectivity for research purpose. The ability of the parasites to survive and proliferating in the presence of lyophilized Rabbit serum indicating that this serum is a good nutritional source. This study opens a new way to make low-cost medium that could be used in cultivation of Leishmanian parasites.

  8. Crystal structure of the Leishmania major phosphodiesterase LmjPDEB1 and insight into the design of the parasite selective inhibitors

    PubMed Central

    Wang, Huanchen; Yan, Zier; Geng, Jie; Kunz, Stefan; Seebeck, Thomas; Ke, Hengming

    2010-01-01

    Human leishmaniasis is a major public health problem in many countries, but chemotherapy is in an unsatisfactory state. Leishmania major phosphodiesterases (LmjPDEs) have been shown to play important roles in cell proliferation and apoptosis of the parasite. Thus LmjPDE inhibitors may potentially represent a novel class of drugs for the treatment of leishmaniasis. Reported here are the kinetic characterization of the LmjPDEB1 catalytic domain and its crystal structure as a complex with 3-isobutyl-1-methylxanthine (IBMX) at 1.55 Å resolution. The structure of LmjPDEB1 is similar to that of human PDEs. IBMX stacks against the conserved phenylalanine and forms a hydrogen bond with the invariant glutamine, in a pattern common to most inhibitors bound to human PDEs. However, an extensive structural comparison reveals subtle but significant differences between the active sites of LmjPDEB1 and human PDEs. In addition, a pocket next to the inhibitor binding site is found to be unique to LmjPDEB1. This pocket is isolated by two gating residues in human PDE families, but constitutes a natural expansion of the inhibitor binding pocket in LmjPDEB1. The structure particularity might be useful for the development of parasite-selective inhibitors for the treatment of leishmaniasis. PMID:17944832

  9. The use of a water-soluble formazan complex to quantitate the cell number and mitochondrial function of Leishmania major promastigotes.

    PubMed

    Berg, K; Zhai, L; Chen, M; Kharazmi, A; Owen, T C

    1994-01-01

    One of the methods to quantitate Leishmania major promastigotes (LmP) has been to utilize the formation of a formazan dye, which in turn is produced via conversion of an artificial substrate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The method has one major drawback in that the formazan complex precipitates inside the parasites and has to be extracted by denaturants before measurements can be performed. By using a new synthetic substrate, 3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4-sulfonyl)-2H-tetrazolium (MTS), the extraction procedure is eliminated as the formazan-like dye is released spontaneously into the medium, making it possible to perform several measurements on the same parasite culture without disturbing or killing the parasites. The measurements were shown to reflect the numbers of parasites as confirmed via comparative experiments using radioactive thymidine uptake and cell counting, respectively. The method is simple, fast, and highly reproducible and is suitable for drug screening, identification of drug-resistant isolates, and growth-kinetics studies. It is therefore contemplated that the MTS method will be a general and useful technique in this field of parasitology.

  10. A glycosylphosphatidylinositol (GPI)-negative phenotype produced in Leishmania major by GPI phospholipase C from Trypanosoma brucei: topography of two GPI pathways

    PubMed Central

    1994-01-01

    The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysaccharide), are glycosylphosphatidylinositol (GPI) anchored. We expressed a cytoplasmic glycosylphosphatidylinositol phospholipase C (GPI-PLC) in L. major in order to examine the topography of the protein- GPI and polysaccharide-GPI pathways. In L. major cells expressing GPI- PLC, cell-associated gp63 could not be detected in immunoblots. Pulse- chase analysis revealed that gp63 was secreted into the culture medium with a half-time of 5.5 h. Secreted gp63 lacked anti-cross reacting determinant epitopes, and was not metabolically labeled with [3H]ethanolamine, indicating that it never received a GPI anchor. Further, the quantity of putative protein-GPI intermediates decreased approximately 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. However, GPI-PLC cleaved polysaccharide-GPI intermediates (glycoinositol phospholipids) in vitro. Thus, reactions specific to the polysaccharide-GPI pathway are compartmentalized in vivo within the endoplasmic reticulum, thereby sequestering polysaccharide-GPI intermediates from GPI-PLC cleavage. On the contrary, protein-GPI synthesis at least up to production of Man(1 alpha 6)Man(1 alpha 4)GlcN-(1 alpha 6)-myo-inositol-1-phospholipid is cytosolic. To our knowledge this represents the first use of a catabolic enzyme in vivo to elucidate the topography of biosynthetic pathways. GPI-PLC causes a protein-GPI-negative phenotype in L. major, even when genes for GPI biosynthesis are functional. This phenotype is remarkably similar to that of some GPI mutants of mammalian cells: implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma are discussed. PMID:8132715

  11. Different Morphologies of Leishmania major Amastigotes with No Molecular Diversity in a Neglected Endemic Area of Zoonotic Cutaneous Leishmaniasis in Iran

    PubMed Central

    Spotin, Adel; Rouhani, Soheila; Ghaemmaghami, Parnazsadat; Haghighi, Ali; Zolfaghari, Mohammad Reza; Amirkhani, Aref; Farahmand, Mahin; Bordbar, Ali; Parvizi, Parviz

    2015-01-01

    Background: Molecular diversity of Leishmania major and its morphological changes have become a controversial issue among researchers. Some aspects of polymorphic shapes of amastigotes in clinical manifestations along with molecular variation were evaluated among suspected patients of some exceptional zoonotic cutaneous leishmaniasis locations in Northern Khuzestan, Southwestern Iran. Methods: Suspected patients (n = 165) were sampled in zoonotic cutaneous leishmaniasis foci over two consecutive years during 2012-2014. Prepared smears were stained, scaled and measured by ocular micrometer. DNA was extracted from smears; ITS-rDNA and Cytochrome b (Cyt b) markers were amplified, and PCR products were digested by BsuR1 restriction enzyme. Then the RFLP and sequencing were employed. Results: Only L. major was identified in patients containing regular amastigotes' shapes (oval or round) with a size of 2-4 µm in each of classical wet, dry, mixed lesions. Meanwhile, irregular shapes (spindle, pear, or cigarette) were observed separately in non-classical wet lesions with more than 4 µm. Interestingly, a few amastigotes with an external flagellum were observed in some lesions. All sequenced ITS-rDNA and Cyt b genes of L. major did not show any molecular variation (χ 2 P > 0.05), including only one common haplotype (GenBank access no. EF413075). Conclusion: Findings proved that unlike other endemic foci, there is not a meaningful correlation between phenotypic and genotypic features of L. major isolates. This study is considered as the first comprehensive report to incriminate morphometric shapes of L. major amastigotes, which enhances our knowledge concerning their relevance with various clinical appearances and genotypic traits. PMID:26081070

  12. Depletion of peritoneal CD5+ B cells has no effect on the course of Leishmania major infection in susceptible and resistant mice

    PubMed Central

    BABAI, B; LOUZIR, H; CAZENAVE, P -A; DELLAGI, K

    1999-01-01

    The mouse peritoneal cavity contains a unique self-renewing population of B cells (B-1) derived from fetal liver precursors and mainly producing polyreactive antibodies. Since B-1 cells are a potential source of IL-10, it has been suggested that these cells may contribute to the susceptibility of BALB/c mice to Leishmania major infection by skewing the T helper cell network towards a Th2 phenotype. Accordingly, L. major infection of B cell-defective BALB/c Xid mice (lacking B-1 cells) induces less severe disease compared with controls. However, in addition to the lack of B-1 cells, the Xid immune deficiency is characterized by high endogenous interferon-gamma (IFN-γ) production. In the present study, the role of B-1 cells during L. major infection was investigated in mice experimentally depleted of peritoneal B-1 cells. Six weeks old C57Bl/6 and BALB/c mice were lethally irradiated and reconstituted with autologous bone marrow which allows systemic depletion of B-1 cells. Untreated BALB/c, C57Bl/6 as well as BALB/c Xid mice were used as controls. After reconstitution, mice were injected with L. major amastigotes and progression was followed using clinical, parasitological and immunological criteria. As previously reported, BALB/c Xid mice showed a significant reduction in disease progression. In contrast, despite the dramatic reduction of B-1 cells, B-1-depleted BALB/c mice showed similar or even worse disease progression compared with control BALB/c mice. No differences were found between B-1-depleted or control C57Bl/6 mice. Our data suggest that the B-1 cells do not contribute to the susceptibility of BALB/c mice to L. major infection. PMID:10403925

  13. Hybridization of different antisense oligonucleotides on the surface of gold nanoparticles to silence zinc metalloproteinase gene after uptake by Leishmania major.

    PubMed

    Jebali, Ali; Anvari-Tafti, Mohammad Hosssein

    2015-05-01

    The use of antisense oligonucleotides is a novel strategy to treat infectious diseases. In this approach, vital mRNAs are targeted by antisense oligonucleotides. The aim of this study was to evaluate the effects of gold nanoparticles hybridized with different antisense oligonucleotides on Leishmania (L) major. In this project, gold nanoparticles were first synthesized, and then conjugated with primary oligonucleotides, 3'-AAA-5'. Next, conjugated gold nanoparticles (NP1) were separately hybridized with three types of antisense oligonucleotide from coding reign of GP63 gene (NP2), non-coding reign of GP63 gene (NP3), and both coding and non-coding reigns of GP63 (NP4). Then, 1mL of L. major suspension was separately added to 1mL of different hybridized gold nanoparticles at serial concentrations (1-200μg/mL), and incubated for 24, 48, and 72h at 37°C. Next, the uptake of each nanoparticle was separately measured by atomic absorption spectroscopy. After incubation, the cell viability was separately evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Also, the expression of GP63 gene was read out by quantitative-real-time PCR. This study showed that NP2 and NP3 had higher (5-fold) uptake than NP1 and NP4. Moreover, NP2 and NP3 led to less cell viability and gene expression, compared with NP1 and NP4. It could be concluded that both sequence and size of antisense oligonucleotide were important for transfection of L. major. Importantly, these antisense oligonucleotides can be obtained from both coding and non-coding reign of GP63 gene. Moreover, hybridized gold nanoparticles not only could silence GP63 gene, but also could kill L. major.

  14. A Listeria monocytogenes-Based Vaccine That Secretes Sand Fly Salivary Protein LJM11 Confers Long-Term Protection against Vector-Transmitted Leishmania major

    PubMed Central

    Abi Abdallah, Delbert S.; Pavinski Bitar, Alan; Oliveira, Fabiano; Meneses, Claudio; Park, Justin J.; Mendez, Susana; Kamhawi, Shaden; Valenzuela, Jesus G.

    2014-01-01

    Cutaneous leishmaniasis is a sand fly-transmitted disease characterized by skin ulcers that carry significant scarring and social stigmatization. Over the past years, there has been cumulative evidence that immunity to specific sand fly salivary proteins confers a significant level of protection against leishmaniasis. In this study, we used an attenuated strain of Listeria monocytogenes as a vaccine expression system for LJM11, a sand fly salivary protein identified as a good vaccine candidate. We observed that mice were best protected against an intradermal needle challenge with Leishmania major and sand fly saliva when vaccinated intravenously. However, this protection was short-lived. Importantly, groups of vaccinated mice were protected long term when challenged with infected sand flies. Protection correlated with smaller lesion size, fewer scars, and better parasite control between 2 and 6 weeks postchallenge compared to the control group of mice vaccinated with the parent L. monocytogenes strain not expressing LJM11. Moreover, protection correlated with high numbers of CD4+, gamma interferon-positive (IFN-γ+), tumor necrosis factor alpha-positive/negative (TNF-α+/−), interleukin-10-negative (IL-10−) cells and low numbers of CD4+ IFN-γ+/− TNF-α− IL-10+ T cells at 2 weeks postchallenge. Overall, our data indicate that delivery of LJM11 by Listeria is a promising vaccination strategy against cutaneous leishmaniasis inducing long-term protection against ulcer formation following a natural challenge with infected sand flies. PMID:24733091

  15. Peganum harmala Aqueous and Ethanol Extracts Effects on Lesions Caused by Leishmania major (MRHO/IR/75/ER) in BALB/c Mice

    PubMed Central

    Khoshzaban, Fariba; Ghaffarifar, Fatemeh; Jamshidi Koohsari, Hamid Reza

    2014-01-01

    Background: Leishmaniasis is one of the six most common parasitic infections in the tropical regions. There are different therapeutic modalities, however therapeutic resistance is developed and resulted in numerous problems. Therefore, evaluation of other therapeutic modalities is performed extensively. Objectives: The current study aimed to compare the therapeutic response of cutaneous leishmaniasis with Glucantime and Peganum harmala extracts (aqueous and ethanol) in the animal model. Materials and Methods: The therapeutic response of Leishmania major to Glucantime and P. harmala extracts (aqueous and ethanol) in animal model was studied in BALB/c mice. These mice were divided into four groups according to receiving either one of these three agents, and the control group. The therapeutic response was evaluated according to the parasitic load before and after treatment and also with measuring the size of the lesions. Results: The results showed that ethanol extract of P. harmala had good therapeutic efficacy in treatment of lesions in mice (P < 0.05), and the efficacy was significant in the eighth week after the treatment. There was also a statistically significant difference between the groups regarding the parasitic load (P < 0.05). Conclusions: According to the current study results, it may be concluded that ethanol extract of P. harmala is efficient in the treatment of cutaneous leishmaniasis, and the efficiency is comparable with that of Glucantime. PMID:25368792

  16. Atenolol Reduces Leishmania major-Induced Hyperalgesia and TNF-α Without Affecting IL-1β or Keratinocyte Derived Chemokines (KC)

    PubMed Central

    Karam, Marc C.; Merckbawi, Rana; Salman, Sara; Mobasheri, Ali

    2016-01-01

    Infection with a high dose of the intracellular parasitic protozoan Leishmania major induces a sustained hyperalgesia in susceptible BALB/c mice accompanied by up-regulation of the pro-inflammatory cytokines IL-1β and IL-6. Interleukin-13 (IL-13) has been shown to reduce this hyperalgesia (despite increased levels of IL-6) and the levels of IL-1β during and after the treatment period. These findings favor the cytokine cascade leading to the production of sympathetic amines (involving TNF-α and KC) over prostaglandins (involving IL-lβ and IL-6) as the final mediators of hyperalgesia. The aim of this study was to investigate the effect of daily treatment with the β-blockers atenolol on L. major-induced inflammation in mice with respect to hyperalgesia as well as the levels of TNF-α and KC (the analog of IL-8 in mice). Our data demonstrates that atenolol is able to reduce the L. major induced sustained peripheral hyperalgesia, which does not seem to involve a direct role for neither IL-lβ nor KC. Moreover, our results show that TNF-α may play a pivotal and direct role in sensitizing the peripheral nerve endings (nociceptors) since its level was reduced during the period of atenolol treatment, which correlates well with the reduction of the observed peripheral, but not central, hyperalgesia. These findings contribute to a better understanding of the cytokine cascade leading to hyperalgesia and may lead to the development of new and more efficient medications for many types of pain. PMID:26913003

  17. Comparative assessment of a DNA and protein Leishmania donovani gamma glutamyl cysteine synthetase vaccine to cross-protect against murine cutaneous leishmaniasis caused by L. major or L. mexicana infection.

    PubMed

    Campbell, S A; Alawa, J; Doro, B; Henriquez, F L; Roberts, C W; Nok, A; Alawa, C B I; Alsaadi, M; Mullen, A B; Carter, K C

    2012-02-08

    Leishmaniasis is a major health problem and it is estimated that 12 million people are currently infected. A vaccine which could cross-protect people against different Leishmania spp. would facilitate control of this disease as more than one species of Leishmania may be present. In this study the ability of a DNA vaccine, using the full gene sequence for L. donovani gamma glutamyl cysteine synthetase (γGCS) incorporated in the pVAX vector (pVAXγGCS), and a protein vaccine, using the corresponding recombinant L. donovani γGCS protein (LdγGCS), to protect against L. major or L. mexicana infection was evaluated. DNA vaccination gave transient protection against L. major and no protection against L. mexicana despite significantly enhancing specific antibody titres in vaccinated infected mice compared to infected controls. Vaccination with the LdγGCS protected against both species but only if the protein was incorporated into non-ionic surfactant vesicles for L. mexicana. The results of this study indicate that a L. donovani γGCS vaccine could be used to vaccinate against more than one Leishmania species but only if the recombinant protein is used.

  18. Brequinar derivatives and species-specific drug design for dihydroorotate dehydrogenase.

    PubMed

    Hurt, Darrell E; Sutton, Amanda E; Clardy, Jon

    2006-03-15

    Therapeutic agents brequinar sodium and leflunomide (Arava) work by binding in a hydrophobic tunnel formed by a highly variable N-terminus of family 2 dihydroorotate dehydrogenase (DHODH). The X-ray crystallographic structure of an analog of brequinar bound to human DHODH was determined. In silico screening of a library of compounds suggested another subset of brequinar analogs that do not inhibit human DHODH as potentially effective inhibitors of Plasmodium falciparum DHODH.

  19. Identification of immunodominant Leishmania major antigenic markers of the early C57BL/6 and BALB/c mice infection stages.

    PubMed

    Sassi, Atfa; Kaak, Olfa; Elgaaied, Amel Benammar

    2015-08-24

    The C57BL/6 mouse strain is resistant to Leishmania (L.) major infection and, unlike susceptible BALB/c, develops small self healing cutaneous lesions. The specific antibody responses of C57BL/6 and BALB/c mice were previously characterized by the predominance of IgG2a ("resistant" isotype associated with Th1) and IgG1 ("pathogenic" isotype associated with Th2) antibodies, respectively. In this study, we looked for the presence of antigens able to elicit an exclusive or predominant IgG1 production during the early stages of C57BL/6 lesion development and checked whether they are recognized or not by BALB/c mice. We demonstrate first that IgG2a predominance in C57BL/6 sera occurs only late after infection whereas in BALB/c, IgG1 antibodies dominate mostly in the early stages. Interestingly, soon after inoculation of live amastigotes, C57BL/6 displayed an exclusive IgG1 reactivity against particular L. major antigens but with MWs different from those identified in BALB/c. Furthermore, mice immunized with killed amastigotes displayed striking differences in their immunodetection profiles, particularly for the IgG1 isotype. Taken together, the observed differences in the specific antibody repertoires between infected mice resulted, at least in part, from immunological events independent from those triggered by the replicating parasite, and bring new insights into the selection of future vaccine candidates. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  20. The double-length tyrosyl-tRNA synthetase from the eukaryote Leishmania major forms an intrinsically asymmetric pseudo-dimer

    PubMed Central

    Larson, Eric T; Kim, Jessica E; Castaneda, Lisa J; Napuli, Alberto J; Zhang, Zhongsheng; Fan, Erkang; Zucker, Frank H; Verlinde, Christophe LMJ; Buckner, Frederick S; Van Voorhis, Wesley C; Hol, Wim G J; Merritt, Ethan A

    2011-01-01

    The single tyrosyl tRNA-synthetase (TyrRS) gene in trypanosomatid genomes codes for a protein that is twice the length of TyrRS from virtually all other organisms. Each half of the double-length TyrRS contains a catalytic domain and an anticodon-binding domain, however the two halves retain only 17% sequence identity to each other. The structural and functional consequences of this duplication and divergence are unclear. TyrRS normally forms a homodimer in which the active site of one monomer pairs with the anticodon-binding domain from the other. However, crystal structures of Leishmania major TyrRS show that instead the two halves of a single molecule form a pseudo-dimer resembling the canonical TyrRS dimer. Curiously, the C-terminal copy of the catalytic domain has lost the catalytically important HIGH and KMSKS motifs characteristic of Class I aminoacyl-tRNA synthetases. Thus the pseudo-dimer contains only one functional active site, contributed by the N-terminal half, and only one functional anticodon recognition site, contributed by the C-terminal half. Despite biochemical evidence for negative cooperativity between the two active sites of the usual TyrRS homodimer, previous structures have captured a crystallographically-imposed symmetric state. As the L. major TyrRS pseudo-dimer is inherently asymmetric, conformational variations observed near the active site may be relevant to understanding how the state of a single active site is communicated across the dimer interface. Furthermore, substantial differences between trypanosomal TyrRS and human homologs are promising for the design of inhibitors that selectively target the parasite enzyme. PMID:21420975

  1. WR279,396, a third generation aminoglycoside ointment for the treatment of Leishmania major cutaneous leishmaniasis: a phase 2, randomized, double blind, placebo controlled study.

    PubMed

    Ben Salah, Afif; Buffet, Pierre A; Morizot, Gloria; Ben Massoud, Nathalie; Zâatour, Amor; Ben Alaya, Nissaf; Haj Hamida, Nabil Bel; El Ahmadi, Zaher; Downs, Matthew T; Smith, Philip L; Dellagi, Koussay; Grögl, Max

    2009-01-01

    Cutaneous leishmaniasis (cl) is a disfiguring disease that confronts clinicians with a quandary: leave patients untreated or engage in a complex or toxic treatment. Topical treatment of CL offers a practical and safe option. Accordingly, the treatment of CL with WR279,396, a formulation of paromomycin and gentamicin in a hydrophilic base, was investigated in a phase 2 clinical study in Tunisia and France. A phase 2, randomized, double blind, vehicle-controlled study was conducted to assess the safety and efficacy of topical WR279,396 when applied twice a day for 20 days as treatment for parasitologically confirmed CL. The study protocol established the primary efficacy end point as complete clinical response (CCR) defined as 50% or greater reduction in the ulceration size of an index lesion by day 50 (D50) followed by complete re-epithelialization by D100, and no relapse through D180. Ninety-two subjects were randomized. Leishmania major was identified in 66 of 68 isolates typed (97%). In the intent-to-treat population, 47 of 50 WR279,396 treated participants (94%) met the definition of CCR, compared with 30 of 42 vehicle-placebo participants (71%) [p = 0.0045]. Erythema occurred in 30% and 24% of participants receiving WR279,396 and placebo, respectively [p = 0.64]. There was no clinical or laboratory evidence of systemic toxicity. Application of WR279,396 for 20 days was found to be safe and effective in treating L. major CL, and offers great potential as a new, simple, easily applicable, and inexpensive topical therapy for this neglected disease. ClinicalTrials.gov NCT00703924.

  2. Structure determination of glycogen synthase kinase-3 from Leishmania major and comparative inhibitor structure-activity relationships with Trypanosoma brucei GSK-3

    SciTech Connect

    Ojo, Kayode K; Arakaki, Tracy L; Napuli, Alberto J; Inampudi, Krishna K; Keyloun, Katelyn R; Zhang, Li; Hol, Wim G.J.; Verlind, Christophe L.M.J.; Merritt, Ethan A; Van Voorhis, Wesley C

    2012-04-24

    Glycogen synthase kinase-3 (GSK-3) is a drug target under intense investigation in pharmaceutical companies and constitutes an attractive piggyback target for eukaryotic pathogens. Two different GSKs are found in trypanosomatids, one about 150 residues shorter than the other. GSK-3 short (GeneDB: Tb927.10.13780) has previously been validated genetically as a drug target in Trypanosoma brucei by RNAi induced growth retardation; and chemically by correlation between enzyme and in vitro growth inhibition. Here, we report investigation of the equivalent GSK-3 short enzymes of L. major (LmjF18.0270) and L. infantum (LinJ18_V3.0270, identical in amino acid sequences to LdonGSK-3 short) and a crystal structure of LmajGSK-3 short at 2 Å resolution. The inhibitor structure-activity relationships (SARs) of L. major and L. infantum are virtually identical, suggesting that inhibitors could be useful for both cutaneous and visceral leishmaniasis. Leishmania spp. GSK-3 short has different inhibitor SARs than TbruGSK-3 short, which can be explained mostly by two variant residues in the ATP-binding pocket. Indeed, mutating these residues in the ATP-binding site of LmajGSK-3 short to the TbruGSK-3 short equivalents results in a mutant LmajGSK-3 short enzyme with SAR more similar to that of TbruGSK-3 short. The differences between human GSK-3β (HsGSK-3β) and LmajGSK-3 short SAR suggest that compounds which selectively inhibit LmajGSK-3 short may be found.

  3. Distinctive cellular immunity in genetically susceptible BALB/c mice recovered from Leishmania major infection or after subcutaneous immunization with killed parasites

    SciTech Connect

    Liew, F.Y.; Dhaliwal, J.S.

    1987-06-15

    Genetically susceptible BALB/c mice are refractory to further infection after recovery from Leishmania major infection after a sublethal dose of gamma-irradiation. In contrast, mice immunized with killed promastigotes s.c. develop exacerbated lesions after infection. Both groups of mice produce only a low level of specific antibody and no detectable cytotoxic T cells, but do have a strong antigen-specific DTH, which is adoptively transferable with Lyt-1+2-, L3T4+ T cells. Kinetic and histological studies revealed that mice immunized s.c. developed Jones-Mote hypersensitivity, peaking at 15 hr. with little mononuclear cell infiltration at the site of antigen administration; whereas mice that had recovered from infection developed tuberculin-type of reactivity, peaking at 24 to 48 hr, with intense mononuclear cell infiltration. Splenic T cells from recovered mice, when injected into the footpads of normal recipients together with live promastigotes, were able to retard lesion development; whereas T cells from s.c. immunized mice, when similarly transferred, accelerated disease progression. Antigen-specific culture supernatant of spleen cells from recovered mice also activated normal resident peritoneal macrophages to kill intracellular L. major amastigotes and tumor cells. Culture supernatants of spleen cells from s.c. immunized or normal mice were devoid of such activities. Part of the macrophage-activating potential can be inhibited by antibody specific for IFN-gamma. These results therefore demonstrate that whereas the Jones-Mote reaction is correlated with disease exacerbation, the tuberculin-type of DTH may be protective. Furthermore, in vivo immunity is directly related to the capacity of T cells to produce macrophage-activating factor.

  4. Expression, purification and crystallization of Trypanosoma cruzi dihydroorotate dehydrogenase complexed with orotate

    SciTech Connect

    Inaoka, Daniel Ken; Takashima, Eizo; Osanai, Arihiro; Shimizu, Hironari; Nara, Takeshi; Aoki, Takashi; Harada, Shigeharu; Kita, Kiyoshi

    2005-10-01

    The Trypanosoma cruzi dihydroorotate dehydrogenase, a key enzyme in pyrimidine de novo biosynthesis and redox homeostasis, was crystallized in complex with its first reaction product, orotate. Dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of dihydroorotate to orotate, the fourth step and the only redox reaction in the de novo biosynthesis of pyrimidine. DHOD from Trypanosoma cruzi (TcDHOD) has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. Crystals of the TcDHOD–orotate complex were grown at 277 K by the sitting-drop vapour-diffusion technique using polyethylene glycol 3350 as a precipitant. The crystals diffract to better than 1.8 Å resolution using synchrotron radiation (λ = 0.900 Å). X-ray diffraction data were collected at 100 K and processed to 1.9 Å resolution with 98.2% completeness and an overall R{sub merge} of 7.8%. The TcDHOD crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 67.87, b = 71.89, c = 123.27 Å. The presence of two molecules in the asymmetric unit (2 × 34 kDa) gives a crystal volume per protein weight (V{sub M}) of 2.2 Å{sup 3} Da{sup −1} and a solvent content of 44%.

