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Sample records for leishmania major dihydroorotate

  1. 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. PMID:26656485

  2. 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. PMID:27062077

  3. The structure of Leishmania major amastigote lipophosphoglycan.

    PubMed

    Moody, S F; Handman, E; McConville, M J; Bacic, A

    1993-09-01

    Intracellular amastigotes of Leishmania major produce 6 x 10(4) copies/cell of a lipophosphoglycan (LPG) that is structurally distinct from the LPG produced by the extracellular promastigote form of L. major, Leishmania donovani, and Leishmania mexicana (reviewed by McConville, M. J. (1991) Cell Biol. Int. Rep. 15, 779-798). L. major amastigote LPG is composed of a lysoalkyl phosphatidylinositol lipid anchor that links via a diphosphorylated hexasaccharide core to a phosphoglycan (6-100 kDa). The structures of the anchor, the core, and the phosphoglycan were determined by monosaccharide and linkage analysis, fast atom bombardment-mass spectrometry, one-dimensional 1H NMR spectroscopy, and exoglycosidase microsequencing. The lipid anchor contains predominantly 1-O-alkylglycerols with 24:0 and 22:0 alkyl chains. The lipids are linked via a glycerol-myo-inositol-PO4 to a core glycan with the structure -PO4-6)Gal(alpha 1-)Gal(alpha 1-) Galf(beta 1-)[Glc(alpha 1-PO4-)]Man(alpha 1-)Man(alpha 1-)GlcN(alpha 1-). The chromatographic characteristics of the core glycan suggest that the saccharide components are linked similarly in amastigote and promastigote LPG. The phosphoglycan attached to the core consists of -PO4-6)Gal(beta 1-4)Man(alpha 1- repeats units which are either unsubstituted (70%) or substituted (30%) at the 3-position of the Gal residues with oligosaccharide side chains containing primarily Gal and some Glc. Thirteen different types of side chains were identified with the structures [Gal(beta 1-3)]x, where x = 1-11, or Glc(1-3)Glc(1-3), or Glc(1-3)Gal(beta 1-3), where glucose is probably in the beta-configuration. All monosaccharides in the phosphoglycan domain are in the pyranose configuration. The average number of repeat units per molecule is 36. The nonreducing terminus of the phosphoglycan chains probably terminates predominantly in the neutral disaccharide Gal(beta 1-4)Man(alpha 1-. Comparison of the structure of L. major amastigote LPG to L. major

  4. 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. PMID:23324218

  5. Structure of the lipophosphoglycan from Leishmania major.

    PubMed

    McConville, M J; Thomas-Oates, J E; Ferguson, M A; Homans, S W

    1990-11-15

    The major cell surface glycoconjugate of the parasitic protozoan Leishmania major is a heterogeneous lipophosphoglycan. It has a tripartite structure, consisting of a phosphoglycan (Mr 5,000-40,000), a variably phosphorylated hexasaccharide glycan core, and a lysoalkylphosphatidylinositol (lysoalkyl-PI) lipid anchor. The structures of the phosphoglycan and the hexasaccharide core were determined by monosaccharide analysis, methylation analysis, fast atom bombardment-mass spectrometry, one- and two-dimensional 500-MHz (correlated spectroscopy (COSY), homonuclear Hartmann-Hahn spectroscopy (HOHAHA] 1H NMR spectroscopy, and exoglycosidase digestions. The phosphoglycan consists of eight types of phosphorylated oligosaccharide repeats which have the general structure, [formula: see text] where R = H, Galp(beta 1-3), Galp(beta 1-3)Galp(beta 1-3), Arap(alpha 1-2)Galp(beta 1-3), Glcp(beta 1-3)Galp(beta 1-3), Galp(beta 1-3)Galp(beta 1-3)Galp(beta 1-3), Arap(alpha 1-2)Galp(beta 1-3)Galp(beta 1-3), or Arap(alpha 1-2)Galp(beta 1-3)Galp(beta 1-3)Galp(beta 1-3)Galp(beta 1-3), and where all the monosaccharides, including arabinose, are in the D-configuration. The average number of repeat units/molecule (n) is 27. Data are presented which suggest that the nonreducing terminus of the phosphoglycan is capped exclusively with the neutral disaccharide Manp(alpha 1-2)Manp alpha 1-. The structure of the glycan core was determined to be, [formula: see text] where approximately 60% of the mannose residues distal to the glucosamine are phosphorylated and where the inositol is part of the lysoalkyl-PI lipid moiety containing predominantly 24:0 and 26:0 alkyl chains. The unusual galactofuranose residue is in the beta-configuration, correcting a previous report where this residue was identified as alpha Galf. Although most of the phosphorylated repeat units are attached to the terminal galactose 6-phosphate of the core to form a linear lipophosphoglycan (LPG) molecule, some of the mannose 6

  6. Wild gorillas as a potential reservoir of Leishmania major.

    PubMed

    Hamad, Ibrahim; Forestier, Claire-Lise; Peeters, Martine; Delaporte, Eric; Raoult, Didier; Bittar, Fadi

    2015-01-15

    Vector-borne parasites of the genus Leishmania are responsible for severe human diseases. Cutaneous leishmaniasis, a common form of the disease, is most often caused by the transmission of Leishmania major to humans by female phlebotomine sand flies. Apes are increasingly being seen as a source of zoonotic diseases, including malaria and rickettsiosis. To examine whether gorillas harbor Leishmania species, we screened fecal samples from wild western lowland gorillas (Gorilla gorilla gorilla) in Cameroon for the presence of these pathogens. Of 91 wild gorilla fecal samples, 12 contained Leishmania parasites, and 4 contained phlebotomine sand fly vectors. The molecular identity was determined by running 3 different polymerase chain reaction tests for detection of L. major. Next, fluorescence in situ hybridization was performed to visualize L. major parasites in fecal samples from the gorillas. Both promastigote and amastigote forms of the parasite were found. This work strongly suggests that wild gorillas carry pathogenic Leishmania parasites. PMID:25001460

  7. Persistence without pathology in phosphoglycan-deficient Leishmania major.

    PubMed

    Späth, Gerald F; Lye, Lon-Fey; Segawa, Hiroaki; Sacks, David L; Turco, Salvatore J; Beverley, Stephen M

    2003-08-29

    Leishmania infections involve an acute phase of replication within macrophages, typically associated with pathology. After recovery parasites persist for long periods, which can lead to severe disease upon reactivation. Unlike the role of host factors, parasite factors affecting persistence are poorly understood. Leishmania major lacking phosphoglycans (lpg2-) were unable to survive in sand flies and macrophages, but retained the ability to persist indefinitely in the mammalian host without inducing disease. The L. major lpg2- thus provides a platform for probing parasite factors implicated in persistence and its role in disease and immunity. PMID:12947201

  8. 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. PMID:27056699

  9. 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. PMID:26277567

  10. LeishCyc: a biochemical pathways database for Leishmania major

    PubMed Central

    Doyle, Maria A; MacRae, James I; De Souza, David P; Saunders, Eleanor C; McConville, Malcolm J; Likić, Vladimir A