  5. Mechanisms of immunity to Leishmania major infection in mice: the contribution of DNA vaccines coding for two novel sets of histones (H2A-H2B or H3-H4).

    PubMed

    Carrión, Javier

    2011-09-01

    The immune phenotype conferred by two different sets of histone genes (H2A-H2B or H3-H4) was assessed. BALB/c mice vaccinated with pcDNA3H2AH2B succumbed to progressive cutaneous leishmaniosis (CL), whereas vaccination with pcDNA3H3H4 resulted in partial resistance to Leishmania major challenge associated with the development of mixed T helper 1 (Th1)/Th2-type response and a reduction in parasite-specific Treg cells number at the site of infection. Therefore, the presence of histones H3 and H4 may be considered essential in the development of vaccine strategies against CL based on the Leishmania histones.

  6. Blocking Junctional Adhesion Molecule C Enhances Dendritic Cell Migration and Boosts the Immune Responses against Leishmania major

    PubMed Central

    Ballet, Romain; Emre, Yalin; Jemelin, Stéphane; Charmoy, Mélanie; Tacchini-Cottier, Fabienne; Imhof, Beat A.

    2014-01-01

    The recruitment of dendritic cells to sites of infections and their migration to lymph nodes is fundamental for antigen processing and presentation to T cells. In the present study, we showed that antibody blockade of junctional adhesion molecule C (JAM-C) on endothelial cells removed JAM-C away from junctions and increased vascular permeability after L. major infection. This has multiple consequences on the output of the immune response. In resistant C57BL/6 and susceptible BALB/c mice, we found higher numbers of innate immune cells migrating from blood to the site of infection. The subsequent migration of dendritic cells (DCs) from the skin to the draining lymph node was also improved, thereby boosting the induction of the adaptive immune response. In C57BL/6 mice, JAM-C blockade after L. major injection led to an enhanced IFN-γ dominated T helper 1 (Th1) response with reduced skin lesions and parasite burden. Conversely, anti JAM-C treatment increased the IL-4-driven T helper 2 (Th2) response in BALB/c mice with disease exacerbation. Overall, our results show that JAM-C blockade can finely-tune the innate cell migration and accelerate the consequent immune response to L. major without changing the type of the T helper cell response. PMID:25474593

  7. De novo assembly and sex-specific transcriptome profiling in the sand fly Phlebotomus perniciosus (Diptera, Phlebotominae), a major Old World vector of Leishmania infantum.

    PubMed

    Petrella, V; Aceto, S; Musacchia, F; Colonna, V; Robinson, M; Benes, V; Cicotti, G; Bongiorno, G; Gradoni, L; Volf, P; Salvemini, M

    2015-10-23

    The phlebotomine sand fly Phlebotomus perniciosus (Diptera: Psychodidae, Phlebotominae) is a major Old World vector of the protozoan Leishmania infantum, the etiological agent of visceral and cutaneous leishmaniases in humans and dogs, a worldwide re-emerging diseases of great public health concern, affecting 101 countries. Despite the growing interest in the study of this sand fly species in the last years, the development of genomic resources has been limited so far. To increase the available sequence data for P. perniciosus and to start studying the molecular basis of the sexual differentiation in sand flies, we performed whole transcriptome Illumina RNA sequencing (RNA-seq) of adult males and females and de novo transcriptome assembly. We assembled 55,393 high quality transcripts, of which 29,292 were unique, starting from adult whole body male and female pools. 11,736 transcripts had at least one functional annotation, including full-length low abundance salivary transcripts, 981 transcripts were classified as putative long non-coding RNAs and 244 transcripts encoded for putative novel proteins specific of the Phlebotominae sub-family. Differential expression analysis identified 8590 transcripts significantly biased between sexes. Among them, some show relaxation of selective constraints when compared to their orthologs of the New World sand fly species Lutzomyia longipalpis. In this paper, we present a comprehensive transcriptome resource for the sand fly species P. perniciosus built from short-read RNA-seq and we provide insights into sex-specific gene expression at adult stage. Our analysis represents a first step towards the identification of sex-specific genes and pathways and a foundation for forthcoming investigations into this important vector species, including the study of the evolution of sex-biased genes and of the sexual differentiation in phlebotomine sand flies.

  8. In Silico Analysis of Six Known Leishmania major Antigens and In Vitro Evaluation of Specific Epitopes Eliciting HLA-A2 Restricted CD8 T Cell Response

    PubMed Central

    Seyed, Negar; Zahedifard, Farnaz; Safaiyan, Shima; Gholami, Elham; Doustdari, Fatemeh; Azadmanesh, Kayhan; Mirzaei, Maryam; Saeedi Eslami, Nasir; Khadem Sadegh, Akbar; Eslami far, Ali; Sharifi, Iraj; Rafati, Sima

    2011-01-01

    Background As a potent CD8+ T cell activator, peptide vaccine has found its way in vaccine development against intracellular infections and cancer, but not against leishmaniasis. The first step toward a peptide vaccine is epitope mapping of different proteins according to the most frequent HLA types in a population. Methods and Findings Six Leishmania (L.) major-related candidate antigens (CPB,CPC,LmsTI-1,TSA,LeIF and LPG-3) were screened for potential CD8+ T cell activating 9-mer epitopes presented by HLA-A*0201 (the most frequent HLA-A allele). Online software including SYFPEITHI, BIMAS, EpiJen, Rankpep, nHLApred, NetCTL and Multipred were used. Peptides were selected only if predicted by almost all programs, according to their predictive scores. Pan-A2 presentation of selected peptides was confirmed by NetMHCPan1.1. Selected peptides were pooled in four peptide groups and the immunogenicity was evaluated by in vitro stimulation and intracellular cytokine assay of PBMCs from HLA-A2+ individuals recovered from L. major. HLA-A2− individuals recovered from L. major and HLA-A2+ healthy donors were included as control groups. Individual response of HLA-A2+ recovered volunteers as percent of CD8+/IFN-γ+ T cells after in vitro stimulation against peptide pools II and IV was notably higher than that of HLA-A2− recovered individuals. Based on cutoff scores calculated from the response of HLA-A2− recovered individuals, 31.6% and 13.3% of HLA-A2+ recovered persons responded above cutoff in pools II and IV, respectively. ELISpot and ELISA results confirmed flow cytometry analysis. The response of HLA-A2− recovered individuals against peptide pools I and III was detected similar and even higher than HLA-A2+ recovered individuals. Conclusion Using in silico prediction we demonstrated specific response to LmsTI-1 (pool II) and LPG-3- (pool IV) related peptides specifically presented in HLA-A*0201 context. This is among the very few reports mapping L. major epitopes for

  9. Depletion of interleukin-4 in BALB/c mice with established Leishmania major infections increases the efficacy of antimony therapy and promotes Th1-like responses.

    PubMed Central

    Nabors, G S; Farrell, J P

    1994-01-01

    Whereas most inbred mouse strains mount a protective Th1 helper T-cell response following infection with Leishmania major, an ineffective Th2 response develops in BALB/c mice, leading to the development of disseminated, ultimately fatal disease. Interleukin-4 (IL-4) production is required for the initiation of the Th2 response, though little is known about the requirements for the long-term maintenance of this response. In order to investigate the role of the expanding parasite population on the Th2 response, mice infected for 2 weeks with L. major, which exhibited a Th2-like cytokine profile, were treated with a leishmanicidal agent (Pentostam) and/or various doses of anti-IL-4 antibody. Untreated mice, mice treated with Pentostam alone, or mice treated with 2.5 mg of anti-IL-4 antibody given at days 13 and 21 of infection developed progressive disease. However, in 8 of 10 mice treated with this dose of anti-IL-4 antibody plus Pentostam lesion development was arrested and lesions were either controlled or eventually healed. Healing was associated with the production of high levels of gamma interferon by spleen cells, and low levels of immunoglobulin E in serum compared with levels for control animals, indicating that a Th1-like response had developed in mice receiving both treatments. Thus, depletion of IL-4 only in combination with a reduction in the parasite burden allowed the expression of a Th1 response. When the dose of anti-IL-4 antibody was increased to 5 mg per injection, all mice treated with this dose of antibody, with or without Pentostam therapy, healed. However, combined therapy with Pentostam in mice treated with this dose of antibody had an additional protective effect. As expected, a Th1 response developed in mice treated with this dose of anti-IL-4 antibody with or without combined therapy with Pentostam, whereas a Th2 response developed in control mice. Thus, a significant effect on the course of disease is noted when mice with established L

  10. Leishmania hijacking of the macrophage intracellular compartments.

    PubMed

    Liévin-Le Moal, Vanessa; Loiseau, Philippe M

    2016-02-01

    Leishmania spp., transmitted to humans by the bite of the sandfly vector, are responsible for the three major forms of leishmaniasis, cutaneous, diffuse mucocutaneous and visceral. Leishmania spp. interact with membrane receptors of neutrophils and macrophages. In macrophages, the parasite is internalized within a parasitophorous vacuole and engages in a particular intracellular lifestyle in which the flagellated, motile Leishmania promastigote metacyclic form differentiates into non-motile, metacyclic amastigote form. This phenomenon is induced by Leishmania-triggered events leading to the fusion of the parasitophorous vacuole with vesicular members of the host cell endocytic pathway including recycling endosomes, late endosomes and the endoplasmic reticulum. Maturation of the parasitophorous vacuole leads to the intracellular proliferation of the Leishmania amastigote forms by acquisition of host cell nutrients while escaping host defense responses. © 2015 FEBS.

  11. LmSmdB: an integrated database for metabolic and gene regulatory network in Leishmania major and Schistosoma mansoni.

    PubMed

    Patel, Priyanka; Mandlik, Vineetha; Singh, Shailza

    2016-03-01

    A database that integrates all the information required for biological processing is essential to be stored in one platform. We have attempted to create one such integrated database that can be a one stop shop for the essential features required to fetch valuable result. LmSmdB (L. major and S. mansoni database) is an integrated database that accounts for the biological networks and regulatory pathways computationally determined by integrating the knowledge of the genome sequences of the mentioned organisms. It is the first database of its kind that has together with the network designing showed the simulation pattern of the product. This database intends to create a comprehensive canopy for the regulation of lipid metabolism reaction in the parasite by integrating the transcription factors, regulatory genes and the protein products controlled by the transcription factors and hence operating the metabolism at genetic level.

  12. LmSmdB: an integrated database for metabolic and gene regulatory network in Leishmania major and Schistosoma mansoni

    PubMed Central

    Patel, Priyanka; Mandlik, Vineetha; Singh, Shailza

    2015-01-01

    A database that integrates all the information required for biological processing is essential to be stored in one platform. We have attempted to create one such integrated database that can be a one stop shop for the essential features required to fetch valuable result. LmSmdB (L. major and S. mansoni database) is an integrated database that accounts for the biological networks and regulatory pathways computationally determined by integrating the knowledge of the genome sequences of the mentioned organisms. It is the first database of its kind that has together with the network designing showed the simulation pattern of the product. This database intends to create a comprehensive canopy for the regulation of lipid metabolism reaction in the parasite by integrating the transcription factors, regulatory genes and the protein products controlled by the transcription factors and hence operating the metabolism at genetic level. PMID:26981382

  13. Optimization of Topical Therapy for Leishmania major Localized Cutaneous Leishmaniasis Using a Reliable C57BL/6 Model

    PubMed Central

    Lecoeur, Hervé; Buffet, Pierre; Morizot, Gloria; Goyard, Sophie; Guigon, Ghislaine; Milon, Geneviève; Lang, Thierry

    2007-01-01

    Background Because topical therapy is easy and usually painless, it is an attractive first-line option for the treatment of localized cutaneous leishmaniasis (LCL). Promising ointments are in the final stages of development. One main objective was to help optimize the treatment modalities of human LCL with WR279396, a topical formulation of aminoglycosides that was recently proven to be efficient and safe for use in humans. Methodology/Principal Findings C57BL/6 mice were inoculated in the ear with luciferase transgenic L. major and then treated with WR279396. The treatment period spanned lesion onset, and the evolution of clinical signs and bioluminescent parasite loads could be followed for several months without killing the mice. As judged by clinical healing and a 1.5-3 log parasite load decrease in less than 2 weeks, the 94% efficacy of 10 daily applications of WR279396 in mice was very similar to what had been previously observed in clinical trials. When WR279396 was applied with an occlusive dressing, parasitological and clinical efficacy was significantly increased and no rebound of parasite load was observed. In addition, 5 applications under occlusion were more efficient when done every other day for 10 days than daily for 5 days, showing that length of therapy is a more important determinant of treatment efficacy than the total dose topically applied. Conclusions/Significance Occlusion has a significant adjuvant effect on aminoglycoside ointment therapy of experimental cutaneaous leishmaniasis (CL), a concept that might apply to other antileishmanial or antimicrobial ointments. Generated in a laboratory mouse-based model that closely mimics the course of LCL in humans, our results support a schedule based on discontinuous applications for a few weeks rather than several daily applications for a few days. PMID:18060082

  14. Alginate microspheres encapsulated with autoclaved Leishmania major (ALM) and CpG-ODN induced partial protection and enhanced immune response against murine model of leishmaniasis.

    PubMed

    Tafaghodi, Mohsen; Eskandari, Maryam; Khamesipour, Ali; Jaafari, Mahmoud R

    2011-10-01

    A suitable adjuvant and delivery system are needed to enhance efficacy of vaccines against leishmaniasis. In this study, alginate microspheres as an antigen delivery system and CpG-ODN as an immunoadjuvant were used to enhance immune response and induce protection against an experimental autoclaved Leishmania major (ALM) vaccine. Alginate microspheres were prepared by an emulsification technique and the characteristics of the preparation such as size, encapsulation efficiency and release profile of encapsulates were studied. Mean diameter of microspheres was determined using SEM (Scanning Electron Microscopy) and particle size analyzer. The encapsulation efficiency was determined using Lowry protein assay method. The integrity of ALM antigens was assessed using SDS-PAGE. Mean diameter of microspheres was 1.8±1.0μm. BALB/c mice were immunized three times in 3-weeks intervals with ALM+CpG-ODN loaded microspheres [(ALM+CpG)(ALG)], ALM encapsulated alginate microspheres [(ALM)(ALG)], (ALM)(ALG)+CpG, ALM+CpG, ALM alone or PBS. The intensity of infection induced by L. major challenge was assessed by measuring size of footpad swelling. The strongest protection was observed in group of mice immunized with (ALM+CpG)(ALG). The groups of mice received (ALM+CpG)(ALG), (ALM)(ALG)+CpG, (ALM)(ALG) and ALM+CpG were also showed a significantly (P<0.05) smaller footpad swelling compared with the group that received either ALM alone or PBS. The mice immunized with (ALM+CpG)(ALG) or ALM+CpG showed the significantly (P<0.05) highest IgG2a/IgG1 ratio. The IFN-γ level was significantly (P<0.0001) highest in group of mice immunized with either (ALM)(ALG)+CpG or ALM+CpG. It is concluded that alginate microspheres and CpG-ODN adjuvant when are used simultaneously induced protection and enhanced immune response against ALM antigen.

  15. C-Terminal Domain Deletion Enhances the Protective Activity of cpa/cpb Loaded Solid Lipid Nanoparticles against Leishmania major in BALB/c Mice

    PubMed Central

    Doroud, Delaram; Zahedifard, Farnaz; Vatanara, Alireza; Taslimi, Yasaman; Vahabpour, Rouholah; Torkashvand, Fatemeh; Vaziri, Behrooz; Rouholamini Najafabadi, Abdolhossein; Rafati, Sima

    2011-01-01

    Background We have demonstrated that vaccination with pDNA encoding cysteine proteinase Type II (CPA) and Type I (CPB) with its unusual C-terminal extension (CTE) can partially protect BALB/c mice against cutaneous leishmanial infection. Unfortunately, this protection is insufficient to completely control infection without booster injection. Furthermore, in developing vaccines for leishmaniasis, it is necessary to consider a proper adjuvant and/or delivery system to promote an antigen specific immune response. Solid lipid nanoparticles have found their way in drug delivery system development against intracellular infections and cancer, but not Leishmania DNA vaccination. Therefore, undefined effect of cationic solid lipid nanoparticles (cSLN) as an adjuvant in enhancing the immune response toward leishmanial antigens led us to refocus our vaccine development projects. Methodology/Principal Findings Three pDNAs encoding L. major cysteine proteinase type I and II (with or without CTE) were formulated by cSLN. BALB/c mice were immunized twice by 3-week interval, with cSLN-pcDNA-cpa/b, pcDNA-cpa/b, cSLN-pcDNA-cpa/b-CTE, pcDNA-cpa/b-CTE, cSLN, cSLN-pcDNA and PBS. Mice vaccinated with cSLN-pcDNA-cpa/b-CTE showed significantly higher levels of parasite inhibition related to protection with specific Th1 immune response development, compared to other groups. Parasite inhibition was determined by different techniques currently available in exploration vacciation efficacy, i.e., flowcytometry on footpad and lymph node, footpad caliper based measurements and imaging as well as lymph node microtitration assay. Among these techniques, lymph node flowcytometry was found to be the most rapid, sensitive and easily reproducible method for discrimination between the efficacy of vaccination strategies. Conclusions/Significance This report demonstrates cSLN's ability to boost immune response magnitude of cpa/cpb-CTE cocktail vaccination against leishmaniasis so that the average

  16. Leishmania metacaspase: an arginine-specific peptidase.

    PubMed

    Martin, Ricardo; Gonzalez, Iveth; Fasel, Nicolas

    2014-01-01

    The purpose of this chapter is to give insights into metacaspase of Leishmania protozoan parasites as arginine-specific cysteine peptidase. The physiological role of metacaspase in Leishmania is still a matter of debate, whereas its peptidase enzymatic activity has been well characterized. Among the different possible expression systems, metacaspase-deficient yeast cells (Δyca1) have been instrumental in studying the activity of Leishmania major metacaspase (LmjMCA). Here, we describe techniques for purification of LmjMCA and its activity measurement, providing a platform for further identification of LmjMCA substrates.

  17. Interconvertible geometric isomers of Plasmodium falciparum dihydroorotate dehydrogenase inhibitors exhibit multiple binding modes.

    PubMed

    McConkey, Glenn A; Bedingfield, Paul T P; Burrell, David R; Chambers, Nicholas C; Cunningham, Fraser; Prior, Timothy J; Fishwick, Colin W G; Boa, Andrew N

    2017-08-15

    Two new tricyclic β-aminoacrylate derivatives (2e and 3e) have been found to be inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) with Ki 0.037 and 0.15μM respectively. (1)H and (13)C NMR spectroscopic data show that these compounds undergo ready cis-trans isomerisation at room temperature in polar solvents. In silico docking studies indicate that for both molecules there is neither conformation nor double bond configuration which bind preferentially to PfDHODH. This flexibility is favourable for inhibitors of this channel that require extensive positioning to reach their binding site. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Evaluation of immune responses and analysis of the effect of vaccination of the Leishmania major recombinant ribosomal proteins L3 or L5 in two different murine models of cutaneous leishmaniasis.

    PubMed

    Ramírez, Laura; Santos, Diego M; Souza, Ana P; Coelho, Eduardo A F; Barral, Aldina; Alonso, Carlos; Escutia, Marta R; Bonay, Pedro; de Oliveira, Camila I; Soto, Manuel

    2013-02-18

    Four new antigenic proteins located in Leishmania ribosomes have been characterized: S4, S6, L3 and L5. Recombinant versions of the four ribosomal proteins from Leishmania major were recognized by sera from human and canine patients suffering different clinical forms of leishmaniasis. The prophylactic properties of these proteins were first studied in the experimental model of cutaneous leishmaniasis caused by L. major inoculation into BALB/c mice. The administration of two of them, LmL3 or LmL5 combined with CpG-oligodeoxynucleotides (CpG-ODN) was able to protect BALB/c mice against L. major infection. Vaccinated mice showed smaller lesions and parasite burden compared to mice inoculated with vaccine diluent or vaccine adjuvant. Protection was correlated with an antigen-specific increased production of IFN-γ paralleled by a decrease of the antigen-specific IL-10 mediated response in protected mice relative to non-protected controls. Further, it was demonstrated that BALB/c mice vaccinated with recombinant LmL3 or LmL5 plus CpG-ODN were also protected against the development of cutaneous lesions following inoculation of L. braziliensis. Together, data presented here indicate that LmL3 or LmL5 ribosomal proteins combined with Th1 inducing adjuvants, may be relevant components of a vaccine against cutaneous leishmaniasis caused by distinct species.

  19. An enhanced method for the identification of Leishmania spp. using real-time polymerase chain reaction and sequence analysis of the 7SL RNA gene region.

    PubMed

    Stevenson, Lindsay G; Fedorko, Daniel P; Zelazny, Adrian M

    2010-04-01

    The accurate identification of Leishmania spp. is important for the treatment of infected patients. Molecular methods offer an alternative to time-consuming traditional laboratory techniques for species determination. We redesigned a 7SL RNA gene-based polymerase chain reaction and sequence assay for increased species identification. DNA extracted from 17 reference strains and 10 cultured clinical isolates was examined. Sequence comparison was used successfully to identify organisms to the complex level with intercomplex similarity ranging from 77.5% to 98.4%. Many species within each complex were discriminated accurately by this method including Leishmania major, Leishmania tropica, Leishmania aethiopica, Leishmania guyanensis, and the previously indistinguishable Leishmania braziliensis and Leishmania panamensis. The Leishmania donovani complex members remain indistinguishable by this method, as are the representatives of Leishmania amazonensis/Leishmania garnhami and Leishmania mexicana/Leishmania pifanoi. Published by Elsevier Inc.

  20. Natural infection of bats with Leishmania in Ethiopia.

    PubMed

    Kassahun, Aysheshm; Sadlova, Jovana; Benda, Petr; Kostalova, Tatiana; Warburg, Alon; Hailu, Asrat; Baneth, Gad; Volf, Petr; Votypka, Jan

    2015-10-01

    The leishmaniases, a group of diseases with a worldwide-distribution, are caused by different species of Leishmania parasites. Both cutaneous and visceral leishmaniasis remain important public health problems in Ethiopia. Epidemiological cycles of these protozoans involve various sand fly (Diptera: Psychodidae) vectors and mammalian hosts, including humans. In recent years, Leishmania infections in bats have been reported in the New World countries endemic to leishmaniasis. The aim of this study was to survey natural Leishmania infection in bats collected from various regions of Ethiopia. Total DNA was isolated from spleens of 163 bats belonging to 23 species and 18 genera. Leishmania infection was detected by real-time (RT) PCR targeting a kinetoplast (k) DNA and internal transcribed spacer one (ITS1) gene of the parasite. Detection was confirmed by sequencing of the PCR products. Leishmania kDNA was detected in eight (4.9%) bats; four of them had been captured in the Aba-Roba and Awash-Methara regions that are endemic for leishmaniasis, while the other four specimens originated from non-endemic localities of Metu, Bedele and Masha. Leishmania isolates from two bats were confirmed by ITS1 PCR to be Leishmania tropica and Leishmania major, isolated from two individual bats, Cardioderma cor and Nycteris hispida, respectively. These results represent the first confirmed observation of natural infection of bats with the Old World Leishmania. Hence, bats should be considered putative hosts of Leishmania spp. affecting humans with a significant role in the transmission.

  1. Fluorescence assay of dihydroorotate dehydrogenase that may become a cancer biomarker

    PubMed Central

    Yin, Sheng; Kabashima, Tsutomu; Zhu, Qinchang; Shibata, Takayuki; Kai, Masaaki

    2017-01-01

    We developed an assay method for measuring dihydroorotate dehydrogenase (DHODH) activity in cultured HeLa cells and fibroblasts, and in stage III stomach cancer and adjacent normal tissues from the same patient. The assay comprised enzymatic reaction of DHODH with a large amount of dihydroorotic acid substrate, followed by fluorescence (FL) detection specific for orotic acid using the 4-trifluoromethyl-benzamidoxime fluorogenic reagent. The DHODH activities in the biologically complex samples were readily measured by the assay method. Our data indicate significantly higher DHODH activity in HeLa cells (340 ± 25.9 pmol/105 cells/h) than in normal fibroblasts (54.1 ± 7.40 pmol/105 cells/h), and in malignant tumour tissue (1.10 ± 0.19 nmol/mg total proteins/h) than in adjacent normal tissue (0.24 ± 0.11 nmol/mg total proteins/h). This is the first report that DHODH activity may be a diagnostic biomarker for cancer. PMID:28084471

  2. Discovery of diverse human dihydroorotate dehydrogenase inhibitors as immunosuppressive agents by structure-based virtual screening.

    PubMed

    Diao, Yanyan; Lu, Weiqiang; Jin, Huangtao; Zhu, Junsheng; Han, Le; Xu, Minghao; Gao, Rui; Shen, Xu; Zhao, Zhenjiang; Liu, Xiaofeng; Xu, Yufang; Huang, Jin; Li, Honglin

    2012-10-11

    This study applied an efficient virtual screening strategy integrating molecular docking with MM-GBSA rescoring to identify diverse human dihydroorotate dehydrogenase (hDHODH) inhibitors. Eighteen compounds with IC(50) values ranging from 0.11 to 18.8 μM were identified as novel hDHODH inhibitors that exhibited overall species-selectivity over Plasmodium falciparum dihydroorotate dehydrogenase (pfDHODH). Compound 8, the most potent one, showed low micromolar inhibitory activity against hDHODH with an IC(50) value of 0.11 μM. Moreover, lipopolysaccharide-induced B-cell assay and mixed lymphocyte reaction assay revealed that most of the hits showed potent antiproliferative activity against B and T cells, which demonstrates their potential application as immunosuppressive agents. In particular, compound 18 exhibited potent B-cell inhibitory activity (IC(50) = 1.78 μM) and presents a B-cell-specific profile with 17- and 26-fold selectivities toward T and Jurkat cells, respectively.