    2009-01-01

    Background Leishmania spp. are sandfly transmitted protozoan parasites that cause a spectrum of diseases in more than 12 million people worldwide. Much research is now focusing on how these parasites adapt to the distinct nutrient environments they encounter in the digestive tract of the sandfly vector and the phagolysosome compartment of mammalian macrophages. While data mining and annotation of the genomes of three Leishmania species has provided an initial inventory of predicted metabolic components and associated pathways, resources for integrating this information into metabolic networks and incorporating data from transcript, protein, and metabolite profiling studies is currently lacking. The development of a reliable, expertly curated, and widely available model of Leishmania metabolic networks is required to facilitate systems analysis, as well as discovery and prioritization of new drug targets for this important human pathogen. Description The LeishCyc database was initially built from the genome sequence of Leishmania major (v5.2), based on the annotation published by the Wellcome Trust Sanger Institute. LeishCyc was manually curated to remove errors, correct automated predictions, and add information from the literature. The ongoing curation is based on public sources, literature searches, and our own experimental and bioinformatics studies. In a number of instances we have improved on the original genome annotation, and, in some ambiguous cases, collected relevant information from the literature in order to help clarify gene or protein annotation in the future. All genes in LeishCyc are linked to the corresponding entry in GeneDB (Wellcome Trust Sanger Institute). Conclusion The LeishCyc database describes Leishmania major genes, gene products, metabolites, their relationships and biochemical organization into metabolic pathways. LeishCyc provides a systematic approach to organizing the evolving information about Leishmania biochemical networks and is

  11. Lipophosphoglycan blocks attachment of Leishmania major amastigotes to macrophages.

    PubMed

    Kelleher, M; Moody, S F; Mirabile, P; Osborn, A H; Bacic, A; Handman, E

    1995-01-01

    Promastigotes of the intracellular protozoan parasite Leishmania major invade mononuclear phagocytes by a direct interaction between the cell surface lipophosphoglycan found on all Leishmania species and macrophage receptors. This interaction is mediated by phosphoglycan repeats containing oligomers of beta (1-3)Gal residues specific to L. major. We show here that although amastigotes also use lipophosphoglycan to bind to both primary macrophages and a cell line, this interaction is independent of the beta (1-3)Gal residues employed by promastigotes. Binding of amastigotes to macrophages could be blocked by intact lipophosphoglycan from L. major amastigotes as well as by lipophosphoglycan from promastigotes of several other Leishmania species, suggesting involvement of a conserved domain. Binding of amastigotes to macrophages could be blocked significantly by the monoclonal antibody WIC 108.3, directed to the lipophosphoglycan backbone. The glycan core of lipophosphoglycan could also inhibit attachment of amastigotes, but to a considerably lesser extent. The glycan core structure is also present in the type 2 glycoinositolphospholipids which are expressed on the surface of amastigotes at 100-fold-higher levels than lipophosphoglycan. However, their inhibitory effect could not be increased even when they were used at a 300-fold-higher concentration than lipophosphoglycan, indicating that lipophosphoglycan is the major macrophage-binding molecule on amastigotes of L. major. In the presence of complement, the attachment of amastigotes to macrophages was not altered, suggesting that lipophosphoglycan interacts directly with macrophage receptors. PMID:7806383

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

    PubMed Central

    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. PMID:26230675

  13. 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].

  14. 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].

  15. 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

  16. Identification of geographically distributed sub-populations of Leishmania (Leishmania) major by microsatellite analysis

    PubMed Central

    2008-01-01

    Background Leishmania (Leishmania) major, one of the agents causing cutaneous leishmaniasis (CL) in humans, is widely distributed in the Old World where different species of wild rodent and phlebotomine sand fly serve as animal reservoir hosts and vectors, respectively. Despite this, strains of L. (L.) major isolated from many different sources over many years have proved to be relatively uniform. To investigate the population structure of the species highly polymorphic microsatellite markers were employed for greater discrimination among it's otherwise closely related strains, an approach applied successfully to other species of Leishmania. Results Multilocus Microsatellite Typing (MLMT) based on 10 different microsatellite markers was applied to 106 strains of L. (L.) major from different regions where it is endemic. On applying a Bayesian model-based approach, three main populations were identified, corresponding to three separate geographical regions: Central Asia (CA); the Middle East (ME); and Africa (AF). This was congruent with phylogenetic reconstructions based on genetic distances. Re-analysis separated each of the populations into two sub-populations. The two African sub-populations did not correlate well with strains' geographical origin. Strains falling into the sub-populations CA and ME did mostly group according to their place of isolation although some anomalies were seen, probably, owing to human migration. Conclusion The model- and distance-based analyses of the microsatellite data exposed three main populations of L. (L.) major, Central Asia, the Middle East and Africa, each of which separated into two sub-populations. This probably correlates with the different species of rodent host. PMID:18577226

  17. 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. PMID:25815025

  18. 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

  19. Immunoregulatory pathways in murine leishmaniasis: different regulatory control during Leishmania mexicana mexicana and Leishmania major infections.