  3. A high-throughput fluorescence-based assay for Plasmodium dihydroorotate dehydrogenase inhibitor screening.

    PubMed

    Caballero, Iván; Lafuente, María José; Gamo, Francisco-Javier; Cid, Concepción

    2016-08-01

    Plasmodium dihydroorotate dehydrogenase (DHODH) is a mitochondrial membrane-associated flavoenzyme that catalyzes the rate-limiting step of de novo pyrimidine biosynthesis. DHODH is a validated target for malaria, and DSM265, a potent inhibitor, is currently in clinical trials. The enzyme catalyzes the oxidation of dihydroorotate to orotate using flavin mononucleotide (FMN) as cofactor in the first half of the reaction. Reoxidation of FMN to regenerate the active enzyme is mediated by ubiquinone (CoQD), which is the physiological final electron acceptor and second substrate of the reaction. We have developed a fluorescence-based high-throughput enzymatic assay to find DHODH inhibitors. In this assay, the CoQD has been replaced by a redox-sensitive fluorogenic dye, resazurin, which changes to a fluorescent state on reduction to resorufin. Remarkably, the assay sensitivity to find competitive inhibitors of the second substrate is higher than that reported for the standard colorimetric assay. It is amenable to 1536-well plates with Z' values close to 0.8. The fact that the human enzyme can also be assayed in the same format opens additional applications of this assay to the discovery of inhibitors to treat cancer, transplant rejection, autoimmune diseases, and other diseases mediated by rapid cellular growth.

  4. Lack of Protection of Pre-Immunization with Saliva of Long-Term Colonized Phlebotomus papatasi against Experimental Challenge with Leishmania major and Saliva of Wild-Caught P. papatasi

    PubMed Central

    Ahmed, Sami Ben Hadj; Kaabi, Belhassen; Chelbi, Ifhem; Derbali, Mohamed; Cherni, Saifedine; Laouini, Dhafer; Zhioua, Elyes

    2010-01-01

    Immunity to saliva of Phlebotomus papatasi protects against Leishmania major infection as determined by co-inoculation of parasites with salivary gland homogenates (SGHs) of this vector. These results were obtained with long-term colonized female P. papatasi. We investigated the effect of pre-immunization with SGH of long-term colonized P. papatasi against L. major infection co-inoculated with SGH of wild-caught P. papatasi. Our results showed that pre-exposure to SGH of long-term, colonized P. papatasi do not confer protection against infection with L. major co-inoculated with SGH of wild-caught P. papatasi. These preliminary results strongly suggest that the effectiveness of a vector saliva-based vaccine derived from colonized sand fly populations may be affected by inconsistent immune response after natural exposure. PMID:20810812

  5. Lack of protection of pre-immunization with saliva of long-term colonized Phlebotomus papatasi against experimental challenge with Leishmania major and saliva of wild-caught P. papatasi.

    PubMed

    Ahmed, Sami Ben Hadj; Kaabi, Belhassen; Chelbi, Ifhem; Derbali, Mohamed; Cherni, Saifedine; Laouini, Dhafer; Zhioua, Elyes

    2010-09-01

    Immunity to saliva of Phlebotomus papatasi protects against Leishmania major infection as determined by co-inoculation of parasites with salivary gland homogenates (SGHs) of this vector. These results were obtained with long-term colonized female P. papatasi. We investigated the effect of pre-immunization with SGH of long-term colonized P. papatasi against L. major infection co-inoculated with SGH of wild-caught P. papatasi. Our results showed that pre-exposure to SGH of long-term, colonized P. papatasi do not confer protection against infection with L. major co-inoculated with SGH of wild-caught P. papatasi. These preliminary results strongly suggest that the effectiveness of a vector saliva-based vaccine derived from colonized sand fly populations may be affected by inconsistent immune response after natural exposure.

  6. The role of CpG ODN in enhancement of immune response and protection in BALB/c mice immunized with recombinant major surface glycoprotein of Leishmania (rgp63) encapsulated in cationic liposome.

    PubMed

    Jaafari, Mahmoud R; Badiee, Ali; Khamesipour, Ali; Samiei, Afshin; Soroush, Dina; Kheiri, Masoumeh Tavassoti; Barkhordari, Farzaneh; McMaster, W Robert; Mahboudi, Fereidoun

    2007-08-10

    CpG oligodeoxynucleotides (CpG ODN) are known to be a potent immunoadjuvant for a wide range of antigens. The aim of this study was to evaluate the role of CpG ODN co-encapsulated with rgp63 antigen in cationic liposomes (Lip-rgp63-CpG ODN) in immune response enhancement and protection in BALB/c mice against leishmaniasis. Lip-rgp63-CpG ODN prepared by using dehydration-rehydration vesicle (DRV) method significantly inhibited (P<0.001) Leishmania major infection in mice measured by footpad swelling compared to Lip-rgp63, rgp63 alone, rgp63 plus CpG ODN, PBS or control liposomes. The mice immunized with Lip-rgp63-CpG ODN also showed the lowest spleen parasite burden, highest IgG2a/IgG1 ratio and IFN-gamma production and the lowest IL-4 production compared to the other groups. The results indicate that co-encapsulation of CpG ODN in liposomes improves the immunogenicity of Leishmania antigen.

  7. In vitro cytocidal effects of the essential oil from Croton cajucara (red sacaca) and its major constituent 7- hydroxycalamenene against Leishmania chagasi

    PubMed Central

    2013-01-01

    Background Visceral leishmaniasis is the most serious form of leishmaniasis and can be lethal if left untreated. Currently available treatments for these parasitic diseases are frequently associated to severe side effects. The leaves of Croton cajucara are used as an infusion in popular medicine to combat several diseases. Previous studies have demonstrated that the linalool-rich essential oil from C. cajucara (white sacaca) is extremely efficient against the tegumentary specie Leishmania amazonensis. In this study, we investigated the effects of the 7-hydroxycalamenene-rich essential oil from the leaves of C. cajucara (red sacaca) against Leishmania chagasi, as well as on the interaction of these parasites with host cells. Methods Promastigotes were treated with different concentrations of the essential oil for determination of its minimum inhibitory concentration (MIC). In addition, the effects of the essential oil on parasite ultrastructure were analyzed by transmission electron microscopy. To evaluate its efficacy against infected cells, mouse peritoneal macrophages infected with L. chagasi promastigotes were treated with the inhibitory and sub-inhibitory concentrations of the essential oil. Results The minimum inhibitory concentrations of the essential oil and its purified component 7-hydroxycalamenene against L. chagasi were 250 and 15.6 μg/mL, respectively. Transmission electron microscopy analysis revealed important nuclear and kinetoplastic alterations in L. chagasi promastigotes. Pre-treatment of macrophages and parasites with the essential oil reduced parasite/macrophage interaction by 52.8%, while it increased the production of nitric oxide by L. chagasi-infected macrophages by 80%. Conclusion These results indicate that the 7-hydroxycalamenene-rich essential oil from C. cajucara is a promising source of leishmanicidal compounds. PMID:24088644

  8. The role of fluorine in stabilizing the bioactive conformation of dihydroorotate dehydrogenase inhibitors.

    PubMed

    Bonomo, Silvia; Tosco, Paolo; Giorgis, Marta; Lolli, Marco; Fruttero, Roberta

    2013-03-01

    Dihydroorotate dehydrogenase (DHODH) is an important drug target due to its prominent role in pyrimidine biosynthesis. Leflunomide and brequinar are two well-known DHODH inhibitors, which bind to the enzyme in the same pocket with different binding modes. We have recently realized a series of new inhibitors based on the 4-hydroxy-1,2,5-oxadiazole ring, whose activity profile was found to be closely dependent on the degree of fluorine substitution at the phenyl ring adjacent to the oxadiazole moiety; a positive influence of fluorine on the DHODH inhibitory potency was observed previously [Baumgartner et al. (2006) J Med Chem 49:1239-1247]. Potential energy surface scans showed that fluorine plays an important role in stabilizing the bioactive conformations; additionally, fluorine influences the balance between leflunomide-like and brequinar-like binding modes. These findings may serve as a guide to design more potent DHODH inhibitors.

  9. Optimization of Potent Inhibitors of P. falciparum Dihydroorotate Dehydrogenase for the Treatment of Malaria

    PubMed Central

    2011-01-01

    Inhibition of dihydroorotate dehydrogenase (DHODH) for P. falciparum potentially represents a new treatment option for malaria, since DHODH catalyzes the rate-limiting step in the pyrimidine biosynthetic pathway and P. falciparum is unable to salvage pyrimidines and must rely on de novo biosynthesis for survival. We report herein the synthesis and structure–activity relationship of a series of 5-(2-methylbenzimidazol-1-yl)-N-alkylthiophene-2-carboxamides that are potent inhibitors against PfDHODH but do not inhibit the human enzyme. On the basis of efficacy observed in three mouse models of malaria, acceptable safety pharmacology risk assessment and safety toxicology profile in rodents, lack of potential drug–drug interactions, acceptable ADME/pharmacokinetic profile, and projected human dose, 5-(4-cyano-2-methyl-1H-benzo[d]imidazol-1-yl)-N-cyclopropylthiophene-2-carboxamide 2q was identified as a potential drug development candidate. PMID:24900364

  10. Susceptibility of Inbred Mice to Leishmania major Infection: Genetic Analysis of Macrophage Activation and Innate Resistance to Disease in Individual Progeny of P/J (Susceptible) and C3H/HeN (Resistant) Mice

    DTIC Science & Technology

    1990-12-01

    mediated immu- ease and defective macrophage activation in Bx mice that nity in mice highly susceptible to Leishmania tropica . J. Exp. could not be...inbred mice to Leishmania tropica infec- tion: correlation of susceptibility with in vitro defective macro- LITERATURE CITED phage microbicidal...probability and phage activation to kill Leishmania tropica : characterization of statistics. Chemical Rubber Co., Cleveland. P/J mouse macrophage defects for

  11. Site directed spin labeling studies of Escherichia coli dihydroorotate dehydrogenase N-terminal extension

    SciTech Connect

    Couto, Sheila G.; Cristina Nonato, M.

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer EcDHODH is a membrane-associated enzyme and a promising target for drug design. Black-Right-Pointing-Pointer Enzyme's N-terminal extension is responsible for membrane association. Black-Right-Pointing-Pointer N-terminal works as a molecular lid regulating access to the protein interior. -- Abstract: Dihydroorotate dehydrogenases (DHODHs) are enzymes that catalyze the fourth step of the de novo synthesis of pyrimidine nucleotides. In this reaction, DHODH converts dihydroorotate to orotate, using a flavine mononucleotide as a cofactor. Since the synthesis of nucleotides has different pathways in mammals as compared to parasites, DHODH has gained much attention as a promising target for drug design. Escherichia coli DHODH (EcDHODH) is a family 2 DHODH that interacts with cell membranes in order to promote catalysis. The membrane association is supposedly made via an extension found in the enzyme's N-terminal. In the present work, we used site directed spin labeling (SDSL) to specifically place a magnetic probe at positions 2, 5, 19, and 21 within the N-terminal and thus monitor, by using Electron Spin Resonance (ESR), dynamics and structural changes in this region in the presence of a membrane model system. Overall, our ESR spectra show that the N-terminal indeed binds to membranes and that it experiences a somewhat high flexibility that could be related to the role of this region as a molecular lid controlling the entrance of the enzyme's active site and thus allowing the enzyme to give access to quinones that are dispersed in the membrane and that are necessary for the catalysis.

  12. The genus Leishmania

    PubMed Central

    Garnham, P. C. C.

    1971-01-01

    The systematic position of the so-called ”species” of Leishmania is examined and an attempt made to determine their phylogenetic relationships. The morphology of the organisms as seen by light- and electron-microscopy is described; neither method provides useful criteria for the determination of species. The behaviour of the parasites in insect and in vertebrate hosts offers a better method of classification. In this way, the species may be divided into 4 main groups, comprising the mammalian species involving man, the distinctive species L. enriettii in the guinea-pig, those infecting lizards, and species apparently in various stages of evolution in phlebotomines. The so-called ”human” group is divided into visceral forms (originating chiefly in wild canidae) and cutaneous forms (probably of rodent origin). The named species of the former group include L. donovani and L. infantum. The cutaneous species include L. tropica tropica (=minor), L. tropica major, L. brasiliensis, L. peruana, L. guyanensis, and L. mexicana. L. pifanoi is probably not a distinct species but represents various forms as modified by the failure of cell-mediated immunity in the host. Leishmanial infections can be identified first by ascertaining the geographical area where the infection was acquired, and then by more or less complicated laboratory investigations including characteristics in culture, serological tests, the response of special hosts in terms of symptomatology, and the behaviour of the parasite in the phlebotomine host. No test is infallible, and an effective simple test is urgently needed. The preservation of Leishmania strains is an important research procedure and a method for conserving parasites by lyophilization is described briefly. PMID:5316250

  13. An imported case of cutaneous leishmaniasis caused by Leishmania (Leishmania) donovani in Japan.

    PubMed

    Ito, Kotaro; Takahara, Masakazu; Ito, Makoto; Oshiro, Minoru; Takahashi, Kenzo; Uezato, Hiroshi; Imafuku, Shinichi

    2014-10-01

    Leishmaniasis is a major world health problem, and 12 million people are estimated to be infected in 88 countries. There have been few reports of leishmaniasis in Japan and all were of foreign origin; therefore diagnosis is difficult for Japanese physicians. There are 21 different pathogenic Leishmania species, and identification is obtained by polymerase chain reaction (PCR). Here we report an imported case of leishmaniasis by Leishmania (Leishmania) donovani infection from Sri Lanka. L. (L.) donovani usually causes visceral leishmaniasis, but in this case, the patient manifested cutaneous leishmaniasis. The identification of Leishmania species by PCR and investigation of the patient's background such as nationality and disease endemicity are important for diagnosis and treatment. This is the first report of cutaneous leishmaniasis by L. (L.) donovani in Japan.

  14. Transcription of Leishmania major U2 small nuclear RNA gene is directed by extragenic sequences located within a tRNA-like and a tRNA-Ala gene.

    PubMed

    Rojas-Sánchez, Saúl; Figueroa-Angulo, Elisa; Moreno-Campos, Rodrigo; Florencio-Martínez, Luis E; Manning-Cela, Rebeca G; Martínez-Calvillo, Santiago

    2016-07-19

    Leishmania and other trypanosomatid parasites possess atypical mechanisms of gene expression, including the maturation of mRNAs by trans-splicing and the involvement of RNA Polymerase III in transcription of all snRNA molecules. Since snRNAs are essential for trans-splicing, we are interested in the study of the sequences that direct their expression. Here we report the characterization of L. major U2 snRNA promoter region. All species of Leishmania possess a single U2 snRNA gene that contains a divergently-oriented tRNA-Ala gene in the upstream region. Between these two genes we found a tRNA-like sequence that possesses conserved boxes A and B. Primer extension and RT-qPCR analyses with RNA from transiently-transfected cells showed that transcription of L. major U2 snRNA is almost abolished when boxes A and B from the tRNA-like are deleted or mutated. The levels of the U2 snRNA were also highly affected when base substitutions were introduced into box B from the tRNA-Ala gene and the first nucleotides of the U2 snRNA gene itself. We also demonstrate that the tRNA-like is transcribed, generating a main transcript of around 109 bases. As pseudouridines in snRNAs are required for splicing in other organisms, we searched for this modified nucleotide in the L. major U2 snRNA. Our results show the presence of six pseudouridines in the U2 snRNA, including one in the Sm site that has not been reported in other organisms. Four different regions control the transcription of the U2 snRNA gene in L. major: boxes A and B from the neighbor tRNA-like, box B from the upstream tRNA-Ala gene and the first nucleotides of the U2 snRNA. Thus, the promoter region of L. major U2 snRNA is different from any other promoter reported for snRNAs. Pseudouridines could play important roles in L. major U2 snRNA, since they were found in functionally important regions, including the branch point recognition region and the Sm binding site.

  15. Novel Leishmania and Malaria Potassium Channels: Candidate Therapeutic Targets

    DTIC Science & Technology

    2005-08-01

    2-each for Plasmodium falciparum [PFK1 & PFK2], Toxoplasma gondii [TGK1 & TGK2], and 3 each for Leishmania major [LMK1, LMK2, & LMK3] and Trypanasoma...cloned from Leishmania major. • Two additional K+ channel genes identified and cloned from Toxoplasma gondii . * Three additional K+ channel genes...falciparum, T. gondii , and 3 each for L. major and T. cruzi). Using a combination of cultured mammalian cells and Xenopus oocytes for heterologous expression

  16. Genetic dissection of pyrimidine biosynthesis and salvage in Leishmania donovani.

    PubMed

    Wilson, Zachary N; Gilroy, Caslin A; Boitz, Jan M; Ullman, Buddy; Yates, Phillip A

    2012-04-13

    Protozoan parasites of the Leishmania genus express the metabolic machinery to synthesize pyrimidine nucleotides via both de novo and salvage pathways. To evaluate the relative contributions of pyrimidine biosynthesis and salvage to pyrimidine homeostasis in both life cycle stages of Leishmania donovani, individual mutant lines deficient in either carbamoyl phosphate synthetase (CPS), the first enzyme in pyrimidine biosynthesis, uracil phosphoribosyltransferase (UPRT), a salvage enzyme, or both CPS and UPRT were constructed. The Δcps lesion conferred pyrimidine auxotrophy and a growth requirement for medium supplementation with one of a plethora of pyrimidine nucleosides or nucleobases, although only dihydroorotate or orotate could circumvent the pyrimidine auxotrophy of the Δcps/Δuprt double knockout. The Δuprt null mutant was prototrophic for pyrimidines but could not salvage uracil or any pyrimidine nucleoside. The capability of the Δcps parasites to infect mice was somewhat diminished but still robust, indicating active pyrimidine salvage by the amastigote form of the parasite, but the Δcps/Δuprt mutant was completely attenuated with no persistent parasites detected after a 4-week infection. Complementation of the Δcps/Δuprt clone with either CPS or UPRT restored infectivity. These data establish that an intact pyrimidine biosynthesis pathway is essential for the growth of the promastigote form of L. donovani in culture, that all uracil and pyrimidine nucleoside salvage in the parasite is mediated by UPRT, and that both the biosynthetic and salvage pathways contribute to a robust infection of the mammalian host by the amastigote. These findings impact potential therapeutic design and vaccine strategies for visceral leishmaniasis.

  17. Genetic Dissection of Pyrimidine Biosynthesis and Salvage in Leishmania donovani*

    PubMed Central

    Wilson, Zachary N.; Gilroy, Caslin A.; Boitz, Jan M.; Ullman, Buddy; Yates, Phillip A.

    2012-01-01

    Protozoan parasites of the Leishmania genus express the metabolic machinery to synthesize pyrimidine nucleotides via both de novo and salvage pathways. To evaluate the relative contributions of pyrimidine biosynthesis and salvage to pyrimidine homeostasis in both life cycle stages of Leishmania donovani, individual mutant lines deficient in either carbamoyl phosphate synthetase (CPS), the first enzyme in pyrimidine biosynthesis, uracil phosphoribosyltransferase (UPRT), a salvage enzyme, or both CPS and UPRT were constructed. The Δcps lesion conferred pyrimidine auxotrophy and a growth requirement for medium supplementation with one of a plethora of pyrimidine nucleosides or nucleobases, although only dihydroorotate or orotate could circumvent the pyrimidine auxotrophy of the Δcps/Δuprt double knockout. The Δuprt null mutant was prototrophic for pyrimidines but could not salvage uracil or any pyrimidine nucleoside. The capability of the Δcps parasites to infect mice was somewhat diminished but still robust, indicating active pyrimidine salvage by the amastigote form of the parasite, but the Δcps/Δuprt mutant was completely attenuated with no persistent parasites detected after a 4-week infection. Complementation of the Δcps/Δuprt clone with either CPS or UPRT restored infectivity. These data establish that an intact pyrimidine biosynthesis pathway is essential for the growth of the promastigote form of L. donovani in culture, that all uracil and pyrimidine nucleoside salvage in the parasite is mediated by UPRT, and that both the biosynthetic and salvage pathways contribute to a robust infection of the mammalian host by the amastigote. These findings impact potential therapeutic design and vaccine strategies for visceral leishmaniasis. PMID:22367196

  18. An overview on Leishmania vaccines: A narrative review article.

    PubMed

    Rezvan, Hossein; Moafi, Mohammad

    2015-01-01

    Leishmaniasis is one of the major health problems and categorized as a class I disease (emerging and uncontrolled) by World Health Organization (WHO), causing highly significant morbidity and mortality. Indeed, more than 350 million individuals are at risk of Leishmania infection, and about 1.6 million new cases occur causing more than 50 thousands death annually. Because of the severe toxicity and drug resistance, present chemotherapy regimen against diverse forms of Leishmania infections is not totally worthwhile. However, sound immunity due to natural infection, implies that vigor cellular immunity against Leishmania parasites, via their live, attenuated or killed forms, can be developed in dogs and humans. Moreover, genetically conserved antigens (in most of Leishmania species), and components of sand fly saliva confer potential immunogenic molecules for Leishmania vaccination. Vaccines successes in animal studies and some clinical trials clearly justify more researches and investments illuminating opportunities in suitable vaccine designation.

  19. An overview on Leishmania vaccines: A narrative review article

    PubMed Central

    Rezvan, Hossein; Moafi, Mohammad

    2015-01-01

    Leishmaniasis is one of the major health problems and categorized as a class I disease (emerging and uncontrolled) by World Health Organization (WHO), causing highly significant morbidity and mortality. Indeed, more than 350 million individuals are at risk of Leishmania infection, and about 1.6 million new cases occur causing more than 50 thousands death annually. Because of the severe toxicity and drug resistance, present chemotherapy regimen against diverse forms of Leishmania infections is not totally worthwhile. However, sound immunity due to natural infection, implies that vigor cellular immunity against Leishmania parasites, via their live, attenuated or killed forms, can be developed in dogs and humans. Moreover, genetically conserved antigens (in most of Leishmania species), and components of sand fly saliva confer potential immunogenic molecules for Leishmania vaccination. Vaccines successes in animal studies and some clinical trials clearly justify more researches and investments illuminating opportunities in suitable vaccine designation. PMID:25992245

  20. Study of Compounds for Activity against Leishmania

    DTIC Science & Technology

    1994-03-27

    Comparative Antileishmanial Activity of Selected Compounds Against Leishmania Leishmania donovani and Leishmania Viannia braziliensis 7 IV. Zn vitro...Studies of Oligonucleotides Against Leishmania Leishmania donovani ............................................................ 9 Discussion...for several years in studies to identify new compounds for antileishmanial activity against both visceral (Lelshmania Leishmania donovani ) and

  1. Validation of Recombinant Salivary Protein PpSP32 as a Suitable Marker of Human Exposure to Phlebotomus papatasi, the Vector of Leishmania major in Tunisia

    PubMed Central

    Bettaieb, Jihene; Abdeladhim, Maha; Hadj Kacem, Saoussen; Abdelkader, Rania; Gritli, Sami; Chemkhi, Jomaa; Aslan, Hamide; Kamhawi, Shaden; Ben Salah, Afif; Louzir, Hechmi; Valenzuela, Jesus G.; Ben Ahmed, Melika

    2015-01-01

    Background During a blood meal, female sand flies, vectors of Leishmania parasites, inject saliva into the host skin. Sand fly saliva is composed of a large variety of components that exert different pharmacological activities facilitating the acquisition of blood by the insect. Importantly, proteins present in saliva are able to elicit the production of specific anti-saliva antibodies, which can be used as markers for exposure to vector bites. Serological tests using total sand fly salivary gland extracts are challenging due to the difficulty of obtaining reproducible salivary gland preparations. Previously, we demonstrated that PpSP32 is the immunodominant salivary antigen in humans exposed to Phlebotomus papatasi bites and established that humans exposed to P. perniciosus bites do not recognize it. Methodology/Principal Findings Herein, we have validated, in a large cohort of 522 individuals, the use of the Phlebotomus papatasi recombinant salivary protein PpSP32 (rPpSP32) as an alternative method for testing exposure to the bite of this sand fly. We also demonstrated that screening for total anti-rPpSP32 IgG antibodies is sufficient, being comparable in efficacy to the screening for IgG2, IgG4 and IgE antibodies against rPpSP32. Additionally, sera obtained from dogs immunized with saliva of P. perniciosus, a sympatric and widely distributed sand fly in Tunisia, did not recognize rPpSP32 demonstrating its suitability as a marker of exposure to P. papatasi saliva. Conclusions/Significance Our data indicate that rPpSP32 constitutes a useful epidemiological tool to monitor the spatial distribution of P. papatasi in a particular region, to direct control measures against zoonotic cutaneous leishmaniasis, to assess the efficiency of vector control interventions and perhaps to assess the risk of contracting the disease. PMID:26368935

  2. Activity of Hydroxyurea against Leishmania mexicana▿

    PubMed Central

    Martinez-Rojano, Hugo; Mancilla-Ramirez, Javier; Quiñonez-Diaz, Laura; Galindo-Sevilla, Norma

    2008-01-01

    Leishmania mexicana is a protozoan parasite that causes a disease in humans with frequent relapses after treatment. It is also highly resistant to the currently available drugs. For this reason, there is an urgent need for more effective antileishmanial drugs. Hydroxyurea, an anticancer drug, is toxic to replicating eukaryotic cells and has been proven to be effective in arresting the Leishmania major cell cycle. In this study, hydroxyurea was tested in an in vitro model of intracellular Leishmania infection in macrophages. The parasite density in infected macrophages was measured by microscopy after incubation for various times and treatment with hydroxyurea at different concentrations. Viable parasites that could be transformed into promastigotes by shifting the temperature to 26°C were counted every other day after the replacement of hydroxyurea with fresh medium. Meglumine antimoniate, the standard drug treatment for Leishmania mexicana, was used as a reference drug under the same experimental conditions. Hydroxyurea completely eliminated Leishmania parasites when it was used at a dosage of 10 or 100 μg/ml. Differences in the length of treatment needed to achieve elimination were as follows: the 10-μg/ml doses required 9 days, while 3 days was sufficient when 100 μg/ml was used. Hydroxyurea had a 50% effective dose of 0.015 μg/ml in vitro, which was observed on day 6 after exposure. Hydroxyurea is highly effective in killing intracellular amastigotes in vitro. PMID:18694950

  3. Searching for New Chemotherapies for Tropical Diseases: Ruthenium-Clotrimazole Complexes Display High in vitro Activity Against Leishmania major and Trypanosoma cruzi and Low Toxicity Toward Normal Mammalian Cells

    PubMed Central

    Martínez, Alberto; Carreon, Teresia; Iniguez, Eva; Anzellotti, Atilio; Sánchez, Antonio; Tyan, Marina; Sattler, Aaron; Herrera, Linda; Maldonado, Rosa A.; Sánchez-Delgado, Roberto A.

    2012-01-01

    Eight new ruthenium complexes of clotrimazole (CTZ) with high antiparasitic activity have been synthesized, cis,fac-[RuIICl2(DMSO)3(CTZ)] (1), cis,cis,trans-[RuIICl2(DMSO)2(CTZ)2] (2), Na[RuIIICl4(DMSO)(CTZ)] (3) and Na[trans-RuIIICl4(CTZ)2] (4), [RuII(η6-p-cymene)Cl2(CTZ)] (5), [RuII(η6-p-cymene)(bipy)(CTZ)][BF4]2 (6), [RuII(η6-p-cymene)(en)(CTZ)][BF4]2 (7) and [RuII(η6-p-cymene)(acac)(CTZ)][BF4] (8) (bipy = bipyridine; en = ethlylenediamine; acac = acetylacetonate). The crystal structures of compounds 4-8 are described. Complexes 1-8 are active against promastigotes of Leishmania major and epimastigotes of Trypanosoma cruzi. Most notably complex 5 increases the activity of CTZ by factors of 110 and 58 against L. major and T. cruzi, with no appreciable toxicity to human osteoblasts, resulting in nanomolar and low micromolar lethal doses and therapeutic indexes of 500 and 75, respectively. In a high-content imaging assay on L. major infected intraperitoneal mice macrophages, complex 5 showed significant inhibition on the proliferation of intracellular amastigotes (IC70 = 29 nM), while complex 8 displayed some effect at a higher concentration (IC40 = 1 μM). PMID:22448965

  4. Hunger tolerance and Leishmania in sandflies.

    PubMed

    Schlein, Y; Jacobson, R L

    2001-11-08

    The sandfly Phlebotomus papatasi transmits Leishmania major, the agent of cutaneous leishmaniasis, in desert and savannah regions of the Old World, where seasonal stress of dehydration and heat reduces the quantity of sugar in plant leaves. Without essential sugar, only a few flies that feed on leaves can survive for long enough to deposit eggs and transmit Leishmania. Accordingly, selection for hunger tolerance may also select for pathogen susceptibility in flies. Here we provide evidence of a link between these advantageous and costly properties by testing the susceptibility of flies selected by sugar deprivation and of flies from irrigated and arid habitats.

  5. A primer for Leishmania population genetic studies.

    PubMed

    Rougeron, V; De Meeûs, T; Bañuls, A-L

    2015-02-01

    Leishmaniases remain a major public health problem. Despite the development of elaborate experimental techniques and sophisticated statistical tools, how these parasites evolve, adapt themselves to new environmental compartments and hosts, and develop resistance to new drugs remains unclear. Leishmania parasites constitute a complex model from a biological, ecological, and epidemiological point of view but also with respect to their genetics and phylogenetics. With this in view, we seek to outline the criteria, caveats, and confounding factors to be considered for Leishmania population genetic studies. We examine how the taxonomic complexity, heterozygosity, intraspecific and interspecific recombination, aneuploidy, and ameiotic recombination of Leishmania intersect with population genetic studies of this parasite. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Leishmania species: Detection and identification by nested PCR assay from skin samples of rodent reservoirs

    PubMed Central

    Akhavan, Amir Ahmad; Mirhendi, Hossein; Khamesipour, Ali; Alimohammadian, Mohammad Hossein; Rassi, Yavar; Bates, Paul; Kamhawi, Shaden; Valenzuela, Jesus G.; Arandian, Mohammad Hossein; Abdoli, Hamid; Jalali-zand, Niloufar; Jafari, Reza; Shareghi, Niloufar; Ghanei, Maryam; Yaghoobi-Ershadi, Mohammad Reza

    2010-01-01

    Many rodent species act as reservoir hosts of zoonotic cutaneous leishmaniasis in endemic areas. In the present study a simple and reliable assay based on nested PCR was developed for the detection and identification of Leishmania parasites from rodent skin samples. We designed Leishmania-specific primers that successfully amplified ITS regions of Leishmania major, Leishmania gerbilli and Leishmania turanica using nested PCR. Out of 95 field collected Rhombomys opimus, 21 were positive by microscopic examination and 48 by nested PCR. The percentage of gerbils infected with L. major, L. gerbilli and L. turanica was 3.2%, 1.1% and 27.4%, respectively. In 15.8% of the rodents, we found mixed natural infections by L. major and L. turanica, 1.1% by L. major and L. gerbilli, and 2.1% by the three species. We concluded that this method is simple and reliable for detecting and identifying Leishmania species circulating in rodent populations. PMID:20566364

  7. Leishmania mexicana amazonensis

    PubMed Central

    Detke, S.; Elsabrouty, R.

    2008-01-01

    Leishmania cannot synthesize purines de novo and rely on their host to furnish these compounds. To accomplish this, they possess multiple purine nucleoside and nucleobase transporters. Subcellular fractionation, immunohistochemical localization with anti adenine nucleotide translocator (ANT) antibodies and surface biotinylation show that the mitochondrial ANT is also present in the plasma membrane of both promastigotes and amastigotes. Leishmania, however, do not appear to rely on this transporter to supplement their purine or energy requirements via preformed ATP from its host. Rather, Leishmania appear to use the plasma membrane ANT as part of a chemotaxis response. ATP is a chemorepellent for Leishmania and cells treated with atractyloside, an inhibitor of ANT, no longer exhibit negative chemotaxis for this compound. PMID:18031742

  8. Leishmania Skin Test

    DTIC Science & Technology

    2010-03-01

    Ninhydrin ), SDS-PAGE and non-viability testing . See Table 3 below: Table 3: Drug Substance Specifications Test Method Specification SDS-PAGE...AD_________________ Award Number: DAMD17-00-C-0030 TITLE: Leishmania Skin Test PRINCIPAL INVESTIGATOR: Nielsen, H.S., Jr...TYPE FINAL, PHASE II ADDENDUM 3. DATES COVERED (From - To) 1 APR 2009 - 28 FEB 2010 4. TITLE AND SUBTITLE Leishmania Skin Test 5a

  9. A long-duration dihydroorotate dehydrogenase inhibitor (DSM265) for prevention and treatment of malaria

    PubMed Central

    Phillips, Margaret A.; Lotharius, Julie; Marsh, Kennan; White, John; Dayan, Anthony; White, Karen L.; Njoroge, Jacqueline W.; El Mazouni, Farah; Lao, Yanbin; Kokkonda, Sreekanth; Tomchick, Diana R.; Deng, Xiaoyi; Laird, Trevor; Bhatia, Sangeeta N.; March, Sandra; Ng, Caroline L.; Fidock, David A.; Wittlin, Sergio; Lafuente-Monasterio, Maria; Benito, Francisco Javier Gamo; Alonso, Laura Maria Sanz; Martinez, Maria Santos; Jimenez-Diaz, Maria Belen; Bazaga, Santiago Ferrer; Angulo-Barturen, Iñigo; Haselden, John N.; Louttit, James; Cui, Yi; Sridhar, Arun; Zeeman, Anna-Marie; Kocken, Clemens; Sauerwein, Robert; Dechering, Koen; Avery, Vicky M.; Duffy, Sandra; Delves, Michael; Sinden, Robert; Ruecker, Andrea; Wickham, Kristina S.; Rochford, Rosemary; Gahagen, Janet; Iyer, Lalitha; Riccio, Ed; Mirsalis, Jon; Bathhurst, Ian; Rueckle, Thomas; Ding, Xavier; Campo, Brice; Leroy, Didier; Rogers, M. John; Rathod, Pradipsinh K.; Burrows, Jeremy N.; Charman, Susan A.

    2015-01-01

    Malaria is one of the most significant causes of childhood mortality but disease control efforts are threatened by resistance of the Plasmodium parasite to current therapies. Continued progress in combating malaria requires development of new, easy to administer drug combinations with broad ranging activity against all manifestations of the disease. DSM265, a triazolopyrimidine-based inhibitor of the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH), is the first DHODH inhibitor to reach clinical development for treatment of malaria. We describe studies profiling the biological activity, pharmacological and pharmacokinetic properties, and safety of DSM265, which supported its advancement to human trials. DSM265 is highly selective towards DHODH of the malaria parasite Plasmodium, efficacious against both blood and liver stages of P. falciparum, and active against drug-resistant parasite isolates. Favorable pharmacokinetic properties of DSM265 are predicted to provide therapeutic concentrations for more than 8 days after a single oral dose in the range of 200–400 mg. DSM265 was well tolerated in repeat dose and cardiovascular safety studies in mice and dogs, was not mutagenic, and was inactive against panels of human enzymes/receptors. The excellent safety profile, blood and liver-stage activity, and predicted long human half-life position DSM265 as a new potential drug combination partner for either single-dose treatment or once weekly chemoprevention. DSM265 has advantages over current treatment options that are dosed daily or are inactive on the parasite liver-stage PMID:26180101

  10. Suppression of immunoglobulin production by a novel dihydroorotate dehydrogenase inhibitor, S-2678.

    PubMed

    Deguchi, Masashi; Kishino, Junji; Hattori, Maki; Furue, Yoko; Yamamoto, Mina; Mochizuki, Izumi; Iguchi, Motofumi; Hirano, Yosuke; Hojou, Kanji; Nagira, Morio; Nishitani, Yoshinori; Okazaki, Kenichi; Yasui, Kiyoshi; Arimura, Akinori

    2008-12-28

    We discovered a novel dihydroorotate dehydrogenase (DHO-DH) inhibitor, S-2678 ([2-fluoro-2',5'-dimethyl-4'-[6-(3-methyl-2-butenyloxy) pyridin-3-yl] biphenyl-4-yl]-(3-methyl-2-butenyl) amine). Its inhibitory activity against DHO-DH was more potent than that of A77 1726, an active metabolite of the anti-rheumatic drug leflunomide. S-2678 suppressed immunoglobulin production in mouse B cells and human peripheral blood mononuclear cells in vitro, with little or no inhibition of cell proliferation, probably through inhibition of class switch recombination in the immunoglobulin heavy chain loci in B cells. In vivo antibody production induced by systemic immunization with ovalbumin was dramatically suppressed by oral administration of S-2678, without any toxicological signs. However, S-2678 did not affect T-cell activation in vitro, and cytokine production induced by intravenous anti-CD3 antibody in mice. S-2678 did not affect host defense in a mouse model of Candida infection, whereas leflunomide severely impaired it. In conclusion, S-2678 selectively acts on B cells, resulting in antibody production, which suggests that it is useful for the treatment of humoral immunity-related diseases.

  11. Characterization of Trypanosoma brucei dihydroorotate dehydrogenase as a possible drug target; structural, kinetic and RNAi studies

    PubMed Central

    Arakaki, Tracy L; Buckner, Frederick S; Gillespie, J Robert; Malmquist, Nicholas A; Phillips, Margaret A; Kalyuzhniy, Oleksandr; Luft, Joseph R; DeTitta, George T; Verlinde, Christophe L M J; Van Voorhis, Wesley C; Hol, Wim G J; Merritt, Ethan A

    2010-01-01

    Nucleotide biosynthesis pathways have been reported to be essential in some protozoan pathogens. Hence, we evaluated the essentiality of one enzyme in the pyrimidine biosyn-thetic pathway, dihydroorotate dehydrogenase (DHODH) from the eukaryotic parasite Trypanosoma brucei through gene knockdown studies. RNAi knockdown of DHODH expression in bloodstream-form T. brucei did not inhibit growth in normal medium, but profoundly retarded growth in pyrimidine-depleted media or in the presence of the known pyrimidine uptake antagonist 5-fluoruracil (5-FU). These results have significant implications for the development of therapeutics to combat T. brucei infection. Specifically, a combination therapy including a T. brucei-specific DHODH inhibitor plus 5-FU may prove to be an effective therapeutic strategy. We also show that this trypanosomal enzyme is inhibited by known inhibitors of bacterial Class 1A DHODH, in distinction to the sensitivity of DHODH from human and other higher eukaryotes. This selectivity is supported by the crystal structure of the T. brucei enzyme, which is reported here at a resolution of 1.95 Å. Additional research, guided by the crystal structure described herein, is needed to identify potent inhibitors of T. brucei DHODH. PMID:18312275

  12. QSAR study on the antimalarial activity of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitors.

    PubMed

    Hou, X; Chen, X; Zhang, M; Yan, A

    2016-01-01

    Plasmodium falciparum, the most fatal parasite that causes malaria, is responsible for over one million deaths per year. P. falciparum dihydroorotate dehydrogenase (PfDHODH) has been validated as a promising drug development target for antimalarial therapy since it catalyzes the rate-limiting step for DNA and RNA biosynthesis. In this study, we investigated the quantitative structure-activity relationships (QSAR) of the antimalarial activity of PfDHODH inhibitors by generating four computational models using a multilinear regression (MLR) and a support vector machine (SVM) based on a dataset of 255 PfDHODH inhibitors. All the models display good prediction quality with a leave-one-out q(2) >0.66, a correlation coefficient (r) >0.85 on both training sets and test sets, and a mean square error (MSE) <0.32 on training sets and <0.37 on test sets, respectively. The study indicated that the hydrogen bonding ability, atom polarizabilities and ring complexity are predominant factors for inhibitors' antimalarial activity. The models are capable of predicting inhibitors' antimalarial activity and the molecular descriptors for building the models could be helpful in the development of new antimalarial drugs.

  13. F901318 represents a novel class of antifungal drug that inhibits dihydroorotate dehydrogenase.

    PubMed

    Oliver, Jason D; Sibley, Graham E M; Beckmann, Nicola; Dobb, Katharine S; Slater, Martin J; McEntee, Laura; du Pré, Saskia; Livermore, Joanne; Bromley, Michael J; Wiederhold, Nathan P; Hope, William W; Kennedy, Anthony J; Law, Derek; Birch, Mike

    2016-10-25

    There is an important medical need for new antifungal agents with novel mechanisms of action to treat the increasing number of patients with life-threatening systemic fungal disease and to overcome the growing problem of resistance to current therapies. F901318, the leading representative of a novel class of drug, the orotomides, is an antifungal drug in clinical development that demonstrates excellent potency against a broad range of dimorphic and filamentous fungi. In vitro susceptibility testing of F901318 against more than 100 strains from the four main pathogenic Aspergillus spp. revealed minimal inhibitory concentrations of ≤0.06 µg/mL-greater potency than the leading antifungal classes. An investigation into the mechanism of action of F901318 found that it acts via inhibition of the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH) in a fungal-specific manner. Homology modeling of Aspergillus fumigatus DHODH has identified a predicted binding mode of the inhibitor and important interacting amino acid residues. In a murine pulmonary model of aspergillosis, F901318 displays in vivo efficacy against a strain of A. fumigatus sensitive to the azole class of antifungals and a strain displaying an azole-resistant phenotype. F901318 is currently in late Phase 1 clinical trials, offering hope that the antifungal armamentarium can be expanded to include a class of agent with a mechanism of action distinct from currently marketed antifungals.

  14. Novel Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase with Anti-malarial Activity in the Mouse Model*

    PubMed Central

    Booker, Michael L.; Bastos, Cecilia M.; Kramer, Martin L.; Barker, Robert H.; Skerlj, Renato; Sidhu, Amar Bir; Deng, Xiaoyi; Celatka, Cassandra; Cortese, Joseph F.; Guerrero Bravo, Jose E.; Crespo Llado, Keila N.; Serrano, Adelfa E.; Angulo-Barturen, Iñigo; Jiménez-Díaz, María Belén; Viera, Sara; Garuti, Helen; Wittlin, Sergio; Papastogiannidis, Petros; Lin, Jing-wen; Janse, Chris J.; Khan, Shahid M.; Duraisingh, Manoj; Coleman, Bradley; Goldsmith, Elizabeth J.; Phillips, Margaret A.; Munoz, Benito; Wirth, Dyann F.; Klinger, Jeffrey D.; Wiegand, Roger; Sybertz, Edmund

    2010-01-01

    Plasmodium falciparum, the causative agent of the most deadly form of human malaria, is unable to salvage pyrimidines and must rely on de novo biosynthesis for survival. Dihydroorotate dehydrogenase (DHODH) catalyzes the rate-limiting step in the pyrimidine biosynthetic pathway and represents a potential target for anti-malarial therapy. A high throughput screen and subsequent medicinal chemistry program identified a series of N-alkyl-5-(1H-benzimidazol-1-yl)thiophene-2-carboxamides with low nanomolar in vitro potency against DHODH from P. falciparum, P. vivax, and P. berghei. The compounds were selective for the parasite enzymes over human DHODH, and x-ray structural data on the analog Genz-667348, demonstrated that species selectivity could be attributed to amino acid differences in the inhibitor-binding site. Compounds from this series demonstrated in vitro potency against the 3D7 and Dd2 strains of P. falciparum, good tolerability and oral exposure in the mouse, and ED50 values in the 4-day murine P. berghei efficacy model of 13–21 mg/kg/day with oral twice-daily dosing. In particular, treatment with Genz-667348 at 100 mg/kg/day resulted in sterile cure. Two recent analogs of Genz-667348 are currently undergoing pilot toxicity testing to determine suitability as clinical development candidates. PMID:20702404

  15. Novel Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase with Anti-malarial Activity in the Mouse Model

    SciTech Connect

    Booker, Michael L.; Bastos, Cecilia M.; Kramer, Martin L.; Barker, Jr., Robert H.; Skerlj, Renato; Sidhu, Amar Bir; Deng, Xiaoyi; Celatka, Cassandra; Cortese, Joseph F.; Guerrero Bravo, Jose E.; Crespo Llado, Keila N.; Serrano, Adelfa E.; Angulo-Barturen, Iñigo; Jiménez-Díaz, María Belén; Viera, Sara; Garuti, Helen; Wittlin, Sergio; Papastogiannidis, Petros; Lin, Jing-wen; Janse, Chris J.; Khan, Shahid M.; Duraisingh, Manoj; Coleman, Bradley; Goldsmith, Elizabeth J.; Phillips, Margaret A.; Munoz, Benito; Wirth, Dyann F.; Klinger, Jeffrey D.; Wiegand, Roger; Sybertz, Edmund

    2010-11-22

    Plasmodium falciparum, the causative agent of the most deadly form of human malaria, is unable to salvage pyrimidines and must rely on de novo biosynthesis for survival. Dihydroorotate dehydrogenase (DHODH) catalyzes the rate-limiting step in the pyrimidine biosynthetic pathway and represents a potential target for anti-malarial therapy. A high throughput screen and subsequent medicinal chemistry program identified a series of N-alkyl-5-(1H-benzimidazol-1-yl)thiophene-2-carboxamides with low nanomolar in vitro potency against DHODH from P. falciparum, P. vivax, and P. berghei. The compounds were selective for the parasite enzymes over human DHODH, and x-ray structural data on the analog Genz-667348, demonstrated that species selectivity could be attributed to amino acid differences in the inhibitor-binding site. Compounds from this series demonstrated in vitro potency against the 3D7 and Dd2 strains of P. falciparum, good tolerability and oral exposure in the mouse, and ED{sub 50} values in the 4-day murine P. berghei efficacy model of 13-21 mg/kg/day with oral twice-daily dosing. In particular, treatment with Genz-667348 at 100 mg/kg/day resulted in sterile cure. Two recent analogs of Genz-667348 are currently undergoing pilot toxicity testing to determine suitability as clinical development candidates.

  16. Target Oriented Drugs against Leishmania

    DTIC Science & Technology

    1981-10-26

    leishmanlal excreted factor (EF) antibody in rabbit sera was developed. The assay, using Leishmania trop ica and Leishmania donovani promastigote EF...tropica LRC L137 L52 Leishmaniia donovani LRC L52 These strains were obtained from the WHO Leishmania Peference Centre collection maintained in the...FO 0 AD M FINAL REPORT0 (N TARGET ORIENTED DRUGS AGAINST LEISHMANIA I URI ZEHAVI, Ph.D. and JOSEPH EL-ON, Ph.D. Supported by U.S. ARMY MEDICAL

  17. Activity of Cuban Plants Extracts against Leishmania amazonensis

    PubMed Central

    García, Marley; Monzote, Lianet; Scull, Ramón; Herrera, Pedro

    2012-01-01

    Natural products have long been providing important drug leads for infectious diseases. Leishmaniasis is a major health problem worldwide that affects millions of people especially in the developing nations. There is no immunoprophylaxis (vaccination) available for Leishmania infections, and conventional treatments are unsatisfactory; therefore, antileishmanial drugs are urgently needed. In this work, 48 alcoholic extracts from 46 Cuban plants were evaluated by an in vitro bioassay against Leishmania amazonensis. Furthermore, their toxicity was assayed against murine macrophage. The three most potent extracts against the amastigote stage of Leishmania amazonensis were from Hura crepitans, Bambusa vulgaris, and Simarouba glauca. PMID:22530133

  18. High Quality Long-Term CD4+ and CD8+ Effector Memory Populations Stimulated by DNA-LACK/MVA-LACK Regimen in Leishmania major BALB/c Model of Infection

    PubMed Central

    Sánchez-Sampedro, Lucas; Gómez, Carmen Elena; Mejías-Pérez, Ernesto; S. Sorzano, Carlos Oscar; Esteban, Mariano

    2012-01-01

    Heterologous vaccination based on priming with a plasmid DNA vector and boosting with an attenuated vaccinia virus MVA recombinant, with both vectors expressing the Leishmania infantum LACK antigen (DNA-LACK and MVA-LACK), has shown efficacy conferring protection in murine and canine models against cutaneus and visceral leishmaniasis, but the immune parameters of protection remain ill defined. Here we performed by flow cytometry an in depth analysis of the T cell populations induced in BALB/c mice during the vaccination protocol DNA-LACK/MVA-LACK, as well as after challenge with L. major parasites. In the adaptive response, there is a polyfunctional CD4+ and CD8+ T cell activation against LACK antigen. At the memory phase the heterologous vaccination induces high quality LACK-specific long-term CD4+ and CD8+ effector memory cells. After parasite challenge, there is a moderate boosting of LACK-specific CD4+ and CD8+ T cells. Anti-vector responses were largely CD8+-mediated. The immune parameters induced against LACK and triggered by the combined vaccination DNA/MVA protocol, like polyfunctionality of CD4+ and CD8+ T cells with an effector phenotype, could be relevant in protection against leishmaniasis. PMID:22715418

  19. Comparing Montanide ISA 720 and 50-V2 adjuvants formulated with LmSTI1 protein of Leishmania major indicated the potential cytokine patterns for induction of protective immune responses in BALB/c mice.

    PubMed

    Shokri, Mahdi; Roohvand, Farzin; Alimohammadian, Mohammad Hossein; Ebrahimirad, Mina; Ajdary, Soheila

    2016-08-01

    Adjuvants have a key role in subunit vaccine formulations to generate protective immune responses. Herein, we present results of a comparative study on mice immunized with E. coli-derived rLmSTI1 antigen formulated with Montanide ISA 720 (Ag-M720) and ISA 50-V2 (Ag-M50) adjuvants against Leishmania major (L. major). Groups of BALB/c mice were immunized with either Ag-M720 or Ag-M50 by 3 subcutaneous injections with 3-week intervals. Three weeks after the last injection mice were challenged by L. major promastigotes. Immune responses were evaluated before, 3 weeks, and 8 weeks after challenge. Results indicated lower parasite and lesion size in vaccinated mice (the lowest for Ag-M720 indicating the best protection) which correlated with higher IFN-γ induction in immunized groups (Ag-M720 and Ag-M50) compared to control (PBS/adjuvant alone) group. Immune assays showed comparable IFN-γ, total IgG, IgG1 and IgG2a levels for Ag-M720 and Ag-M50 immunized mice but higher induction of IL-4, IL-10 and IL-17 in Ag-M50 and the highest IL-10/IL-17 ratio in Ag-M720 group followed by Ag-M50 and control groups. Altogether, results indicated that lower induction of IL-4, IL-10 and IL-17 cytokines (and/or higher ratio of IL-10/IL-17) despite comparable IFN-γ might be the reason for the superior protection in Ag-M720 group. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Characterization of Leishmania (Leishmania) tropica axenic amastigotes.

    PubMed

    Nasereddin, Abedelmajeed; Schweynoch, Carola; Schonian, Gabriele; Jaffe, Charles L

    2010-01-01

    Optimum conditions for generating Leishmania (Leishmania) tropica axenic amastigotes (AxA) in culture were determined, pH 5.5/36 degrees C, and the parasites characterized by different techniques, including light microscopy, macrophage infection, stage specific antigen expression and differential display. AxA were morphologically similar to amastigotes and 15.5-fold more infective than stationary phase promastigotes for mouse peritoneal macrophages. Western blotting with promastigote stage specific monoclonal antibodies to either lipophosphoglycan (T2) or a 60 kDa flagella antigen (F3) showed a dramatic decrease in antigen expression when AxA were compared to promastigotes. Similarly F3 gave strong immune fluorescent staining of the promastigote flagellum, but no fluorescence was detected when AxA were examined. Conversely, Western blotting with the amastigote specific monoclonal antibody (T16) showed that this antigen is more highly expressed in AxA than promastigotes. Differential display-PCR was used to identify several parasite genes showing stage specific expression. One gene selectively expressed by AxA was partially sequenced and identified as Leishmania (L.) tropicaamastin. Amastigote specific expression of this gene was further confirmed by reverse transcriptase-PCR (RT-PCR) using AxA and infected macrophages. No amastin expression was observed with promastigotes. Expression of the cysteine protease B (cpb) and protein kinase A catalytic isoform 1 subunit (pkac1) in promastigotes and AxA was also examined by RT-PCR. Pkac1 was strongly expressed by promastigotes, while cpb expression was only seen with AxA or infected macrophages. L. (L.) tropica AxA will prove useful for further studies on parasite differentiation and gene regulation, as well as for drug screening.

  1. In vitro analysis regarding the safety of components used in a film-based therapeutic system loaded with meglumine antimoniate and its activity toward Leishmania major experimental infections: a preliminary study.

    PubMed

    Gutiérrez, Jennifer; Vallejo, Bibiana; Barbosa, Helber; Pinzón, John; Delgado, Gabriela

    2013-06-01

    Owing to its biocompatibility properties and its ability to promote the scar healing process, chitosan is employed in tissue engineering for the manufacture of formulations. To control the characteristic skin ulcers of cutaneous leishmaniasis (CL), the use of a biopolymeric system that favors the scar healing process and releases an active agent such as meglumine antimoniate may be a better option. For these reasons, here we analyzed the cytotoxic capabilities of excipients [medium molecular weight chitosan (MMWC), lactic acid (LA) and polyvinylpyrrolidone (PVP)], used for the formulation of a film-based therapeutic system that releases meglumine antimoniate and were evaluated on human macrophages [monocyte-derived macrophages (MDMs)], L929 fibroblasts and parasites (Leishmania major promastigotes and intracellular amastigotes). The ability of excipients to modulate the cytokines production involved in the scar healing process was compared with film-based therapeutic system. The efficiency of a film-based therapeutic system loaded with meglumine antimoniate was compared with conventional formulation (Albiventriz(®)). We found that MMWC was toxic for two parasite forms. In contrast, measurement of interleukin levels did not show any evidence of preferential secretion as a side effect of treating human macrophages with MMWC. Finally, the efficiency of a polymeric film-based therapeutic system that was loaded with meglumine antimoniate could not be determined due to the high degree of toxicity observed in infected MDMs; moreover, these compounds do not induce any apparent immunomodulatory effects. Our findings suggest that the final concentrations of each excipients (MMWC, LA and PVP) that were used in the polymeric film were suitable vehicles for active pharmaceutical compound delivery and did not selectively affect (enhancing or diminishing immune activity) macrophages.