    PubMed Central

    Alexander, J; Kaye, P M

    1985-01-01

    The effect of whole body sublethal gamma irradiation on the subsequent growth of Leishmania mexicana mexicana and Leishmania major was studied in CBA/Ca and BALB/c mice. Whereas BALB/c mice are highly susceptible to both parasites developing non healing progressively growing lesions at the site of cutaneous infection, CBA/Ca mice develop small healing cutaneous ulcers following subcutaneous infection with L. major but non healing lesions following subcutaneous infection with L.m. mexicana. Prior whole body sublethal irradiation of CBA/Ca mice, but not BALB/c mice, resulted in strong resistance against infection with L.m. mexicana: no lesions developed at the site of cutaneous infection. Irradiated BALB/c mice did, however, develop small lesions which healed when infected with L. major. The protective effects of irradiation coincided with the development of delayed type hypersensitivity. Both naive and sensitized nylon wool purified lymphocytes could restore susceptibility to L. major in irradiated BALB/c mice but only lymphocytes from long term infected donor mice adoptively transferred a non healing response to irradiated CBA/Ca mice infected with L.m. mexicana. Non-irradiated, L. major infected, CBA/Ca mice, but not similarly treated BALB/c mice, were found to be resistant to subsequent infection with L.m. mexicana. On the other hand, irradiated BALB/c mice infected with L. major were resistant to subsequent infectious challenge with L.m. mexicana. We suggest that the susceptibility of CBA/Ca mice to L.m. mexicana is under the control of an as yet unidentified gene which is not dependent on the generation of T suppressor cells and is bypassed by previous infection with L. major. Therefore, BALB/c mice immunized against L. major by prior sublethal irradiation are also resistant to L.m. mexicana. PMID:3907906

  20. Synthetic fragments of antigenic lipophosphoglycans from Leishmania major and Leishmania mexicana and their use for characterisation of the Leishmania elongating alpha-D-mannopyranosylphosphate transferase.

    PubMed

    Higson, Adrian P; Ross, Andrew J; Tsvetkov, Yury E; Routier, Françoise H; Sizova, Olga V; Ferguson, Michael A J; Nikolaev, Andrei V

    2005-03-18

    The phosphorylated branched heptasaccharides 7 and 8, the octasaccharide 9 and the phosphorylated trisaccharides 5 and 6, which are fragments of the phosphoglycan portion of the surface lipophosphoglycans from Leishmania mexicana (5) or L. major (6-9), were synthesised by using the glycosyl hydrogenphosphonate method for the preparation of phosphodiester bridges. The compounds were tested as acceptor substrates/putative inhibitors for the Leishmania elongating alpha-D-mannosylphosphate transferase. PMID:15685582

  1. Tamoxifen Induces Apoptosis of Leishmania major Promastigotes in Vitro

    PubMed Central

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

    2016-01-01

    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. PMID:26951973

  2. Aurapten, a coumarin with growth inhibition against Leishmania major promastigotes.

    PubMed

    Napolitano, H B; Silva, M; Ellena, J; Rodrigues, B D G; Almeida, A L C; Vieira, P C; Oliva, G; Thiemann, O H

    2004-12-01

    Several natural compounds have been identified for the treatment of leishmaniasis. Among them are some alkaloids, chalcones, lactones, tetralones, and saponins. The new compound reported here, 7-geranyloxycoumarin, called aurapten, belongs to the chemical class of the coumarins and has a molecular weight of 298.37. The compound was extracted from the Rutaceae species Esenbeckia febrifuga and was purified from a hexane extract starting from 407.7 g of dried leaves and followed by four silica gel chromatographic fractionation steps using different solvents as the mobile phase. The resulting compound (47 mg) of shows significant growth inhibition with an LD50 of 30 microM against the tropical parasite Leishmania major, which causes severe clinical manifestations in humans and is endemic in the tropical and subtropical regions. In the present study, we investigated the atomic structure of aurapten in order to determine the existence of common structural motifs that might be related to other coumarins and potentially to other identified inhibitors of Leishmania growth and viability. This compound has a comparable inhibitory activity of other isolated molecules. The aurapten is a planar molecule constituted of an aromatic system with electron delocalization. A hydrophobic side chain consisting of ten carbon atoms with two double bonds and negative density has been identified and may be relevant for further compound synthesis. PMID:15558191

  3. Leishmania major and Trypanosoma cruzi present distinct DNA damage responses.

    PubMed

    Garcia, Juliana B F; Rocha, João P Vieira da; Costa-Silva, Héllida M; Alves, Ceres L; Machado, Carlos R; Cruz, Angela K

    2016-05-01

    Leishmania major and Trypanosoma cruzi are medically relevant parasites and interesting model organisms, as they present unique biological processes. Despite increasing data regarding the mechanisms of gene expression regulation, there is little information on how the DNA damage response (DDR) occurs in trypanosomatids. We found that L. major presented a higher radiosensitivity than T. cruzi. L. major showed G1 arrest and displayed high mortality in response to ionizing radiation as a result of the inefficient repair of double-strand breaks (DSBs). Conversely, T. cruzi exhibited arrest in the S/G2 cell cycle phase, was able to efficiently repair DSBs and did not display high rates of cell death after exposure to gamma irradiation. L. major showed higher resistance to alkylating DNA damage, and only L. major was able to promote DNA repair and growth recovery in the presence of MMS. ASF1c overexpression did not interfere with the efficiency of DNA repair in either of the parasites but did accentuate the DNA damage checkpoint response, thereby delaying cell fate after damage. The observed differences in the DNA damage responses of T. cruzi and L. major may originate from the distinct preferred routes of genetic plasticity of the two parasites, i.e., DNA recombination versus amplification. PMID:27188657

  4. 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.

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

    PubMed

    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

  6. [Spread of Leishmania major to the north of Algeria].

    PubMed

    Boudrissa, A; Cherif, K; Kherrachi, I; Benbetka, S; Bouiba, L; Boubidi, S C; Benikhlef, R; Arrar, L; Hamrioui, B; Harrat, Z

    2012-02-01

    Since a long time, Leishmania major and L. infantum foci in Algeria were geographically separated by the mountains of the Tell Atlas which represent a natural barrier. Recently, a new focus of cutaneous leishmaniasis (CL) has emerged in the village of El M'hir, located on the north side of the chain of the Tell Atlas, in the basin of the Soummam. During the period 2004-2010, 152 CL cases have been registered and 12 isolates were obtained from patients who declared never having been outside the village the last years. The identification of the parasites showed that all strains belonged to L major MON-25. Investigations on the reservoir hosts showed the presence of the sand rat (Psammomys obesus), for the first time, in this locality. Five strains isolated from this rodent belonged to L. major MON-25. The sand rat, which is usually observed around the chotts in the Saharan and steppe areas, acts as the main reservoir of L. major in Algeria. Its presence in the new focus of El M'hir is reported for the first time. Entomological surveys carried out in 2009 showed the predominance of two sandfly species: Phlebotomus papatasi and P. perniciosus. The first one is known as a vector of L major in the Algerian Sahara. This study highlights the spread of L. major from the arid zones towards the semi arid areas, particularly in the Soummam valley. Climate changes and desertification observed in the steppe area northern Sahara could play a role in the extension of the disease. PMID:22170408