  2. Mice from a genetically resistant background lacking the interferon gamma receptor are susceptible to infection with Leishmania major but mount a polarized T helper cell 1-type CD4+ T cell response

    PubMed Central

    1995-01-01

    Mice with homologous disruption of the gene coding for the ligand- binding chain of the interferon (IFN) gamma receptor and derived from a strain genetically resistant to infection with Leishmania major have been used to study further the role of this cytokine in the differentiation of functional CD4+ T cell subsets in vivo and resistance to infection. Wild-type 129/Sv/Ev mice are resistant to infection with this parasite, developing only small lesions, which resolve spontaneously within 6 wk. In contrast, mice lacking the IFN- gamma receptor develop large, progressing lesions. After infection, lymph nodes (LN) and spleens from both wild-type and knockout mice showed an expansion of CD4+ cells producing IFN-gamma as revealed by measuring IFN-gamma in supernatants of specifically stimulated CD4+ T cells, by enumerating IFN-gamma-producing T cells, and by Northern blot analysis of IFN-gamma transcripts. No biologically active interleukin (IL) 4 was detected in supernatants of in vitro-stimulated LN or spleen cells from infected wild-type or deficient mice. Reverse transcription polymerase chain reaction analysis with primers specific for IL-4 showed similar IL-4 message levels in LN from both types of mice. The IL-4 message levels observed were comparable to those found in similarly infected C57BL/6 mice and significantly lower than the levels found in BALB/c mice. Anti-IFN-gamma treatment of both types of mice failed to alter the pattern of cytokines produced after infection. These data show that even in the absence of IFN-gamma receptors, T helper cell (Th) 1-type responses still develop in genetically resistant mice with no evidence for the expansion of Th2 cells. PMID:7869054

  3. Alum-precipitated autoclaved Leishmania major plus bacille Calmette-Guérrin, a candidate vaccine for visceral leishmaniasis: safety, skin-delayed type hypersensitivity response and dose finding in healthy volunteers.

    PubMed

    Kamil, A A; Khalil, E A G; Musa, A M; Modabber, F; Mukhtar, M M; Ibrahim, M E; Zijlstra, E E; Sacks, D; Smith, P G; Zicker, F; El-Hassan, A M

    2003-01-01

    In a previous efficacy study, autoclaved Leishmania major (ALM) + bacille Calmette-Guérrin (BCG) vaccine was shown to be safe, but not superior to BCG alone, in protecting against visceral leishmaniasis. From June 1999 to June 2000, we studied the safety and immunogenicity of different doses of alum-precipitated ALM + BCG vaccine mixture administered intradermally to evaluate whether the addition of alum improved the immunogenicity of ALM. Twenty-four healthy adult volunteers were recruited and sequentially allocated to receive either 10 microg, 100 microg, 200 microg, or 400 microg of leishmanial protein in the alum-precipitated ALM + BCG vaccine mixture. Side effects were minimal for all doses and confined to the site of injection. All volunteers in the 10 microg, 100 microg, and 400 microg groups had a leishmanin skin test (LST) reaction of > or = 5 mm by day 42 and this response was maintained when tested after 90 d. Only 1 volunteer out of 5 in the 200 microg group had a LST reaction of > or = 5 mm by day 42 and the reasons for the different LST responses in this group are unclear. This is the first time that an alum adjuvant with ALM has been in used in humans and the vaccine mixture was safe and induced a strong delayed type hypersensitivity (DTH) reaction in the study volunteers. On the basis of this study we suggest that 100 1 microg of leishmanial protein in the vaccine mixture is a suitable dose for future efficacy studies, as it induced the strongest DTH reaction following vaccination.

  4. Proteomic analysis of the secretome of Leishmania donovani

    PubMed Central

    Silverman, J Maxwell; Chan, Simon K; Robinson, Dale P; Dwyer, Dennis M; Nandan, Devki; Foster, Leonard J; Reiner, Neil E

    2008-01-01

    Background Leishmania and other intracellular pathogens have evolved strategies that support invasion and persistence within host target cells. In some cases the underlying mechanisms involve the export of virulence factors into the host cell cytosol. Previous work from our laboratory identified one such candidate leishmania effector, namely elongation factor-1α, to be present in conditioned medium of infectious leishmania as well as within macrophage cytosol after infection. To investigate secretion of potential effectors more broadly, we used quantitative mass spectrometry to analyze the protein content of conditioned medium collected from cultures of stationary-phase promastigotes of Leishmania donovani, an agent of visceral leishmaniasis. Results Analysis of leishmania conditioned medium resulted in the identification of 151 proteins apparently secreted by L. donovani. Ratios reflecting the relative amounts of each leishmania protein secreted, as compared to that remaining cell associated, revealed a hierarchy of protein secretion, with some proteins secreted to a greater extent than others. Comparison with an in silico approach defining proteins potentially exported along the classic eukaryotic secretion pathway suggested that few leishmania proteins are targeted for export using a classic eukaryotic amino-terminal secretion signal peptide. Unexpectedly, a large majority of known eukaryotic exosomal proteins was detected in leishmania conditioned medium, suggesting a vesicle-based secretion system. Conclusion This analysis shows that protein secretion by L. donovani is a heterogeneous process that is unlikely to be determined by a classical amino-terminal secretion signal. As an alternative, L. donovani appears to use multiple nonclassical secretion pathways, including the release of exosome-like microvesicles. PMID:18282296

  5. Identification of inhibitors that dually target the new permeability pathway and dihydroorotate dehydrogenase in the blood stage of Plasmodium falciparum

    PubMed Central

    Dickerman, Benjamin K.; Elsworth, Brendan; Cobbold, Simon A.; Nie, Catherine Q.; McConville, Malcolm J.; Crabb, Brendan S.; Gilson, Paul R.

    2016-01-01

    Plasmodium parasites are responsible for the devastating disease malaria that affects hundreds of millions of people each year. Blood stage parasites establish new permeability pathways (NPPs) in infected red blood cell membranes to facilitate the uptake of nutrients and removal of parasite waste products. Pharmacological inhibition of the NPPs is expected to lead to nutrient starvation and accumulation of toxic metabolites resulting in parasite death. Here, we have screened a curated library of antimalarial compounds, the MMV Malaria Box, identifying two compounds that inhibit NPP function. Unexpectedly, metabolic profiling suggested that both compounds also inhibit dihydroorotate dehydrogense (DHODH), which is required for pyrimidine synthesis and is a validated drug target in its own right. Expression of yeast DHODH, which bypasses the need for the parasite DHODH, increased parasite resistance to these compounds. These studies identify two potential candidates for therapeutic development that simultaneously target two essential pathways in Plasmodium, NPP and DHODH. PMID:27874068

  6. 3D-QSAR Studies on a Series of Dihydroorotate Dehydrogenase Inhibitors: Analogues of the Active Metabolite of Leflunomide

    PubMed Central

    Li, Shun-Lai; He, Mao-Yu; Du, Hong-Guang

    2011-01-01

    The active metabolite of the novel immunosuppressive agent leflunomide has been shown to inhibit the enzyme dihydroorotate dehydrogenase (DHODH). This enzyme catalyzes the fourth step in de novo pyrimidine biosynthesis. Self-organizing molecular field analysis (SOMFA), a simple three-dimensional quantitative structure-activity relationship (3D-QSAR) method is used to study the correlation between the molecular properties and the biological activities of a series of analogues of the active metabolite. The statistical results, cross-validated rCV2 (0.664) and non cross-validated r2 (0.687), show a good predictive ability. The final SOMFA model provides a better understanding of DHODH inhibitor-enzyme interactions, and may be useful for further modification and improvement of inhibitors of this important enzyme. PMID:21686163

  7. Use of parasitological culture to detect Leishmania (Leishmania) chagasi in naturally infected dogs.

    PubMed

    de Almeida, Arleana do Bom Parto Ferreira; Sousa, Valéria Régia Franco; Sorte, Eveline da Cruz Boa; Figueiredo, Fabiano Borges; de Paula, Daphine Ariadne Jesus; Pimentel, Maria Fernanda Aranega; Dutra, Valéria; Madeira, Maria de Fátima

    2011-12-01

    In Brazil, although the domestic dog is a major target for the control actions for visceral leishmaniasis, knowledge gaps of the Leishmania species present in those animals still exist in many endemic areas. The objective of this study was the use of parasitological culture as a diagnosis tool and identification of species of Leishmania and other trypanosomatids in the canine population in the city of Cuiaba/Mato Grosso. Biological samples such as blood, intact skin fragments, cutaneous ulcers, and bone marrow were collected during a cross-sectional study and cultured on biphasic medium (Novy-MacNeil-Nicolle [NNN]/Schneider's). Leishmania isolates were characterized through isoenzyme electrophoresis. Isolates were obtained from 11.2% (n=54) of the 482 animals studied considering the different anatomical sites investigated. Leishmania chagasi was confirmed in 8.3% (n=40) dogs and Trypanosoma caninum in 2.9% (n=14). The sample of intact skin presented a higher chance of isolation of L. chagasi in symptomatic dogs and bone marrow in asymptomatic dogs (p<0.05). The results presented in this study emphasize the value of culture and confirm, for the first time, the circulation of L. chagasi in the canine population in different neighborhoods of the city of Cuiaba and broaden the knowledge of the geographical distribution of T. caninum in Brazil.

  8. Autophagic digestion of Leishmania major by host macrophages is associated with differential expression of BNIP3, CTSE, and the miRNAs miR-101c, miR-129, and miR-210.

    PubMed

    Frank, Benjamin; Marcu, Ana; de Oliveira Almeida Petersen, Antonio Luis; Weber, Heike; Stigloher, Christian; Mottram, Jeremy C; Scholz, Claus Juergen; Schurigt, Uta

    2015-07-31

    Autophagy participates in innate immunity by eliminating intracellular pathogens. Consequently, numerous microorganisms have developed strategies to impair the autophagic machinery in phagocytes. In the current study, interactions between Leishmania major (L. m.) and the autophagic machinery of bone marrow-derived macrophages (BMDM) were analyzed. BMDM were generated from BALB/c mice, and the cells were infected with L. m. promastigotes. Transmission electron microscopy (TEM) and electron tomography were used to investigate the ultrastructure of BMDM and the intracellular parasites. Affymetrix chip analyses were conducted to identify autophagy-related messenger RNAs (mRNAs) and microRNAs (miRNAs). The protein expression levels of autophagy related 5 (ATG5), BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), cathepsin E (CTSE), mechanistic target of rapamycin (MTOR), microtubule-associated proteins 1A/1B light chain 3B (LC3B), and ubiquitin (UB) were investigated through western blot analyses. BMDM were transfected with specific small interfering RNAs (siRNAs) against autophagy-related genes and with mimics or inhibitors of autophagy-associated miRNAs. The infection rates of BMDM were determined by light microscopy after a parasite-specific staining. The experiments demonstrated autophagy induction in BMDM after in vitro infection with L. m.. The results suggested a putative MTOR phosphorylation-dependent counteracting mechanism in the early infection phase and indicated that intracellular amastigotes were cleared by autophagy in BMDM in the late infection phase. Transcriptomic analyses and specific downregulation of protein expression with siRNAs suggested there is an association between the infection-specific over expression of BNIP3, as well as CTSE, and the autophagic activity of BMDM. Transfection with mimics of mmu-miR-101c and mmu-miR-129-5p, as well as with an inhibitor of mmu-miR-210-5p, demonstrated direct effects of the respective miRNAs on

  9. A Historical Overview of the Classification, Evolution, and Dispersion of Leishmania Parasites and Sandflies

    PubMed Central

    Akhoundi, Mohammad; Kuhls, Katrin; Cannet, Arnaud; Votýpka, Jan; Marty, Pierre; Delaunay, Pascal; Sereno, Denis

    2016-01-01

    Background The aim of this study is to describe the major evolutionary historical events among Leishmania, sandflies, and the associated animal reservoirs in detail, in accordance with the geographical evolution of the Earth, which has not been previously discussed on a large scale. Methodology and Principal Findings Leishmania and sandfly classification has always been a controversial matter, and the increasing number of species currently described further complicates this issue. Despite several hypotheses on the origin, evolution, and distribution of Leishmania and sandflies in the Old and New World, no consistent agreement exists regarding dissemination of the actors that play roles in leishmaniasis. For this purpose, we present here three centuries of research on sandflies and Leishmania descriptions, as well as a complete description of Leishmania and sandfly fossils and the emergence date of each Leishmania and sandfly group during different geographical periods, from 550 million years ago until now. We discuss critically the different approaches that were used for Leishmana and sandfly classification and their synonymies, proposing an updated classification for each species of Leishmania and sandfly. We update information on the current distribution and dispersion of different species of Leishmania (53), sandflies (more than 800 at genus or subgenus level), and animal reservoirs in each of the following geographical ecozones: Palearctic, Nearctic, Neotropic, Afrotropical, Oriental, Malagasy, and Australian. We propose an updated list of the potential and proven sandfly vectors for each Leishmania species in the Old and New World. Finally, we address a classical question about digenetic Leishmania evolution: which was the first host, a vertebrate or an invertebrate? Conclusions and Significance We propose an updated view of events that have played important roles in the geographical dispersion of sandflies, in relation to both the Leishmania species they

  10. The Dynamics of Lateral Gene Transfer in Genus Leishmania - A Route for Adaptation and Species Diversification

    PubMed Central

    Vikeved, Elisabet; Backlund, Anders; Alsmark, Cecilia

    2016-01-01

    Background The genome of Leishmania major harbours a comparably high proportion of genes of prokaryote origin, acquired by lateral gene transfer (LGT). Some of these are present in closely related trypanosomatids, while some are detected in Leishmania only. We have evaluated the impact and destiny of LGT in genus Leishmania. Methodology/Principal Findings To study the dynamics and fate of LGTs we have performed phylogenetic, as well as nucleotide and amino acid composition analyses within orthologous groups of LGTs detected in Leishmania. A set of universal trypanosomatid LGTs was added as a reference group. Both groups of LGTs have, to some extent, ameliorated to resemble the recipient genomes. However, while virtually all of the universal trypanosomatid LGTs are distributed and conserved in the entire genus Leishmania, the LGTs uniquely present in genus Leishmania are more prone to gene loss and display faster rates of evolution. Furthermore, a PCR based approach has been employed to ascertain the presence of a set of twenty LGTs uniquely present in genus Leishmania, and three universal trypanosomatid LGTs, in ten additional strains of Leishmania. Evolutionary rates and predicted expression levels of these LGTs have also been estimated. Ten of the twenty LGTs are distributed and conserved in all species investigated, while the remainder have been subjected to modifications, or undergone pseudogenization, degradation or loss in one or more species. Conclusions/Significance LGTs unique to the genus Leishmania have been acquired after the divergence of Leishmania from the other trypanosomatids, and are evolving faster than their recipient genomes. This implies that LGT in genus Leishmania is a continuous and dynamic process contributing to species differentiation and speciation. This study also highlights the importance of carefully evaluating these dynamic genes, e.g. as LGTs have been suggested as potential drug targets. PMID:26730948

  11. The Dynamics of Lateral Gene Transfer in Genus Leishmania - A Route for Adaptation and Species Diversification.

    PubMed

    Vikeved, Elisabet; Backlund, Anders; Alsmark, Cecilia

    2016-01-01

    The genome of Leishmania major harbours a comparably high proportion of genes of prokaryote origin, acquired by lateral gene transfer (LGT). Some of these are present in closely related trypanosomatids, while some are detected in Leishmania only. We have evaluated the impact and destiny of LGT in genus Leishmania. To study the dynamics and fate of LGTs we have performed phylogenetic, as well as nucleotide and amino acid composition analyses within orthologous groups of LGTs detected in Leishmania. A set of universal trypanosomatid LGTs was added as a reference group. Both groups of LGTs have, to some extent, ameliorated to resemble the recipient genomes. However, while virtually all of the universal trypanosomatid LGTs are distributed and conserved in the entire genus Leishmania, the LGTs uniquely present in genus Leishmania are more prone to gene loss and display faster rates of evolution. Furthermore, a PCR based approach has been employed to ascertain the presence of a set of twenty LGTs uniquely present in genus Leishmania, and three universal trypanosomatid LGTs, in ten additional strains of Leishmania. Evolutionary rates and predicted expression levels of these LGTs have also been estimated. Ten of the twenty LGTs are distributed and conserved in all species investigated, while the remainder have been subjected to modifications, or undergone pseudogenization, degradation or loss in one or more species. LGTs unique to the genus Leishmania have been acquired after the divergence of Leishmania from the other trypanosomatids, and are evolving faster than their recipient genomes. This implies that LGT in genus Leishmania is a continuous and dynamic process contributing to species differentiation and speciation. This study also highlights the importance of carefully evaluating these dynamic genes, e.g. as LGTs have been suggested as potential drug targets.

  12. A Historical Overview of the Classification, Evolution, and Dispersion of Leishmania Parasites and Sandflies.

    PubMed

    Akhoundi, Mohammad; Kuhls, Katrin; Cannet, Arnaud; Votýpka, Jan; Marty, Pierre; Delaunay, Pascal; Sereno, Denis

    2016-03-01

    The aim of this study is to describe the major evolutionary historical events among Leishmania, sandflies, and the associated animal reservoirs in detail, in accordance with the geographical evolution of the Earth, which has not been previously discussed on a large scale. Leishmania and sandfly classification has always been a controversial matter, and the increasing number of species currently described further complicates this issue. Despite several hypotheses on the origin, evolution, and distribution of Leishmania and sandflies in the Old and New World, no consistent agreement exists regarding dissemination of the actors that play roles in leishmaniasis. For this purpose, we present here three centuries of research on sandflies and Leishmania descriptions, as well as a complete description of Leishmania and sandfly fossils and the emergence date of each Leishmania and sandfly group during different geographical periods, from 550 million years ago until now. We discuss critically the different approaches that were used for Leishmana and sandfly classification and their synonymies, proposing an updated classification for each species of Leishmania and sandfly. We update information on the current distribution and dispersion of different species of Leishmania (53), sandflies (more than 800 at genus or subgenus level), and animal reservoirs in each of the following geographical ecozones: Palearctic, Nearctic, Neotropic, Afrotropical, Oriental, Malagasy, and Australian. We propose an updated list of the potential and proven sandfly vectors for each Leishmania species in the Old and New World. Finally, we address a classical question about digenetic Leishmania evolution: which was the first host, a vertebrate or an invertebrate? We propose an updated view of events that have played important roles in the geographical dispersion of sandflies, in relation to both the Leishmania species they transmit and the animal reservoirs of the parasites.

  13. Leishmania(Leishmania) chagasi in captive wild felids in Brazil.

    PubMed

    Dahroug, Magyda A A; Almeida, Arleana B P F; Sousa, Valéria R F; Dutra, Valéria; Turbino, Nívea C M R; Nakazato, Luciano; de Souza, Roberto L

    2010-01-01

    This study used a PCR-RFLP test to determine the presence of Leishmania (Leishmania) chagasi in 16 captive wild felids [seven Puma concolor (Linnaeus, 1771); five Panthera onca (Linnaeus, 1758) and four Leopardus pardalis (Linnaeus, 1758)] at the zoological park of the Federal University of Mato Grosso, Brazil. Amplification of Leishmania spp. DNA was seen in samples from five pumas and one jaguar, and the species was characterized as L. chagasi using restriction enzymes. It is already known that domestic felids can act as a reservoir of L. chagasi in endemic areas, and further studies are necessary to investigate their participation in the epidemiological chain of leishmaniasis.

  14. Efficacy of Recombinant Canine Distemper Virus Expressing Leishmania Antigen against Leishmania Challenge in Dogs.

    PubMed

    Miura, Ryuichi; Kooriyama, Takanori; Yoneda, Misako; Takenaka, Akiko; Doki, Miho; Goto, Yasuyuki; Sanjoba, Chizu; Endo, Yasuyuki; Fujiyuki, Tomoko; Sugai, Akihiro; Tsukiyama-Kohara, Kyoko; Matsumoto, Yoshitsugu; Sato, Hiroki; Kai, Chieko

    2015-01-01

    Canine distemper virus (CDV) vaccination confers long-term protection against CDV reinfection. To investigate the utility of CDV as a polyvalent vaccine vector for Leishmania, we generated recombinant CDVs, based on an avirulent Yanaka strain, that expressed Leishmania antigens: LACK, TSA, or LmSTI1 (rCDV-LACK, rCDV-TSA, and rCDV-LmSTI1, respectively). Dogs immunized with rCDV-LACK were protected against challenge with lethal doses of virulent CDV, in the same way as the parental Yanaka strain. To evaluate the protective effects of the recombinant CDVs against cutaneous leishmaniasis in dogs, dogs were immunized with one recombinant CDV or a cocktail of three recombinant CDVs, before intradermal challenge (in the ears) with infective-stage promastigotes of Leishmania major. Unvaccinated dogs showed increased nodules with ulcer formation after 3 weeks, whereas dogs immunized with rCDV-LACK showed markedly smaller nodules without ulceration. Although the rCDV-TSA- and rCDV-LmSTI1-immunized dogs showed little protection against L. major, the cocktail of three recombinant CDVs more effectively suppressed the progression of nodule formation than immunization with rCDV-LACK alone. These results indicate that recombinant CDV is suitable for use as a polyvalent live attenuated vaccine for protection against both CDV and L. major infections in dogs.

  15. Efficacy of Recombinant Canine Distemper Virus Expressing Leishmania Antigen against Leishmania Challenge in Dogs

    PubMed Central

    Yoneda, Misako; Takenaka, Akiko; Doki, Miho; Goto, Yasuyuki; Sanjoba, Chizu; Endo, Yasuyuki; Fujiyuki, Tomoko; Sugai, Akihiro; Tsukiyama-Kohara, Kyoko; Matsumoto, Yoshitsugu; Sato, Hiroki; Kai, Chieko

    2015-01-01

    Canine distemper virus (CDV) vaccination confers long-term protection against CDV reinfection. To investigate the utility of CDV as a polyvalent vaccine vector for Leishmania, we generated recombinant CDVs, based on an avirulent Yanaka strain, that expressed Leishmania antigens: LACK, TSA, or LmSTI1 (rCDV–LACK, rCDV–TSA, and rCDV–LmSTI1, respectively). Dogs immunized with rCDV-LACK were protected against challenge with lethal doses of virulent CDV, in the same way as the parental Yanaka strain. To evaluate the protective effects of the recombinant CDVs against cutaneous leishmaniasis in dogs, dogs were immunized with one recombinant CDV or a cocktail of three recombinant CDVs, before intradermal challenge (in the ears) with infective-stage promastigotes of Leishmania major. Unvaccinated dogs showed increased nodules with ulcer formation after 3 weeks, whereas dogs immunized with rCDV–LACK showed markedly smaller nodules without ulceration. Although the rCDV–TSA- and rCDV–LmSTI1-immunized dogs showed little protection against L. major, the cocktail of three recombinant CDVs more effectively suppressed the progression of nodule formation than immunization with rCDV–LACK alone. These results indicate that recombinant CDV is suitable for use as a polyvalent live attenuated vaccine for protection against both CDV and L. major infections in dogs. PMID:26162094

  16. The genomic fingerprinting of the coding region of the beta-tubulin gene in Leishmania identification.

    PubMed

    Luis, L; Ramírez, A; Aguilar, C M; Eresh, S; Barker, D C; Mendoza-León, A

    1998-06-01

    We have demonstrated the polymorphism of the beta-tubulin gene region in Leishmania and its value in the identification of the parasite. In this work we have shown that the coding region of the gene has sufficient variation to accurately discriminate these parasites at the subgenus level. Nevertheless, intrasubgenus diversity, for particular restriction enzymes, was found in New World Leishmania belonging to the Leishmania subgenus. For instance, differences were found between mexicana and amazonensis strains. A unique pattern at the species level was found in particular species of both subgenera, e.g. L. (L.) major strain P and L. (L.) tropica belonging to the Leishmania subgenus, and L. (V.) panamensis strain LS94 from the Viannia subgenus. Particular endonucleases are diagnostic in Leishmania species discrimination as in the case of PvuII for the mexicana and amazonensis. This variation evidenced in the beta-tubulin gene region of Leishmania also occurred in other Kinetoplastida e.g. Trypanosoma cruzi, Leptomonas spp. and Crithidia spp. Moreover, these organisms showed a different genomic fingerprinting for the beta-tubulin gene among them and also Leishmania. Thus, the polymorphism of the coding region of the beta-tubulin gene can be used as a molecular marker for the identification of Leishmania.

  17. Cross-protective efficacy from a immunogen firstly identified in Leishmania infantum against tegumentary leishmaniasis.

    PubMed

    Martins, V T; Lage, D P; Duarte, M C; Costa, L E; Chávez-Fumagalli, M A; Roatt, B M; Menezes-Souza, D; Tavares, C A P; Coelho, E A F

    2016-02-01

    Experimental vaccine candidates have been evaluated to prevent leishmaniasis, but no commercial vaccine has been proved to be effective against more than one parasite species. LiHyT is a Leishmania-specific protein that was firstly identified as protective against Leishmania infantum. In this study, LiHyT was evaluated as a vaccine to against two Leishmania species causing tegumentary leishmaniasis (TL): Leishmania major and Leishmania braziliensis. BALB/c mice were immunized with rLiHyT plus saponin and lately challenged with promastigotes of the two parasite species. The immune response generated was evaluated before and 10 weeks after infection, as well as the parasite burden at this time after infection. The vaccination induced a Th1 response, which was characterized by the production of IFN-γ, IL-12 and GM-CSF, as well as by high levels of IgG2a antibodies, after in vitro stimulation using both the protein and parasite extracts. After challenge, vaccinated mice showed significant reductions in their infected footpads, as well as in the parasite burden in the tissue and organs evaluated, when compared to the control groups. The anti-Leishmania Th1 response was maintained after infection, being the IFN-γ production based mainly on CD4(+) T cells. We described one conserved Leishmania-specific protein that could compose a pan-Leishmania vaccine.

  18. NEW PRIMERS FOR DETECTION OF Leishmania infantumUSING POLYMERASE CHAIN REACTION

    PubMed Central

    GUALDA, Kézia Peres; MARCUSSI, Lílian Mathias; NEITZKE-ABREU, Herintha Coeto; ARISTIDES, Sandra Mara Alessi; LONARDONI, Maria Valdrinez Campana; CARDOSO, Rosilene Fressatti; SILVEIRA, Thaís Gomes Verzignassi

    2015-01-01

    SUMMARY Leishmania infantum causes visceral leishmaniasis (VL) in the New World. The diagnosis of VL is confirmed by parasitological and serological tests, which are not always sensitive or specific. Our aim was to design new primers to perform a Polymerase Chain Reaction (PCR) for detecting L. infantum. Sequences of the minicircle kinetoplast DNA (kDNA) were obtained from GenBank, and the FLC2/RLC2 primers were designed. Samples of DNA from L. infantum, Leishmania amazonensis,Leishmania braziliensis, Leishmania guyanensis, Leishmania naiffi, Leishmania lainsoni, Leishmania panamensis,Leishmania major and Trypanosoma cruzi were used to standardize the PCR. PCR with FLC2/RLC2 primers amplified a fragment of 230 bp and the detection limit was 0.2 fg of L. infantum DNA. Of the parasite species assayed, only L. infantum DNA was amplified. After sequencing, the fragment was aligned to GenBank sequences, and showed (99%) homology with L. infantum. In the analysis of blood samples and lesion biopsy from a dog clinically suspected to have VL, the PCR detected DNA from L. infantum. In biopsy lesions from humans and dogs with cutaneous leishmaniasis, the PCR was negative. The PCR with FLC2/RLC2 primers showed high sensitivity and specificity, and constitutes a promising technique for the diagnosis of VL. PMID:26603223

  19. Novel selective inhibitor of Leishmania (Leishmania) amazonensis arginase.

    PubMed

    da Silva, Edson R; Boechat, Nubia; Pinheiro, Luiz C S; Bastos, Monica M; Costa, Carolina C P; Bartholomeu, Juliana C; da Costa, Talita H

    2015-11-01

    Arginase is a glycosomal enzyme in Leishmania that is involved in polyamine and trypanothione biosynthesis. The central role of arginase in Leishmania (Leishmania) amazonensis was demonstrated by the generation of two mutants: one with an arginase lacking the glycosomal addressing signal and one in which the arginase-coding gene was knocked out. Both of these mutants exhibited decreased infectivity. Thus, arginase seems to be a potential drug target for Leishmania treatment. In an attempt to search for arginase inhibitors, 29 derivatives of the [1,2,4]triazolo[1,5-a]pyrimidine system were tested against Leishmania (Leishmania) amazonensis arginase in vitro. The [1,2,4]triazolo[1,5-a]pyrimidine scaffold containing R1  = CF3 exhibited greater activity against the arginase rather than when the substituent R1  = CH3 in the 2-position. The novel compound 2-(5-methyl-2-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)hydrazinecarbothioamide (30) was the most potent, inhibiting arginase by a non-competitive mechanism, with the Ki and IC50 values for arginase inhibition estimated to be 17 ± 1 μm and 16.5 ± 0.5 μm, respectively. These results can guide the development of new drugs against leishmaniasis based on [1,2,4]triazolo[1,5-a]pyrimidine derivatives targeting the arginase enzyme.

  20. Guns, germs and dogs: On the origin of Leishmania chagasi.

    PubMed

    Leblois, Raphaël; Kuhls, Katrin; François, Olivier; Schönian, Gabriele; Wirth, Thierry

    2011-07-01

    The evolutionary history of Leishmania chagasi, the aetiological agent of visceral leishmaniasis in South America has been widely debated. This study addresses the problem of the origin of L. chagasi, its timing and demography with fast evolving genetic markers, a suite of Bayesian clustering algorithms and coalescent modelling. Here, using 14 microsatellite markers, 450 strains from the Leishmania donovani complex, we show that the vast majority of the Central and South American L. chagasi were nested within the Portuguese Leishmania infantum clade. Moreover, L. chagasi allelic richness was half that of their Old World counterparts. The bottleneck signature was estimated to be about 500 years old and the settlement of L. chagasi in the New World, probably via infected dogs, was accompanied by a thousand-fold population decrease. Visceral leishmaniasis, lethal if untreated, is therefore one more disease that the Conquistadores brought to the New World. Copyright © 2011 Elsevier B.V. All rights reserved.

  1. Leishmania carbon metabolism in the macrophage phagolysosome- feast or famine?

    PubMed Central

    McConville, Malcolm J.; Saunders, Eleanor C.; Kloehn, Joachim; Dagley, Michael J.

    2015-01-01

    A number of medically important microbial pathogens target and proliferate within macrophages and other phagocytic cells in their mammalian hosts. While the majority of these pathogens replicate within the host cell cytosol or non-hydrolytic vacuolar compartments, a few, including protists belonging to the genus Leishmania, proliferate long-term within mature lysosome compartments.  How these parasites achieve this feat remains poorly defined. In this review, we highlight recent studies that suggest that Leishmania virulence is intimately linked to programmed changes in the growth rate and carbon metabolism of the obligate intra-macrophage stages. We propose that activation of a slow growth and a stringent metabolic response confers resistance to multiple stresses (oxidative, temperature, pH), as well as both nutrient limitation and nutrient excess within this niche. These studies highlight the importance of metabolic processes as key virulence determinants in Leishmania. PMID:26594352

  2. Evolution of the genus Leishmania as revealed by comparisons of nuclear DNA restriction fragment patterns.

    PubMed Central

    Beverley, S M; Ismach, R B; Pratt, D M

    1987-01-01

    Restriction endonuclease DNA fragment patterns have been used to examine the relationships among 28 isolates of Leishmania as well as Crithidia, Endotrypanum, and Trypanosoma cruzi. Fragments of nuclear DNA were generated with six restriction enzymes, and blots were hybridized with probes from three loci. Among the major lineages the fragment patterns are essentially completely different, while within the major lineages various degrees of divergence are found. Molecular evolutionary trees were constructed using the method of Nei and Li to estimate the percent nucleotide sequence divergence among strains from the fraction of fragments shared. Defined groups, such as species or subspecies within the major lineages, are also grouped by nuclear DNA comparisons. Within the donovani complex, we find Leishmania donovani chagasi and Leishmania donovani infantum to be as similar as strains within Leishmania donovani donovani, consistent with the proposal by other workers that New World visceral leishmaniasis originated quite recently. Images PMID:3025876

  3. Glycoprotein 63 (gp63) genes show gene conversion and reveal the evolution of Old World Leishmania.

    PubMed

    Mauricio, Isabel L; Gaunt, Michael W; Stothard, J Russell; Miles, Michael A

    2007-04-01

    Species of the subgenus Leishmania (Leishmania) cause the debilitating disease leishmaniasis on four continents. Species grouped within the Leishmania donovani complex cause visceral leishmaniasis, a life-threatening disease, often associated with poverty, and affecting some 0.5 million people each year. The Leishmania glycoprotein GP63, or major surface protease, is a metalloprotease involved in parasite survival, infectivity and virulence. Here, we show that evolution of the gp63 multigene family is influenced by mosaic or fragmental gene conversion. This is a major evolutionary force for both homogenisation and for generating diversity, even in the absence of sexual reproduction. We propose here that the high GC content at the third codon position in the gp63 family of Old World Leishmania may be higher in multicopy regions, under the biased gene conversion model, because increased copy numbers may lead to increased rates of recombination. We confirm that one class of gp63 genes with an extended 3'end signal, gp63(EXT), reveals genetic groups within the complex and gives insights into evolution and host associations. Gp63(EXT) genes can also provide the basis for rapid and reliable genotyping of strains in the L. donovani complex. Our results confirmed that a more stringent definition of Leishmania infantum is required and that the species Leishmania archibaldi should be suppressed.

  4. Vector transmission of leishmania abrogates vaccine-induced protective immunity.

    PubMed

    Peters, Nathan C; Kimblin, Nicola; Secundino, Nagila; Kamhawi, Shaden; Lawyer, Phillip; Sacks, David L

    2009-06-01

    Numerous experimental vaccines have been developed to protect against the cutaneous and visceral forms of leishmaniasis caused by infection with the obligate intracellular protozoan Leishmania, but a human vaccine still does not exist. Remarkably, the efficacy of anti-Leishmania vaccines has never been fully evaluated under experimental conditions following natural vector transmission by infected sand fly bite. The only immunization strategy known to protect humans against natural exposure is "leishmanization," in which viable L. major parasites are intentionally inoculated into a selected site in the skin. We employed mice with healed L. major infections to mimic leishmanization, and found tissue-seeking, cytokine-producing CD4+ T cells specific for Leishmania at the site of challenge by infected sand fly bite within 24 hours, and these mice were highly resistant to sand fly transmitted infection. In contrast, mice vaccinated with a killed vaccine comprised of autoclaved L. major antigen (ALM)+CpG oligodeoxynucleotides that protected against needle inoculation of parasites, showed delayed expression of protective immunity and failed to protect against infected sand fly challenge. Two-photon intra-vital microscopy and flow cytometric analysis revealed that sand fly, but not needle challenge, resulted in the maintenance of a localized neutrophilic response at the inoculation site, and removal of neutrophils following vector transmission led to increased parasite-specific immune responses and promoted the efficacy of the killed vaccine. These observations identify the critical immunological factors influencing vaccine efficacy following natural transmission of Leishmania.

  5. Effects of dihydroorotate dehydrogenase (DHODH) inhibitors on the growth of Theileria equi and Babesia caballi in vitro.

    PubMed

    Kamyingkird, Ketsarin; Cao, Shinuo; Tuvshintulga, Bumduuren; Salama, Akram; Mousa, Ahmed Abdelmoniem; Efstratiou, Artemis; Nishikawa, Yoshifumi; Yokoyama, Naoaki; Igarashi, Ikuo; Xuan, Xuenan

    2017-05-01

    Theileria equi and Babesia caballi are the causative agents of equine piroplasmosis (EP), which affects equine production in various parts of the world. However, a safe and effective drug is not currently available for treatment of EP. Dihydroorotate dehydrogenase (DHODH) is the fourth enzyme in the de novo pyrimidine synthesis pathway and has been known as a novel drug target for several apicomplexan protozoan parasites. In this study, we evaluated four DHODH inhibitors; atovaquone (ATV), leflunomide (LFN), brequinar (Breq), and 7-hydroxy-5-[1,2,4] triazolo [1,5,a] pyrimidine (TAZ) on the growth of T. equi and B. caballi in vitro and compared them to diminacene aceturate (Di) as the control drug. The growth of T. equi and B. caballi was significantly hindered by all inhibitors except TAZ. The half maximal inhibitory concentration (IC50) of ATV, LFN, Breq and Di against T. equi was approximately 0.028, 109, 11 and 40 μM, respectively, whereas the IC50 of ATV, LFN, Breq and Di against B. caballi was approximately 0.128, 193, 5.2 and 16.2 μM, respectively. Using bioinformatics and Western blot analysis, we showed that TeDHODH was similar to other Babesia parasite DHODHs, and confirmed that targeting DHODHs could be useful for the development of novel chemotherapeutics for treatment of EP.

  6. Original 2-(3-Alkoxy-1H-pyrazol-1-yl)azines Inhibitors of Human Dihydroorotate Dehydrogenase (DHODH)

    PubMed Central

    2016-01-01

    Following our discovery of human dihydroorotate dehydrogenase (DHODH) inhibition by 2-(3-alkoxy-1H-pyrazol-1-yl)pyrimidine derivatives as well as 2-(4-benzyl-3-ethoxy-5-methyl-1H-pyrazol-1-yl)-5-methylpyridine, we describe here the syntheses and evaluation of an array of azine-bearing analogues. As in our previous report, the structure–activity study of this series of human DHODH inhibitors was based on a phenotypic assay measuring measles virus replication. Among other inhibitors, this round of syntheses and biological evaluation iteration led to the highly active 5-cyclopropyl-2-(4-(2,6-difluorophenoxy)-3-isopropoxy-5-methyl-1H-pyrazol-1-yl)-3-fluoropyridine. Inhibition of DHODH by this compound was confirmed in an array of in vitro assays, including enzymatic tests and cell-based assays for viral replication and cellular growth. This molecule was found to be more active than the known inhibitors of DHODH, brequinar and teriflunomide, thus opening perspectives for its use as a tool or for the design of an original series of immunosuppressive agent. Moreover, because other series of inhibitors of human DHODH have been found to also affect Plasmodium falciparum DHODH, all the compounds were assayed for their effect on P. falciparum growth. However, the modest in vitro inhibition solely observed for two compounds did not correlate with their inhibition of P. falciparum DHODH. PMID:26079043

  7. Tetrahydro-2-naphthyl and 2-Indanyl Triazolopyrimidines Targeting Plasmodium falciparum Dihydroorotate Dehydrogenase Display Potent and Selective Antimalarial Activity

    PubMed Central

    2016-01-01

    Malaria persists as one of the most devastating global infectious diseases. The pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH) has been identified as a new malaria drug target, and a triazolopyrimidine-based DHODH inhibitor 1 (DSM265) is in clinical development. We sought to identify compounds with higher potency against Plasmodium DHODH while showing greater selectivity toward animal DHODHs. Herein we describe a series of novel triazolopyrimidines wherein the p-SF5-aniline was replaced with substituted 1,2,3,4-tetrahydro-2-naphthyl or 2-indanyl amines. These compounds showed strong species selectivity, and several highly potent tetrahydro-2-naphthyl derivatives were identified. Compounds with halogen substitutions displayed sustained plasma levels after oral dosing in rodents leading to efficacy in the P. falciparum SCID mouse malaria model. These data suggest that tetrahydro-2-naphthyl derivatives have the potential to be efficacious for the treatment of malaria, but due to higher metabolic clearance than 1, they most likely would need to be part of a multidose regimen. PMID:27127993

  8. A Triazolopyrimidine-Based Dihydroorotate Dehydrogenase Inhibitor with Improved Drug-like Properties for Treatment and Prevention of Malaria.

    PubMed

    Phillips, Margaret A; White, Karen L; Kokkonda, Sreekanth; Deng, Xiaoyi; White, John; El Mazouni, Farah; Marsh, Kennan; Tomchick, Diana R; Manjalanagara, Krishne; Rudra, Kakali Rani; Wirjanata, Grennady; Noviyanti, Rintis; Price, Ric N; Marfurt, Jutta; Shackleford, David M; Chiu, Francis C K; Campbell, Michael; Jimenez-Diaz, Maria Belen; Bazaga, Santiago Ferrer; Angulo-Barturen, Iñigo; Martinez, Maria Santos; Lafuente-Monasterio, Maria; Kaminsky, Werner; Silue, Kigbafori; Zeeman, Anne-Marie; Kocken, Clemens; Leroy, Didier; Blasco, Benjamin; Rossignol, Emilie; Rueckle, Thomas; Matthews, Dave; Burrows, Jeremy N; Waterson, David; Palmer, Michael J; Rathod, Pradipsinh K; Charman, Susan A

    2016-12-09

    The emergence of drug-resistant malaria parasites continues to hamper efforts to control this lethal disease. Dihydroorotate dehydrogenase has recently been validated as a new target for the treatment of malaria, and a selective inhibitor (DSM265) of the Plasmodium enzyme is currently in clinical development. With the goal of identifying a backup compound to DSM265, we explored replacement of the SF5-aniline moiety of DSM265 with a series of CF3-pyridinyls while maintaining the core triazolopyrimidine scaffold. This effort led to the identification of DSM421, which has improved solubility, lower intrinsic clearance, and increased plasma exposure after oral dosing compared to DSM265, while maintaining a long predicted human half-life. Its improved physical and chemical properties will allow it to be formulated more readily than DSM265. DSM421 showed excellent efficacy in the SCID mouse model of P. falciparum malaria that supports the prediction of a low human dose (<200 mg). Importantly DSM421 showed equal activity against both P. falciparum and P. vivax field isolates, while DSM265 was more active on P. falciparum. DSM421 has the potential to be developed as a single-dose cure or once-weekly chemopreventative for both P. falciparum and P. vivax malaria, leading to its advancement as a preclinical development candidate.

  9. Dihydro-orotate dehydrogenase is physically associated with the respiratory complex and its loss leads to mitochondrial dysfunction

    PubMed Central

    Fang, JingXian; Uchiumi, Takeshi; Yagi, Mikako; Matsumoto, Shinya; Amamoto, Rie; Takazaki, Shinya; Yamaza, Haruyoshi; Nonaka, Kazuaki; Kang, Dongchon

    2012-01-01

    Some mutations of the DHODH (dihydro-orotate dehydrogenase) gene lead to postaxial acrofacial dysostosis or Miller syndrome. Only DHODH is localized at mitochondria among enzymes of the de novo pyrimidine biosynthesis pathway. Since the pyrimidine biosynthesis pathway is coupled to the mitochondrial RC (respiratory chain) via DHODH, impairment of DHODH should affect the RC function. To investigate this, we used siRNA (small interfering RNA)-mediated knockdown and observed that DHODH knockdown induced cell growth retardation because of G2/M cell-cycle arrest, whereas pyrimidine deficiency usually causes G1/S arrest. Inconsistent with this, the cell retardation was not rescued by exogenous uridine, which should bypass the DHODH reaction for pyrimidine synthesis. DHODH depletion partially inhibited the RC complex III, decreased the mitochondrial membrane potential, and increased the generation of ROS (reactive oxygen species). We observed that DHODH physically interacts with respiratory complexes II and III by IP (immunoprecipitation) and BN (blue native)/SDS/PAGE analysis. Considering that pyrimidine deficiency alone does not induce craniofacial dysmorphism, the DHODH mutations may contribute to the Miller syndrome in part through somehow altered mitochondrial function. PMID:23216091

  10. Dihydroorotate dehydrogenase is required for N-(4-hydroxyphenyl)retinamide-induced reactive oxygen species production and apoptosis

    PubMed Central

    Hail, Numsen; Chen, Ping; Kepa, Jadwiga J.; Bushman, Lane R.; Shearn, Colin

    2010-01-01

    The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) exhibits anticancer activity in vivo and triggers apoptosis in transformed cells in vitro. Thus, apoptosis induction is acknowledged as a mechanistic underpinning for 4HPR's cancer preventive and therapeutic effects. Apoptosis induction by 4HPR is routinely preceded by and dependent on the production of reactive oxygen species (ROS) in transformed cells. Very little evidence exists outside the possible involvement of the mitochondrial electron transport chain or the plasma membrane NADPH oxidase complex, which would pinpoint the predominant site of 4HPR-induced ROS production in transformed cells. Here, we investigated the role of dihydroorotate dehydrogenase (DHODH, an enzyme associated with the mitochondrial electron transport chain and required for de novo pyrimidine synthesis) in 4HPR-induced ROS production and attendant apoptosis in transformed skin and prostate epithelial cells. In premalignant prostate epithelial cells and malignant cutaneous keratinocytes the suppression of DHODH activity by the chemical inhibitor teriflunomide or the reduction in DHODH protein expression by RNA interference markedly reduced 4HPR-induced ROS generation and apoptosis. Conversely, colon carcinoma cells that lacked DHODH expression were markedly resistant to the prooxidant and cytotoxic effects of 4HPR. Together, these results strongly implicate DHODH in 4HPR-induced ROS production and apoptosis. PMID:20399851

  11. Dihydroorotate dehydrogenase is required for N-(4-hydroxyphenyl)retinamide-induced reactive oxygen species production and apoptosis.

    PubMed

    Hail, Numsen; Chen, Ping; Kepa, Jadwiga J; Bushman, Lane R; Shearn, Colin

    2010-07-01

    The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) exhibits anticancer activity in vivo and triggers apoptosis in transformed cells in vitro. Thus, apoptosis induction is acknowledged as a mechanistic underpinning for 4HPR's cancer preventive and therapeutic effects. Apoptosis induction by 4HPR is routinely preceded by and dependent on the production of reactive oxygen species (ROS) in transformed cells. Very little evidence exists, outside the possible involvement of the mitochondrial electron transport chain or the plasma membrane NADPH oxidase complex, that would pinpoint the predominant site of 4HPR-induced ROS production in transformed cells. Here, we investigated the role of dihydroorotate dehydrogenase (DHODH; an enzyme associated with the mitochondrial electron transport chain and required for de novo pyrimidine synthesis) in 4HPR-induced ROS production and attendant apoptosis in transformed skin and prostate epithelial cells. In premalignant prostate epithelial cells and malignant cutaneous keratinocytes the suppression of DHODH activity by the chemical inhibitor teriflunomide or the reduction in DHODH protein expression by RNA interference markedly reduced 4HPR-induced ROS generation and apoptosis. Conversely, colon carcinoma cells that lacked DHODH expression were markedly resistant to the pro-oxidant and cytotoxic effects of 4HPR. Together, these results strongly implicate DHODH in 4HPR-induced ROS production and apoptosis. (c) 2010 Elsevier Inc. All rights reserved.

  12. Identification of New Human Malaria Parasite Plasmodium Falciparum Dihydroorotate Dehydrogenase Inhibitors by Pharmacophore and Structure-Based Virtual Screening

    PubMed Central

    Pavadai, Elumalai; El Mazouni, Farah; Wittlin, Sergio; de Kock, Carmen; Phillips, Margaret A.; Chibale, Kelly

    2016-01-01

    Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH), a key enzyme in the de novo pyrimidine biosynthesis pathway, which the Plasmodium falciparum relies on exclusively for survival, has emerged as a promising target for antimalarial drugs. In an effort to discover new and potent PfDHODH inhibitors, 3D-QSAR pharmacophore models were developed based on the structures of known PfDHODH inhibitors and the validated Hypo1 model was used as a 3D search query for virtual screening of the National Cancer Institute database. The virtual hit compounds were further filtered based on molecular docking and Molecular Mechanics/Generalized Born Surface Area binding energy calculations. The combination of the pharmacophore and structure-based virtual screening resulted in the identification of nine new compounds that showed >25% inhibition of PfDHODH at a concentration of 10 μM, three of which exhibited IC50 values in the range of 0.38–20 μM. The most active compound, NSC336047, displayed species-selectivity for PfDHODH over human DHODH and inhibited parasite growth with an IC50 of 26 μM. In addition to this, thirteen compounds inhibited parasite growth with IC50 values of ≤ 50 μM, four of which showed IC50 values in the range of 5–12 μM. These compounds could be further explored in the identification and development of more potent PfDHODH and parasite growth inhibitors. PMID:26915022

  13. Identification of New Human Malaria Parasite Plasmodium falciparum Dihydroorotate Dehydrogenase Inhibitors by Pharmacophore and Structure-Based Virtual Screening.

    PubMed

    Pavadai, Elumalai; El Mazouni, Farah; Wittlin, Sergio; de Kock, Carmen; Phillips, Margaret A; Chibale, Kelly

    2016-03-28

    Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH), a key enzyme in the de novo pyrimidine biosynthesis pathway, which the Plasmodium falciparum relies on exclusively for survival, has emerged as a promising target for antimalarial drugs. In an effort to discover new and potent PfDHODH inhibitors, 3D-QSAR pharmacophore models were developed based on the structures of known PfDHODH inhibitors and the validated Hypo1 model was used as a 3D search query for virtual screening of the National Cancer Institute database. The virtual hit compounds were further filtered based on molecular docking and Molecular Mechanics/Generalized Born Surface Area binding energy calculations. The combination of the pharmacophore and structure-based virtual screening resulted in the identification of nine new compounds that showed >25% inhibition of PfDHODH at a concentration of 10 μM, three of which exhibited IC50 values in the range of 0.38-20 μM. The most active compound, NSC336047, displayed species-selectivity for PfDHODH over human DHODH and inhibited parasite growth with an IC50 of 26 μM. In addition to this, 13 compounds inhibited parasite growth with IC50 values of ≤ 50 μM, 4 of which showed IC50 values in the range of 5-12 μM. These compounds could be further explored in the identification and development of more potent PfDHODH and parasite growth inhibitors.

  14. Perifosine Mechanisms of Action in Leishmania Species.

    PubMed

    López-Arencibia, Atteneri; Martín-Navarro, Carmen; Sifaoui, Ines; Reyes-Batlle, María; Wagner, Carolina; Lorenzo-Morales, Jacob; Maciver, Sutherland K; Piñero, José E

    2017-04-01

    Here the mechanism by which perifosine induced cell death in Leishmania donovani and Leishmania amazonensis is described. The drug reduced Leishmania mitochondrial membrane potential and decreased cellular ATP levels while increasing phosphatidylserine externalization. Perifosine did not increase membrane permeabilization. We also found that the drug inhibited the phosphorylation of Akt in the parasites. These results highlight the potential use of perifosine as an alternative to miltefosine against Leishmania.

  15. Perifosine Mechanisms of Action in Leishmania Species

    PubMed Central

    Martín-Navarro, Carmen; Sifaoui, Ines; Reyes-Batlle, María; Wagner, Carolina; Lorenzo-Morales, Jacob; Maciver, Sutherland K.; Piñero, José E.

    2017-01-01

    ABSTRACT Here the mechanism by which perifosine induced cell death in Leishmania donovani and Leishmania amazonensis is described. The drug reduced Leishmania mitochondrial membrane potential and decreased cellular ATP levels while increasing phosphatidylserine externalization. Perifosine did not increase membrane permeabilization. We also found that the drug inhibited the phosphorylation of Akt in the parasites. These results highlight the potential use of perifosine as an alternative to miltefosine against Leishmania. PMID:28096161

  16. Structure-Based Design of Potent and Selective LeishmaniaN-Myristoyltransferase Inhibitors

    PubMed Central

    2014-01-01

    Inhibitors of LeishmaniaN-myristoyltransferase (NMT), a potential target for the treatment of leishmaniasis, obtained from a high-throughput screen, were resynthesized to validate activity. Crystal structures bound to Leishmania major NMT were obtained, and the active diastereoisomer of one of the inhibitors was identified. On the basis of structural insights, enzyme inhibition was increased 40-fold through hybridization of two distinct binding modes, resulting in novel, highly potent Leishmania donovani NMT inhibitors with good selectivity over the human enzyme. PMID:25238611

  17. Immunogenicity Evaluation of a Rationally Designed Polytope Construct Encoding HLA-A*0201 Restricted Epitopes Derived from Leishmania major Related Proteins in HLA-A2/DR1 Transgenic Mice: Steps toward Polytope Vaccine

    PubMed Central

    Seyed, Negar; Taheri, Tahereh; Vauchy, Charline; Dosset, Magalie; Godet, Yann; Eslamifar, Ali; Sharifi, Iraj; Adotevi, Olivier; Borg, Christophe; Rohrlich, Pierre Simon; Rafati, Sima

    2014-01-01

    Background There are several reports demonstrating the role of CD8 T cells against Leishmania species. Therefore peptide vaccine might represent an effective approach to control the infection. We developed a rational polytope-DNA construct encoding immunogenic HLA-A2 restricted peptides and validated the processing and presentation of encoded epitopes in a preclinical mouse model humanized for the MHC-class-I and II. Methods and Findings HLA-A*0201 restricted epitopes from LPG-3, LmSTI-1, CPB and CPC along with H-2Kd restricted peptides, were lined-up together as a polytope string in a DNA construct. Polytope string was rationally designed by harnessing advantages of ubiquitin, spacers and HLA-DR restricted Th1 epitope. Endotoxin free pcDNA plasmid expressing the polytope was inoculated into humanized HLA-DRB1*0101/HLA-A*0201 transgenic mice intramuscularly 4 days after Cardiotoxin priming followed by 2 boosters at one week interval. Mice were sacrificed 10 days after the last booster, and splenocytes were subjected to ex-vivo and in-vitro evaluation of specific IFN-γ production and in-vitro cytotoxicity against individual peptides by ELISpot and standard chromium-51(51Cr) release assay respectively. 4 H-2Kd and 5 HLA-A*0201 restricted peptides were able to induce specific CD8 T cell responses in BALB/C and HLA-A2/DR1 mice respectively. IFN-γ and cytolytic activity together discriminated LPG-3-P1 as dominant, LmSTI-1-P3 and LmSTI-1-P6 as subdominant with both cytolytic activity and IFN-γ production, LmSTI-1-P4 and LPG-3-P5 as subdominant with only IFN-γ production potential. Conclusions Here we described a new DNA-polytope construct for Leishmania vaccination encompassing immunogenic HLA-A2 restricted peptides. Immunogenicity evaluation in HLA-transgenic model confirmed CD8 T cell induction with expected affinities and avidities showing almost efficient processing and presentation of the peptides in relevant preclinical model. Further evaluation will determine

  18. In vivo antileishmanial efficacy of miltefosine against Leishmania (Leishmania) amazonensis.

    PubMed

    García Bustos, María F; Barrio, Alejandra; Prieto, Gabriela G; de Raspi, Emma M; Cimino, Rubén O; Cardozo, Rubén M; Parada, Luis A; Yeo, Matthew; Soto, Jaime; Uncos, Delfor A; Parodi, Cecilia; Basombrío, Miguel A

    2014-12-01

    Leishmaniasis, a disease caused by parasites of the Leishmania genus, constitutes a significant health and social problem in many countries and is increasing worldwide. The conventional treatment, meglumine antimoniate (MA), presents numerous disadvantages, including invasiveness, toxicity, and frequent therapeutic failure, justifying the attempts at finding alternatives to the first-line therapy. We have studied the comparative long-term efficacy of MA against miltefosine (MF) in Leishmania infection in experimental mice. The criteria for efficacy evaluation were footpad lesion size, anti-Leishmania antibodies level, histopathology of the site of inoculation (right footpad, RFP), splenic index (SI), and the presence of parasites in RFP, spleen, and liver, determined by polymerase chain reaction (PCR). Swiss mice, infected with Leishmania (Leishmania) amazonensis were treated, at different time points (5 and 40 days after infection) with either MA or MF. The efficacy of MF was better than that of MA for inhibiting lesions and for reducing tissue damage and presence/load of amastigotes in spleen and liver. Moreover, early administration of MF produced a clear reduction in splenomegaly and was equal in reducing antibody titles in comparison with MA. Our results demonstrated that MF is an effective and safe therapeutic alternative for leishmaniasis by L. (L.) amazonensis and is more efficacious than MA.

  19. Mucosal Leishmaniasis Caused by Leishmania (Viannia) braziliensis and Leishmania (Viannia) guyanensis in the Brazilian Amazon

    PubMed Central

    de Oliveira Guerra, Jorge Augusto; Prestes, Suzane Ribeiro; Silveira, Henrique; Coelho, Leila Inês de Aguiar Raposo Câmara; Gama, Pricila; Moura, Aristoteles; Amato, Valdir; Barbosa, Maria das Graças Vale; de Lima Ferreira, Luiz Carlos

    2011-01-01

    Background Leishmania (Viannia) braziliensis is a parasite recognized as the most important etiologic agent of mucosal leishmaniasis (ML) in the New World. In Amazonia, seven different species of Leishmania, etiologic agents of human Cutaneous Leishmaniasis, have been described. Isolated cases of ML have been described for several different species of Leishmania: L. (V.) panamensis, L. (V.) guyanensis and L. (L.) amazonensis. Methodology Leishmania species were characterized by polymerase chain reaction (PCR) of tissues taken from mucosal biopsies of Amazonian patients who were diagnosed with ML and treated at the Tropical Medicine Foundation of Amazonas (FMTAM) in Manaus, Amazonas state, Brazil. Samples were obtained retrospectively from the pathology laboratory and prospectively from patients attending the aforementioned tertiary care unit. Results This study reports 46 cases of ML along with their geographical origin, 30 cases caused by L. (V.) braziliensis and 16 cases by L. (V.) guyanensis. This is the first record of ML cases in 16 different municipalities in the state of Amazonas and of simultaneous detection of both species in 4 municipalities of this state. It is also the first record of ML caused by L. (V.) guyanensis in the states of Pará, Acre, and Rondônia and cases of ML caused by L. (V.) braziliensis in the state of Rondônia. Conclusions/Significance L. (V.) braziliensis is the predominant species that causes ML in the Amazon region. However, contrary to previous studies, L. (V.) guyanensis is also a significant causative agent of ML within the region. The clinical and epidemiological expression of ML in the Manaus region is similar to the rest of the country, although the majority of ML cases are found south of the Amazon River. PMID:21408116

  20. Anti-Leishmania Activity of Osthole

    PubMed Central

    Kermani, Elaheh Kordzadeh; Sajjadi, Seyed Ebrahim; Hejazi, Seyed Hossein; Arjmand, Reza; Saberi, Sedigheh; Eskandarian, Abbas Ali

    2016-01-01

    Background: Treatment of cutaneous leishmaniasis (CL) is occasionally highly resistant to pentavalent antimonials, the gold standard in pharmacotherapy of CL. Since there is no effective vaccine, the discovery of natural antileishmanial products as complementary therapeutic agents could be used to improve the current regimens. Objective: In this study in vitro and in vivo antileishmanial activities of osthole, a natural coumarin known to possess antibacterial and parasiticidal activities are evaluated. Materials and Methods: Leishmania major infected J774.A1 macrophages were treated with increasing concentrations of osthole. CL lesions of BALB/c mice were treated topically with 0.2% osthole. Results: Osthole exhibited dose-dependent leishmanicidal activity against intracellular amastigotes with IC50 value of 14.95 μg/ml. Treatment of CL lesions in BALB/c mice with osthole significantly declined lesion progression compared to untreated mice (P < 0.05), however did not result in recovery. Conclusion: Osthole demonstrated remarkable leishmanicidal activity in vitro. Higher concentrations of osthole may demonstrate the therapeutic property in vivo. SUMMARY In vitro and in vivo antileishmanial activities of osthole, a pernylated coumarin extracted from Prangos asperula Boiss., are studied against Leishmania major. PMID:27114685

  1. HIV proteinase inhibitors target the Ddi1-like protein of Leishmania parasites

    PubMed Central

    White, Rhian E.; Powell, David J.; Berry, Colin

    2011-01-01

    HIV proteinase inhibitors reduce the levels of Leishmania parasites in vivo and in vitro, but their biochemical target is unknown. We have identified an ortholog of the yeast Ddi1 protein as the only member of the aspartic proteinase family in Leishmania parasites, and in this study we investigate this protein as a potential target for the drugs. To date, no enzyme assay has been developed for the Ddi1 proteins, but Saccharomyces cerevisiae lacking the DDI1 gene secrete high levels of protein into the medium. We developed an assay in which these knockout yeast were functionally complemented to low secretion by introduction of genes encoding Ddi1 orthologs from Leishmania major or humans. Plasmid alone controls gave no complementation. Treatment of the Ddi1 transformants with HIV proteinase inhibitors showed differential effects dependent on the origin of the Ddi1. Dose responses allowed calculation of IC50 values; e.g., for nelfinavir, of 3.4 μM (human Ddi1) and 0.44 μM (Leishmania Ddi1). IC50 values with Leishmania constructs mirror the potency of inhibitors against parasites. Our results show that Ddi1 proteins are targets of HIV proteinase inhibitors and indicates the Leishmania Ddi1 as the likely target for these drugs and a potential target for antiparasitic therapy.—White, R. E., Powell, D. J., Berry, C. HIV proteinase inhibitors target the Ddi1-Like protein of Leishmania parasites. PMID:21266539

  2. [Risk assessment on laboratory biosafety of Leishmania].

    PubMed

    Xiong, Yan-Hong; Guan, Ya-Yi; Cao, Jian-Ping

    2012-06-01

    To provide the evidence for improving the risk assessment and personal protective equipment and techniques to laboratory staff related to Leishmania. The laboratory biosafety of Leishmania was preliminarily assessed based on the biological background information, potential hazards in experimental activities, the risk analyses of laboratory personnel and other relevant factors. The risk assessment on laboratory biosafety of Leishmania was helpful for the establishment of the laboratory standard operating procedure, and was helpful for protecting the staff from infection of Leishmania. The risk assessment on laboratory biosafety is important to the safety of laboratory activity related to Leishmania, and is of a great significance to protect the laboratory staff.

  3. Experimental Acquisition, Development, and Transmission of Leishmania tropica by Phlebotomus duboscqi

    DTIC Science & Technology

    2013-01-01

    comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden, to Washington ...transmission of Leishmania major to man by the bite of a naturally infected Phlebotomus duboscqi. Transactions of the Royal Society of Tropical Medicine and...Leishmania tropica in Morocco: infection in dogs. Transactions of the Royal Society of Tropical Medicine and Hygiene 85, 595. 42 H.A. Hanafi et al

  4. Experimental Acquisition, Development, and Transmission of Leishmania tropica by Phlebotomus duboscqi

    DTIC Science & Technology

    2012-09-10

    for reducing this burden, to Washington Headquarters Setviccs, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite...transmission of Leishmania major to man by the bite of a naturally infected Phlebotomus duboscqi. Transactions of the Royal Society of Tropical Medicine...1991. Leishmania tropica in Morocco: infection in dogs. Transactions of the Royal Society ofTropical Medicine and Hygiene 85, 595. 42 H.A. Hanafi et al

  5. The flagellar protein FLAG1/SMP1 is a candidate for Leishmania-sand fly interaction.

    PubMed

    Di-Blasi, Tatiana; Lobo, Amanda R; Nascimento, Luanda M; Córdova-Rojas, Jose L; Pestana, Karen; Marín-Villa, Marcel; Tempone, Antonio J; Telleria, Erich L; Ramalho-Ortigão, Marcelo; McMahon-Pratt, Diane; Traub-Csekö, Yara M

    2015-03-01

    Leishmaniasis is a serious problem that affects mostly poor countries. Various species of Leishmania are the agents of the disease, which take different clinical manifestations. The parasite is transmitted by sandflies, predominantly from the Phlebotomus genus in the Old World and Lutzomyia in the New World. During development in the gut, Leishmania must survive various challenges, which include avoiding being expelled with blood remnants after digestion. It is believed that attachment to the gut epithelium is a necessary step for vector infection, and molecules from parasites and sand flies have been implicated in this attachment. In previous work, monoclonal antibodies were produced against Leishmania. Among these an antibody was obtained against Leishmania braziliensis flagella, which blocked the attachment of Leishmania panamensis flagella to Phlebotomus papatasi guts. The protein recognized by this antibody was identified and named FLAG1, and the complete FLAG1 gene sequence was obtained. This protein was later independently identified as a small, myristoylated protein and called SMP1, so from now on it will be denominated FLAG1/SMP1. The FLAG1/SMP1 gene is expressed in all developmental stages of the parasite, but has higher expression in promastigotes. The anti-FLAG1/SMP1 antibody recognized the flagellum of all Leishmania species tested and generated the expected band by western blots. This antibody was used in attachment and infection blocking experiments. Using the New World vector Lutzomyia longipalpis and Leishmania infantum chagasi, no inhibition of attachment ex vivo or infection in vivo was seen. On the other hand, when the Old World vectors P. papatasi and Leishmania major were used, a significant decrease of both attachment and infection were seen in the presence of the antibody. We propose that FLAG1/SMP1 is involved in the attachment/infection of Leishmania in the strict vector P. papatasi and not the permissive vector L. longipalpis.

  6. Passive transfer of leishmania lipopolysaccharide confers parasite survival in macrophages

    SciTech Connect

    Handman, E.; Schnur, L.F.; Spithill, T.W.; Mitchell, G.F.

    1986-12-01

    Infection of macrophages by the intracellular protozoan parasite Leishmania involves specific attachment to the host membrane, followed by phagocytosis and intracellular survival and growth. Two parasite molecules have been implicated in the attachment event: Leishmania lipopolysaccharide (L-LPS) and a glycoprotein (gp63). This study was designed to clarify the role of L-LPS in infection and the stage in the process of infection at which it operates. The authors have recently identified a Leishmania major strain (LRC-L119) which lacks the L-LPS molecule and is not infective for hamsters or mice. This parasite was isolated from a gerbil in Kenya and was identified phenotypically as L. major by isoenzyme and fatty acid analysis. In this study they have confirmed at the genotype level that LRC-L119 is L. major by analyzing and comparing the organization of cloned DNA sequences in the genome of different strains of L. major. Here they show that LRC-L119 promastigotes are phagocytosed rapidly by macrophages in vitro, but in contrast to virulent strains of L. major, they are then killed over a period of 18 hr. In addition, they show that transfer of purified L-LPS from a virulent clone of L. major (V121) into LRC-L119 promastigotes confers on them the ability to survive in macrophages in vitro.

  7. Genomic organization and expression of the HSP70 locus in New and Old World Leishmania species.

    PubMed

    Folgueira, C; Cañavate, C; Chicharro, C; Requena, J M

    2007-03-01

    Heat shock is believed to be a developmental inductor of differentiation in Leishmania. Furthermore, heat shock genes are extensively studied as gene models to decipher mechanisms of gene regulation in kinetoplastids. Here, we describe the organization and expression of the HSP70 loci in representative Leishmania species (L. infantum, L. major, L. tropica, L. mexicana, L. amazonensis and L. braziliensis). With the exception of L. braziliensis, the organization of the HSP70 loci was found to be well conserved among the other Leishmania species. Two types of genes, HSP70-I and HSP70-II, were found to be present in these Leishmania species except for L. braziliensis that lacks HSP70-II gene. Polymorphisms in the HSP70 locus allow the differentiation of the Old and New World species within the subgenus Leishmania. A notable discrepancy between our data and those of the L. major genome database in relation to the gene copy number composing the L. major HSP70 locus was revealed. The temperature-dependent accumulation of the HSP70-I mRNAs is also conserved among the different Leishmania species with the exception of L. braziliensis. In spite of these differences, analysis of the HSP70 synthesis indicated that the HSP70 mRNAs are also preferentially translated during heat shock in L. braziliensis.

  8. Prevalence and Distribution of Leishmania RNA Virus 1 in Leishmania Parasites from French Guiana

    PubMed Central

    Ginouvès, Marine; Simon, Stéphane; Bourreau, Eliane; Lacoste, Vincent; Ronet, Catherine; Couppié, Pierre; Nacher, Mathieu; Demar, Magalie; Prévot, Ghislaine

    2016-01-01

    In South America, the presence of the Leishmania RNA virus type 1 (LRV1) was described in Leishmania guyanensis and Leishmania braziliensis strains. The aim of this study was to determine the prevalence distribution of LRV1 in Leishmania isolates in French Guiana given that, in this French overseas department, most Leishmania infections are due to these parasite species. The presence of the virus was observed in 74% of Leishmania sp. isolates, with a highest presence in the internal areas of the country. PMID:26598572

  9. Prevalence and Distribution of Leishmania RNA Virus 1 in Leishmania Parasites from French Guiana.

    PubMed

    Ginouvès, Marine; Simon, Stéphane; Bourreau, Eliane; Lacoste, Vincent; Ronet, Catherine; Couppié, Pierre; Nacher, Mathieu; Demar, Magalie; Prévot, Ghislaine

    2016-01-01

    In South America, the presence of the Leishmania RNA virus type 1 (LRV1) was described in Leishmania guyanensis and Leishmania braziliensis strains. The aim of this study was to determine the prevalence distribution of LRV1 in Leishmania isolates in French Guiana given that, in this French overseas department, most Leishmania infections are due to these parasite species. The presence of the virus was observed in 74% of Leishmania spp. isolates, with a highest presence in the internal areas of the country. © The American Society of Tropical Medicine and Hygiene.

  10. Attenuation of Leishmania infantum chagasi metacyclic promastigotes by sterol depletion.

    PubMed

    Yao, Chaoqun; Gaur Dixit, Upasna; Barker, Jason H; Teesch, Lynn M; Love-Homan, Laurie; Donelson, John E; Wilson, Mary E

    2013-07-01

    The infectious metacyclic promastigotes of Leishmania protozoa establish infection in a mammalian host after they are deposited into the dermis by a sand fly vector. Several Leishmania virulence factors promote infection, including the glycosylphosphatidylinositol membrane-anchored major surface protease (MSP). Metacyclic Leishmania infantum chagasi promastigotes were treated with methyl-beta-cyclodextrin (MβCD), a sterol-chelating reagent, causing a 3-fold reduction in total cellular sterols as well as enhancing MSP release without affecting parasite viability in vitro. MβCD-treated promastigotes were more susceptible to complement-mediated lysis than untreated controls and reduced the parasite load 3-fold when inoculated into BALB/c mice. Paradoxically, MβCD-treated promastigotes caused a higher initial in vitro infection rate in human or murine macrophages than untreated controls, although their intracellular multiplication was hindered upon infection establishment. There was a corresponding larger amount of covalently bound C3b than iC3b on the parasite surfaces of MβCD-treated promastigotes exposed to healthy human serum in vitro, as well as loss of MSP, a protease that enhances C3b cleavage to iC3b. Mass spectrometry showed that MβCD promotes the release of proteins into the extracellular medium, including both MSP and MSP-like protein (MLP), from virulent metacyclic promastigotes. These data support the hypothesis that plasma membrane sterols are important for the virulence of Leishmania protozoa at least in part through retention of membrane virulence proteins.

  11. Attenuation of Leishmania infantum chagasi Metacyclic Promastigotes by Sterol Depletion

    PubMed Central

    Gaur Dixit, Upasna; Barker, Jason H.; Teesch, Lynn M.; Love-Homan, Laurie; Donelson, John E.; Wilson, Mary E.

    2013-01-01

    The infectious metacyclic promastigotes of Leishmania protozoa establish infection in a mammalian host after they are deposited into the dermis by a sand fly vector. Several Leishmania virulence factors promote infection, including the glycosylphosphatidylinositol membrane-anchored major surface protease (MSP). Metacyclic Leishmania infantum chagasi promastigotes were treated with methyl-beta-cyclodextrin (MβCD), a sterol-chelating reagent, causing a 3-fold reduction in total cellular sterols as well as enhancing MSP release without affecting parasite viability in vitro. MβCD-treated promastigotes were more susceptible to complement-mediated lysis than untreated controls and reduced the parasite load 3-fold when inoculated into BALB/c mice. Paradoxically, MβCD-treated promastigotes caused a higher initial in vitro infection rate in human or murine macrophages than untreated controls, although their intracellular multiplication was hindered upon infection establishment. There was a corresponding larger amount of covalently bound C3b than iC3b on the parasite surfaces of MβCD-treated promastigotes exposed to healthy human serum in vitro, as well as loss of MSP, a protease that enhances C3b cleavage to iC3b. Mass spectrometry showed that MβCD promotes the release of proteins into the extracellular medium, including both MSP and MSP-like protein (MLP), from virulent metacyclic promastigotes. These data support the hypothesis that plasma membrane sterols are important for the virulence of Leishmania protozoa at least in part through retention of membrane virulence proteins. PMID:23630964

  12. Specific anti-Leishmania spp. antibodies in stray cats in Greece.

    PubMed

    Diakou, Anastasia; Papadopoulos, Elias; Lazarides, Kostantinos

    2009-08-01

    Greece is a highly endemic country for Leishmania species. Canine cases of leishmaniosis are recorded in different parts of the country. However, no case of feline leishmaniosis has been reported yet. In the present study, the seroprevalence in cats was investigated as a first approach to measuring Leishmania spp. infection of this animal species, in Greece. For this purpose, blood serum samples from 284 stray adult cats, living in the major area of Thessaloniki (Northern Greece), were examined by enzyme-linked immunosorbent assay for the detection of anti-Leishmania spp. IgG. Eleven (3.87%) of the examined animals were found positive. The prevalence was lower in cats than in dogs coming from the same area, based on previous studies. Despite the low seroprevalence for Leishmania spp. in cats, leishmaniosis may be taken into consideration concerning the differential diagnosis of the feline diseases, especially in endemic areas.

  13. Chemotherapy and Biochemistry of Leishmania

    DTIC Science & Technology

    1985-12-01

    Leishmania- sis is initiated when sandflies inject the extracellular prcmastigate form of the parasite into the skin . The promastigotes are...arginine 1 mg folic acid 100 mg L-histidine 2 mg i-inositol 100 mg L-iosleucine 1 mg niacinamide 300 mg L-leucine 1 mg D-pantothenic acid 250 mg L-lysine

  14. Leishmania and human immunodeficiency virus coinfection: the first 10 years.

    PubMed Central

    Alvar, J; Cañavate, C; Gutiérrez-Solar, B; Jiménez, M; Laguna, F; López-Vélez, R; Molina, R; Moreno, J

    1997-01-01

    Over 850 Leishmania-human immunodeficiency virus (HIV) coinfection cases have been recorded, the majority in Europe, where 7 to 17% of HIV-positive individuals with fever have amastigotes, suggesting that Leishmania-infected individuals without symptoms will express symptoms of leishmaniasis if they become immunosuppressed. However, there are indirect reasons and statistical data demonstrating that intravenous drug addiction plays a specific role in Leishmania infantum transmission: an anthroponotic cycle complementary to the zoonotic one has been suggested. Due to anergy in patients with coinfection, L. infantum dermotropic zymodemes are isolated from patient viscera and a higher L. infantum phenotypic variability is seen. Moreover, insect trypanosomatids that are currently considered nonpathogenic have been isolated from coinfected patients. HIV infection and Leishmania infection each induce important analogous immunological changes whose effects are multiplied if they occur concomitantly, such as a Th1-to-Th2 response switch; however, the consequences of the viral infection predominate. In fact, a large proportion of coinfected patients have no detectable anti-Leishmania antibodies. The microorganisms share target cells, and it has been demonstrated in vitro how L. infantum induces the expression of latent HIV-1. Bone marrow culture is the most useful diagnostic technique, but it is invasive. Blood smears and culture are good alternatives. PCR, xenodiagnosis, and circulating-antigen detection are available only in specialized laboratories. The relationship with low levels of CD4+ cells conditions the clinical presentation and evolution of disease. Most patients have visceral leishmaniasis, but asymptomatic, cutaneous, mucocutaneous, diffuse cutaneous, and post-kala-azar dermal leishmaniasis can be produced by L. infantum. The digestive and respiratory tracts are frequently parasitized. The course of coinfection is marked by a high relapse rate. There is a lack

  15. A general classification of New World Leishmania using numerical zymotaxonomy.

    PubMed

    Cupolillo, E; Grimaldi, G; Momen, H

    1994-03-01

    More than 250 strains of Leishmania isolated from different localities and hosts in the New World were analyzed by enzyme electrophoresis, and their electromorphic profiles were compared with 19 reference strains representing most of the described species of this parasite. The 18 enzymic loci analyzed were very polymorphic, and the strains were classified into 44 zymodemes, each grouping strains with the same enzyme profiles. Each zymodeme was considered as an elementary taxon and the phenetic and phylogenetic relationships were determined by agglomerative hierarchical, ordination, and cladistic techniques. The different classification methods produced very similar results. The 44 zymodemes could be clustered into two groups, corresponding to the subgenera Leishmania and Viannia, by the numerical methods. The subgenus Viannia was shown to be monophyletic and could be further divided into species complexes representing L. braziliensis, L. naiffi, and L. guyanensis/L. panamensis/L. shawi, as well as some isolated taxa including L. lainsoni. The subgenus Leishmania, on the other hand, was polyphyletic, with New World isolates related to L. major clustered separately from the L. mexicana species complex. Most of the other zymodemes in this group represented independent taxa. The results confirm Viannia as a valid taxon but suggest that the status of the subgenus Leishmania should be further investigated. Leishmania braziliensis and L. naiffi were shown to be the most polymorphic species, while L. guyanensis, in spite of being the most common species found in this study, was remarkably homogeneous. The only variants were found south of the Amazon river. North of this river, the species was monomorphic.

  16. First Isolation of Leishmania from Northern Thailand: Case Report, Identification as Leishmania martiniquensis and Phylogenetic Position within the Leishmania enriettii Complex

    PubMed Central

    Pothirat, Thatawan; Tantiworawit, Adisak; Chaiwarith, Romanee; Jariyapan, Narissara; Wannasan, Anchalee; Siriyasatien, Padet; Supparatpinyo, Khuanchai; Bates, Michelle D.; Kwakye-Nuako, Godwin; Bates, Paul A.

    2014-01-01

    Since 1996, there have been several case reports of autochthonous visceral leishmaniasis in Thailand. Here we report a case in a 52-year-old Thai male from northern Thailand, who presented with subacute fever, huge splenomegaly and pancytopenia. Bone marrow aspiration revealed numerous amastigotes within macrophages. Isolation of Leishmania LSCM1 into culture and DNA sequence analysis (ribosomal RNA ITS-1 and large subunit of RNA polymerase II) revealed the parasites to be members of the Leishmania enriettii complex, and apparently identical to L. martiniquensis previously reported from the Caribbean island of Martinique. This is the first report of visceral leishmaniasis caused by L. martiniquensis from the region. Moreover, the majority of parasites previously identified as “L. siamensis” also appear to be L. martiniquensis. PMID:25474647

  17. Investigations of Cross Immunity between Leishmania tropica (Jericho) and Leishmania braziliensis in Experimentally Infected Mystromys albacaudatus.

    DTIC Science & Technology

    1979-09-01

    AD-AL15 528 VIR61NIA UNIV CHARLOTTESVILLE DEPT OF DERMATOLOGY F/G 6/5 INVESTIGATIONS OF CROSS IMMUNITY BETWEEN LEISHMANIA TROPICA (JE--ETC(U) SEP 79...Investigations of Cross Immunity Between First Annual -- Leishmania tropica (Jericho) and Leishmania Feburary 1979-September 1979 braziliensis in... Leishmania tropica (Jericho) and LeisLmania braziliensis panamensis in Experimentally Infected Mystromys albacaudatus" First Annual Report Bruce E

  18. Leishmania in synanthropic rodents (Rattus rattus): new evidence for the urbanization of Leishmania (Leishmania) amazonensis.

    PubMed

    Caldart, Eloiza Teles; Freire, Roberta Lemos; Ferreira, Fernanda Pinto; Ruffolo, Bruno Bergamo; Sbeghen, Mônica Raquel; Mareze, Marcelle; Garcia, João Luis; Mitsuka-Breganó, Regina; Navarro, Italmar Teodorico

    2017-02-06

    This study aimed to detect parasites from Leishmania genus, to determine the prevalence of anti-Leishmania spp. antibodies, to identify circulating species of the parasite, and to determine epidemiological variables associated with infection in rats caught in urban area of Londrina, Paraná, Brazil. Animal capture was carried out from May to December 2006, serological and molecular methods were performed. DNA was extracted from total blood, and nested-PCR, targeting SSu rRNA from Leishmania genus, was performed in triplicate. The positive samples were sequenced twice by Sanger method to species determination. In total, 181 rodents were captured, all were identified as Rattus rattus and none showed clinical alterations. Forty-one of the 176 (23.3%) animals were positive for Leishmania by ELISA and 6/181 (3.3%) were positive by IFAT. Nine of 127 tested animals (7.1%) were positive by PCR; seven were identified as L. (L.) amazonensis, one as L. (L.) infantum. Four rats were positive using more than one test. This was the first description of synanthropic rodents naturally infected by L. (L.) amazonensis (in the world) and by L. (L.) infantum (in South Brazil). Regarding L. (L.) amazonensis, this finding provides new evidence of the urbanization of this etiological agent.

  19. The use of kDNA minicircle subclass relative abundance to differentiate between Leishmania (L.) infantum and Leishmania (L.) amazonensis.

    PubMed

    Ceccarelli, Marcello; Galluzzi, Luca; Diotallevi, Aurora; Andreoni, Francesca; Fowler, Hailie; Petersen, Christine; Vitale, Fabrizio; Magnani, Mauro

    2017-05-16

    Leishmaniasis is a neglected disease caused by many Leishmania species, belonging to subgenera Leishmania (Leishmania) and Leishmania (Viannia). Several qPCR-based molecular diagnostic approaches have been reported for detection and quantification of Leishmania species. Many of these approaches use the kinetoplast DNA (kDNA) minicircles as the target sequence. These assays had potential cross-species amplification, due to sequence similarity between Leishmania species. Previous works demonstrated discrimination between L. (Leishmania) and L. (Viannia) by SYBR green-based qPCR assays designed on kDNA, followed by melting or high-resolution melt (HRM) analysis. Importantly, these approaches cannot fully distinguish L. (L.) infantum from L. (L.) amazonensis, which can coexist in the same geographical area. DNA from 18 strains/isolates of L. (L.) infantum, L. (L.) amazonensis, L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, and 62 clinical samples from L. (L.) infantum-infected dogs were amplified by a previously developed qPCR (qPCR-ML) and subjected to HRM analysis; selected PCR products were sequenced using an ABI PRISM 310 Genetic Analyzer. Based on the obtained sequences, a new SYBR-green qPCR assay (qPCR-ama) intended to amplify a minicircle subclass more abundant in L. (L.) amazonensis was designed. The qPCR-ML followed by HRM analysis did not allow discrimination between L. (L.) amazonensis and L. (L.) infantum in 53.4% of cases. Hence, the novel SYBR green-based qPCR (qPCR-ama) has been tested. This assay achieved a detection limit of 0.1 pg of parasite DNA in samples spiked with host DNA and did not show cross amplification with Trypanosoma cruzi or host DNA. Although the qPCR-ama also amplified L. (L.) infantum strains, the Cq values were dramatically increased compared to qPCR-ML. Therefore, the combined analysis of Cq values from qPCR-ML and qPCR-ama allowed to distinguish L. (L.) infantum and L. (L.) amazonensis in 100% of tested samples. A

  20. Unraveling the genetic diversity and phylogeny of Leishmania RNA virus 1 strains of infected Leishmania isolates circulating in French Guiana.

    PubMed

    Tirera, Sourakhata; Ginouves, Marine; Donato, Damien; Caballero, Ignacio S; Bouchier, Christiane; Lavergne, Anne; Bourreau, Eliane; Mosnier, Emilie; Vantilcke, Vincent; Couppié, Pierre; Prevot, Ghislaine; Lacoste, Vincent

    2017-07-01

    Leishmania RNA virus type 1 (LRV1) is an endosymbiont of some Leishmania (Vianna) species in South America. Presence of LRV1 in parasites exacerbates disease severity in animal models and humans, related to a disproportioned innate immune response, and is correlated with drug treatment failures in humans. Although the virus was identified decades ago, its genomic diversity has been overlooked until now. We subjected LRV1 strains from 19 L. (V.) guyanensis and one L. (V.) braziliensis isolates obtained from cutaneous leishmaniasis samples identified throughout French Guiana with next-generation sequencing and de novo sequence assembly. We generated and analyzed 24 unique LRV1 sequences over their full-length coding regions. Multiple alignment of these new sequences revealed variability (0.5%-23.5%) across the entire sequence except for highly conserved motifs within the 5' untranslated region. Phylogenetic analyses showed that viral genomes of L. (V.) guyanensis grouped into five distinct clusters. They further showed a species-dependent clustering between viral genomes of L. (V.) guyanensis and L. (V.) braziliensis, confirming a long-term co-evolutionary history. Noteworthy, we identified cases of multiple LRV1 infections in three of the 20 Leishmania isolates. Here, we present the first-ever estimate of LRV1 genomic diversity that exists in Leishmania (V.) guyanensis parasites. Genetic characterization and phylogenetic analyses of these viruses has shed light on their evolutionary relationships. To our knowledge, this study is also the first to report cases of multiple LRV1 infections in some parasites. Finally, this work has made it possible to develop molecular tools for adequate identification and genotyping of LRV1 strains for diagnostic purposes. Given the suspected worsening role of LRV1 infection in the pathogenesis of human leishmaniasis, these data have a major impact from a clinical viewpoint and for the management of Leishmania-infected patients.

  1. Natural infection of Algerian hedgehog, Atelerix algirus (Lereboullet 1842) with Leishmania parasites in Tunisia.

    PubMed

    Chemkhi, Jomaa; Souguir, Hejer; Ali, Insaf Bel Hadj; Driss, Mehdi; Guizani, Ikram; Guerbouj, Souheila

    2015-10-01

    In Tunisia, Leishmania parasites are responsible of visceral leishmaniasis, caused by Leishmania infantum species while three cutaneous disease forms are documented: chronic cutaneous leishmaniasis due to Leishmania killicki, sporadic cutaneous form (SCL) caused by L. infantum and the predominant zoonotic cutaneous leishmanaisis (ZCL) due to Leishmania major. ZCL reservoirs are rodents of the Psammomys and Meriones genera, while for SCL the dog is supposed to be a reservoir. Ctenodactylus gundii is involved in the transmission of L. killicki. However, other mammals could constitute potential reservoir hosts in Tunisia and other North African countries. In order to explore the role of hedgehogs as potential reservoirs of leishmaniasis, specimens (N=6) were captured during July-November period in 2011-2013 in an SCL endemic area in El Kef region, North-Western Tunisia. Using morphological characteristics, all specimens were described and measured. Biopsies from liver, heart, kidney and spleen of each animal were used to extract genomic DNA, which was further used in PCR assays to assess the presence of Leishmania parasites. Different PCRs targeting kinetoplast minicircles, ITS1, mini-exon genes and a repetitive Leishmania- specific sequence, were applied. To further identify Leishmania species involved, RFLP analysis of amplified fragments was performed with appropriate restriction enzymes. Using morphological characters, animals were identified as North African hedgehogs, also called Algerian hedgehogs, that belong to the Erinaceidae family, genus Atelerix Pomel 1848, and species algirus (Lereboullet, 1842). PCR results showed in total that all specimens were Leishmania infected, with different organs incriminated, mainly liver and spleen. Results were confirmed by direct sequencing of amplified fragments. Species identification showed that all specimens were infected with L. major, three of which were additionally co-infected with L. infantum. The present study

  2. Ultrastructural and cytochemical identification of megasome in Leishmania (Leishmania) chagasi.

    PubMed

    Alberio, Sanny O; Dias, Suzana S; Faria, Flávio P; Mortara, Renato A; Barbiéri, Clara L; Freymüller Haapalainen, Edna

    2004-02-01

    The present work showed the presence of a megasome in Leishmania (Leishmania) chagasi amastigotes. Transmission electron microscopy analysis of ultrathin serial sections and three-dimensional reconstruction allowed visualization of large structures in amastigote forms of L. (L.) chagasi and a multivesicular tubule-lysosome structure in metacyclic promastigotes. Morphometric data showed that the relative volume occupied by the megasome and the multivesicular tubule (MVT)-lysosome structures was about 5% and 3.2%, respectively, in amastigotes and promastigotes of L. (L.) chagasi. Further characterization of the megasome in L. (L.) chagasi amastigotes was carried out by immunolabeling of cysteine proteinase, whereas the lysosomal content of amastigotes and promastigotes was confirmed by arylsulfatase cytochemistry.

  3. Molecular detection of Leishmania infection in sand flies in border line of Iran-Turkmenistan: restricted and permissive vectors.

    PubMed

    Bakhshi, H; Oshaghi, M A; Abai, M R; Rassi, Y; Akhavan, A A; Sheikh, Z; Mohtarami, F; Saidi, Z; Mirzajani, H; Anjomruz, M

    2013-10-01

    A molecular study was carried out to incriminate sand fly vectors of cutaneous leishmaniasis (CL) in rural areas of Sarakhs district, Khorassane-Razavi Province, northeastern Iran, in 2011. Sand flies of Sergentomyia with three species and Phlebotomus with six species respectively comprised 73.3% and 26.7% of the specimens. Phlebotomus papatasi was the most common Phlebotomine species in outdoor and indoor resting places. Leishmania infection was found at least in 17 (22%) specimens including Ph. papatasi (n=9 pool samples), Phlebotomus caucasicus (n=6), Phlebotomus alexandri (n=1), and Sergentomyia sintoni (n=1). The parasites were found comprised Leishmania major (n=5), Leishmania turanica (n=10), and Leishmania gerbilli (n=4). Infection of Ph. papatasi with both L. major and L. turanica supporting the new suggestion indicating that it is not restricted only with L. major. Circulation of L. major by Ph. alexandri, and both L. gerbilli and L. turanica by Ph. caucasicus, in addition to previous data indicating the ability of Ph. alexandri to circulate Leishmania infantum and Leishmania donovani, and Ph. caucasicus to circulate L. major, suggests that these two species can be permissive vectors. The results suggest that Ph. papatasi and Ph. alexandri are the primary and secondary vectors of CL where circulating L. major between human and reservoirs, whereas Ph. caucasicus is circulating L. turanica and L. gerbilli between the rodents in the region.

  4. Detection of Leishmania RNA Virus in Leishmania Parasites

    PubMed Central

    Desponds, Chantal; Kuhlmann, F. Matthew; Robinson, John; Hartley, Mary-Anne; Prevel, Florence; Castiglioni, Patrik; Pratlong, Francine; Bastien, Patrick; Müller, Norbert; Parmentier, Laurent; Saravia, Nancy Gore; Beverley, Stephen M.; Fasel, Nicolas

    2013-01-01

    Background Patients suffering from cutaneous leishmaniasis (CL) caused by New World Leishmania (Viannia) species are at high risk of developing mucosal (ML) or disseminated cutaneous leishmaniasis (DCL). After the formation of a primary skin lesion at the site of the bite by a Leishmania-infected sand fly, the infection can disseminate to form secondary lesions. This metastatic phenotype causes significant morbidity and is often associated with a hyper-inflammatory immune response leading to the destruction of nasopharyngeal tissues in ML, and appearance of nodules or numerous ulcerated skin lesions in DCL. Recently, we connected this aggressive phenotype to the presence of Leishmania RNA virus (LRV) in strains of L. guyanensis, showing that LRV is responsible for elevated parasitaemia, destructive hyper-inflammation and an overall exacerbation of the disease. Further studies of this relationship and the distribution of LRVs in other Leishmania strains and species would benefit from improved methods of viral detection and quantitation, especially ones not dependent on prior knowledge of the viral sequence as LRVs show significant evolutionary divergence. Methodology/Principal Findings This study reports various techniques, among which, the use of an anti-dsRNA monoclonal antibody (J2) stands out for its specific and quantitative recognition of dsRNA in a sequence-independent fashion. Applications of J2 include immunofluorescence, ELISA and dot blot: techniques complementing an arsenal of other detection tools, such as nucleic acid purification and quantitative real-time-PCR. We evaluate each method as well as demonstrate a successful LRV detection by the J2 antibody in several parasite strains, a freshly isolated patient sample and lesion biopsies of infected mice. Conclusions/Significance We propose that refinements of these methods could be transferred to the field for use as a diagnostic tool in detecting the presence of LRV, and potentially assessing the LRV

  5. Dihydroorotate dehydrogenase (DHODH) inhibitors affect ATP depletion, endogenous ROS and mediate S-phase arrest in breast cancer cells.

    PubMed

    Mohamad Fairus, A K; Choudhary, B; Hosahalli, S; Kavitha, N; Shatrah, O

    2017-04-01

    Dihydroorotate dehydrogenase (DHODH) is the key enzyme in de novo biosynthesis of pyrimidine in both prokaryotes and eukaryotes. The de novo pathway of pyrimidine biosynthesis is essential in cancer cells proliferation. Leflunomide is an approved DHODH inhibitor that has been widely used for the treatment of arthritis. Similarly, brequinar sodium is another DHODH inhibitor that showed anti-tumour effect in MC38 colon carcinoma cells when used in combination with fluorouracil. Despite the potential role of DHODH inhibitors in cancer therapy, their mechanisms of action remain obscure and await further elucidation. Here, we evaluated the effect of DHODH inhibitors on the production of ATP and ROS in sensitive and non-sensitive breast cancer cells. Subsequently, the effects of DHODH inhibitors on cell cycle as well as on signalling molecules such as p53, p65 and STAT6 were evaluated in sensitive T-47D and non-sensitive MDAMB-436 cells. The correlations between DHODH protein expression, proliferation speed and sensitivity to DHODH inhibitors were also investigated in a panel of cancer cell lines. DHODH inhibitors-sensitive T-47D and MDAMB-231 cells appeared to preserve ROS production closely to endogenous ROS level whereas the opposite was observed in non-sensitive MDAMB-436 and W3.006 cells. In addition, we observed approximately 90% of intracellular ATP depletion in highly sensitive T-47D and MDAMB-231 cells compared to non-sensitive MDAMB-436 cells. There was significant over-expression of p53, p65 and STAT6 signalling molecules in sensitive cells which may be involved in mediating the S-phase arrest in cell cycle progression. The current study suggests that DHODH inhibitors are most effective in cells that express high levels of DHODH enzyme. The inhibition of cell proliferation by these inhibitors appears to be accompanied by ROS production as well as ATP depletion. The increase in expression of signalling molecules observed may be due to pyrimidine depletion

  6. Serine protease activities in Leishmania (Leishmania) chagasi promastigotes.

    PubMed

    da Silva-López, Raquel Elisa; dos Santos, Tatiana Resende; Morgado-Díaz, José Andrés; Tanaka, Marcelo Neves; de Simone, Salvatore Giovanni

    2010-10-01

    The present work reports the isolation, biochemical characterization, and subcellular location of serine proteases from aqueous, detergent soluble, and culture supernatant of Leishmania chagasi promastigote extracts, respectively, LCSII, LCSI, and LCSIII. The active enzyme molecular masses of LCSII were about 105, 66, and 60 kDa; of LCSI, 60 and 58 kDa; and of LCSIII, approximately 76 and 68 kDa. Optimal pH for the enzymes was 7.0 for LCSI and LCSIII and 8.5 for LCSII, and the optimal temperature for all enzymes was 37°C, using α-N-ρ-tosyl-L: -arginine methyl ester as substrate. Assay of thermal stability indicated that LCSIII is the more stable enzyme. Hemoglobin, bovine serum albumin, and ovalbumin were hydrolyzed by LCSII and LCSI but not by LCSIII. Inhibition studies suggested that enzymes belong to the serine protease class modulated by divalent cations. Rabbit antiserum against 56-kDa serine protease of Leishmania amazonensis identified proteins in all extracts of L. chagasi. Furthermore, immunocytochemistry demonstrated that serine proteases are located in flagellar pocket region and cytoplasmic vesicles of L. chagasi promastigotes. These findings indicate that L. chagasi serine proteases differ from L. amazonensis proteases and all known flagellate proteases, but display some similarities with serine proteases from other Leishmania species, suggesting a conservation of this enzymatic activity in the genus.

  7. Molecular and Cellular Characterization of an AT-Hook Protein from Leishmania

    PubMed Central

    Kelly, Ben L.; Singh, Gyanendra; Aiyar, Ashok

    2011-01-01

    AT-rich DNA, and the proteins that bind it (AT-hook proteins), modulate chromosome structure and function in most eukaryotes. Unlike other trypanosomatids, the genome of Leishmania species is unusually GC-rich, and the regulation of Leishmania chromosome structure, replication, partitioning is not fully understood. Because AT-hook proteins modulate these functions in other eukaryotes, we examined whether AT-hook proteins are encoded in the Leishmania genome, to test their potential functions. Several Leishmania ORFs predicted to be AT-hook proteins were identified using in silico approaches based on sequences shared between eukaryotic AT-hook proteins. We have used biochemical, molecular and cellular techniques to characterize the L. amazonensis ortholog of the L. major protein LmjF06.0720, a potential AT-hook protein that is highly conserved in Leishmania species. Using a novel fusion between the AT-hook domain encoded by LmjF06.0720 and a herpesviral protein, we have demonstrated that LmjF06.0720 functions as an AT-hook protein in mammalian cells. Further, as observed for mammalian and viral AT-hook proteins, the AT-hook domains of LmjF06.0720 bind specific regions of condensed mammalian metaphase chromosomes, and support the licensed replication of DNA in mammalian cells. LmjF06.0720 is nuclear in Leishmania, and this localization is disrupted upon exposure to drugs that displace AT-hook proteins from AT-rich DNA. Coincidentally, these drugs dramatically alter the cellular physiology of Leishmania promastigotes. Finally, we have devised a novel peptido-mimetic agent derived from the sequence of LmjF06.0720 that blocks the proliferation of Leishmania promastigotes, and lowers amastigote parasitic burden in infected macrophages. Our results indicate that AT-hook proteins are critical for the normal biology of Leishmania. In addition, we have described a simple technique to examine the function of Leishmania chromatin-binding proteins in a eukaryotic context

  8. Genomic and proteomic expression analysis of Leishmania promastigote and amastigote life stages: the Leishmania genome is constitutively expressed.

    PubMed

    Leifso, Kirk; Cohen-Freue, Gabriela; Dogra, Nisha; Murray, Angus; McMaster, W Robert

    2007-03-01

    Leishmania are protozoan parasites that cause a wide spectrum of clinical diseases in humans and are a major public health risk in several countries. Leishmania life cycle consists of an extracellular flagellated promastigote stage within the midgut of a sandfly vector, and a morphological distinct intracellular amastigote stage within macrophages of a mammalian host. This study reports the use of DNA oligonucleotide genome microarrays representing 8160 genes to analyze the mRNA expression profiles of L. major promastigotes and lesion derived amastigotes. Over 94% of the genes were expressed in both life stages. Advanced statistical analysis identified a surprisingly low degree of differential mRNA expression: 1.4% of the total genes in amastigotes and 1.5% in promastigotes. These microarray results demonstrate that the L. major genome is essentially constitutively expressed in both life stages and suggest that Leishmania is constitutively adapted for survival and replication in either the sandfly vector or macrophage host utilizing an appropriate set of genes for each vastly different environment. Quantitative proteomics, using the isotope coded affinity tag (ICAT) technology and mass spectrometry, was used to identify L. infantum promastigote and axenic amastigote differentially expressed proteins. Of the 91 distinct proteins identified, 8% were differentially expressed in the amastigote stage, 20% were differentially expressed in the promastigote stage, and the remaining 72% were considered constitutively expressed. The differential expression was validated by the identification of previously reported stage specific proteins and identified several amastigote and promastigote novel stage specific proteins.

  9. Molecular Survey on Detection of Leishmania Infection in Rodent Reservoirs in Jahrom District, Southern Iran

    PubMed Central

    Davami, Mohammad Hassan; Motazedian, Mohammad Hossein; Kalantari, Mohsen; Asgari, Qasem; Mohammadpour, Iraj; Sotoodeh-Jahromi, Abdolreza; Solhjoo, Kavous; Pourahmad, Morteza

    2014-01-01

    Background: Zoonotic Cutaneous Leishmaniasis (ZCL) is endemic in many parts of Iran. Recently its incidence is considerable in different parts of Jahrom district, in Fars Province, southern Iran. The aims of our study were to investigate the prevalence of leishmania infection, and identify and characterize the Leishmania species present, among the rodents by molecular methods in a new endemic focus of ZCL, in an urban and rural area of the Jahrom district, Fars Province, southern Iran. Methods: From May to November 2010), 55 rodents in four regions of Jahrom focus were caught and checked for leishmania infection by the microscopical examination of liver, spleen, ears, and footpads’ smears. Results: Overall 18 Meriones persicus, 15 Tatera indica, 14 Mus musculus, and 8 Rattus rattus were caught. Totally, four (16.5%) and two (13.3%) of the Me. persicus and Ta. indica, but only one of Mu. musculus and Ra. rattus were found smear-positive for leishmania amastigotes, respectively. In the nested-PCR assay 8 (14.6%) smears were found positive for Leishmania major, none was found positive for any other Leishmania species. Sequencing based detection of Leishmania confirmed the microscopic and PCR findings. All positive specimens were shown 95–96% similarity with L. major Friedlin. Conclusion: Tatera indica and Me. persicus are incriminated as the main ‘reservoir’ hosts of L. major in the rural area of Jahrom, moreover, Mu. musculus and Ra. rattus have the minor but remarkable role in the maintenance of the disease in the urban regions of Jahrom focus. PMID:26114127

  10. Leishmania-based expression systems.

    PubMed

    Taheri, Tahereh; Seyed, Negar; Mizbani, Amir; Rafati, Sima

    2016-09-01

    Production of therapeutic or medical recombinant proteins, such as monoclonal antibodies, proteins, or active enzymes, requires a highly efficient system allowing natural folding and perfect post-translation modifications of the expressed protein. These requirements lead to the generation of a variety of gene expression systems from bacteria to eukaryotes. To achieve the best form of eukaryotic proteins, two factors need to be taken into consideration: choosing a suitable organism to express the protein of interest, and selecting an efficient delivery system. For this reason, the expression of recombinant proteins in eukaryotic nonpathogenic Leishmania parasites is an interesting approach which meets both criteria. Here, new Leishmania-based expression systems are compared with current systems that have long histories in research and industry.

  11. Folate metabolic pathways in Leishmania

    PubMed Central

    Vickers, Tim J.; Beverley, Stephen M.

    2012-01-01

    Trypanosomatid parasitic protozoans of the genus Leishmania are autotrophic for both folate and unconjugated pteridines. Leishmania salvage these metabolites from their mammalian hosts and insect vectors through multiple transporters. Within the parasite, folates are reduced by a bifunctional DHFR (dihydrofolate reductase)-TS (thymidylate synthase) and by a novel PTR1 (pteridine reductase 1), which reduces both folates and unconjugated pteridines. PTR1 can act as a metabolic bypass of DHFR inhibition, reducing the effectiveness of existing antifolate drugs. Leishmania possess a reduced set of folate-dependent metabolic reactions and can salvage many of the key products of folate metabolism from their hosts. For example, they lack purine synthesis, which normally requires 10-formyltetrahydrofolate, and instead rely on a network of purine salvage enzymes. Leishmania elaborate at least three pathways for the synthesis of the key metabolite 5,10-methylene-tetrahydrofolate, required for the synthesis of thymidylate, and for 10-formyltetrahydrofolate, whose presumptive function is for methionyl-tRNAMet formylation required for mitochondrial protein synthesis. Genetic studies have shown that the synthesis of methionine using 5-methyltetrahydrofolate is dispensable, as is the activity of the glycine cleavage complex, probably due to redundancy with serine hydroxymethyltransferase. Although not always essential, the loss of several folate metabolic enzymes results in attenuation or loss of virulence in animal models, and a null DHFR-TS mutant has been used to induce protective immunity. The folate metabolic pathway provides numerous opportunities for targeted chemotherapy, with strong potential for ‘repurposing’ of compounds developed originally for treatment of human cancers or other infectious agents. PMID:22023442

  12. Folate metabolic pathways in Leishmania.

    PubMed

    Vickers, Tim J; Beverley, Stephen M

    2011-01-01

    Trypanosomatid parasitic protozoans of the genus Leishmania are autotrophic for both folate and unconjugated pteridines. Leishmania salvage these metabolites from their mammalian hosts and insect vectors through multiple transporters. Within the parasite, folates are reduced by a bifunctional DHFR (dihydrofolate reductase)-TS (thymidylate synthase) and by a novel PTR1 (pteridine reductase 1), which reduces both folates and unconjugated pteridines. PTR1 can act as a metabolic bypass of DHFR inhibition, reducing the effectiveness of existing antifolate drugs. Leishmania possess a reduced set of folate-dependent metabolic reactions and can salvage many of the key products of folate metabolism from their hosts. For example, they lack purine synthesis, which normally requires 10-formyltetrahydrofolate, and instead rely on a network of purine salvage enzymes. Leishmania elaborate at least three pathways for the synthesis of the key metabolite 5,10-methylene-tetrahydrofolate, required for the synthesis of thymidylate, and for 10-formyltetrahydrofolate, whose presumptive function is for methionyl-tRNAMet formylation required for mitochondrial protein synthesis. Genetic studies have shown that the synthesis of methionine using 5-methyltetrahydrofolate is dispensable, as is the activity of the glycine cleavage complex, probably due to redundancy with serine