  7. The Multiple Forms of Leishmania major in BALB/C Mice Lung in Iran

    PubMed Central

    Shirbazou, SH; Jafari, M

    2012-01-01

    Cutaneous leishmaniasis is one of the most important parasitic diseases, which are endemic in different parts of Iran. Leishmania major and L. tropica are the primary causative agents of this disease. The aim of the present study was to detect the multiple forms of L. major in lung. Ppromastigotes of L. major at stationary phase were injected to BALB/c mice. After 60 days, the different forms of Leishmania parasites were checked in lung tissue. Promastigote and amastigote forms of Leishmania parasites were detected. PMID:23109953

  8. 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

  9. Exploring biochemical and functional features of Leishmania major phosphoenolpyruvate carboxykinase.

    PubMed

    Sosa, Máximo Hernán; Giordana, Lucila; Nowicki, Cristina

    2015-10-01

    This work reports the first functional characterization of leishmanial PEPCK. The recombinant Leishmania major enzyme (Lmj_PEPCK) exhibits equivalent kcat values for the phosphoenolpyruvate (PEP) and oxaloacetate (OAA) forming reactions. The apparent Km towards OAA is 10-fold lower than that for PEP, while the Km values for ADP and ATP are equivalent. Mutagenesis studies showed that D241, D242 and H205 of Lmj_PEPCK like the homologous residues of all known PEPCKs are implicated in metal ions binding. In contrast, the replacement of R43 for Q nearly abolishes Lmj_PEPCK activity. Moreover, the Y180F variant exhibits unchanged Km values for PEP, Mn(2+), and [Formula: see text] , being the kcat for PEP- but not that for OAA-forming reaction more notably decreased. Instead, the Y180A mutant displays an increase in the Km value towards Mn(2+). Therefore in Lmj_PEPCK, Y180 seems to exert different functions to those of the analogous residue in ATP- and GTP-dependant enzymes. Besides, the guanidinium group of R43 appears to play an essential but yet unknown role. These findings promote the need for further structural studies to disclose whether Y180 and R43 participate in the catalytic mechanism or/and in the transitions between the open and the catalytically competent (closed) forms of Lmj_PEPCK. PMID:26271440

  10. 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. PMID:26976272

  11. Structural and Kinetic Characterization of Thymidine Kinase from Leishmania major

    PubMed Central

    Recio, Eliseo; Nettleship, Joanne E.; Rada, Heather; González-Pacanowska, Dolores; Wilson, Keith S.

    2015-01-01

    Leishmania spp. is a protozoan parasite and the causative agent of leishmaniasis. Thymidine kinase (TK) catalyses the transfer of the γ-phosphate of ATP to 2’-deoxythymidine (dThd) forming thymidine monophosphate (dTMP). L. major Type II TK (LmTK) has been previously shown to be important for infectivity of the parasite and therefore has potential as a drug target for anti-leishmanial therapy. In this study, we determined the enzymatic properties and the 3D structures of holo forms of the enzyme. LmTK efficiently phosphorylates dThd and dUrd and has high structural homology to TKs from other species. However, it significantly differs in its kinetic properties from Trypanosoma brucei TK since purines are not substrates of the enzyme and dNTPs such as dUTP inhibit LmTK. The enzyme had Km and kcat values for dThd of 1.1 μM and 2.62 s-1 and exhibits cooperative binding for ATP. Additionally, we show that the anti-retroviral prodrug zidovudine (3-azido-3-deoxythymidine, AZT) and 5’-modified dUrd can be readily phosphorylated by LmTK. The production of recombinant enzyme at a level suitable for structural studies was achieved by the construction of C-terminal truncated versions of the enzyme and the use of a baculoviral expression system. The structures of the catalytic core of LmTK in complex with dThd, the negative feedback regulator dTTP and the bi-substrate analogue AP5dT, were determined to 2.74, 3.00 and 2.40 Å, respectively, and provide the structural basis for exclusion of purines and dNTP inhibition. The results will aid the process of rational drug design with LmTK as a potential target for anti-leishmanial drugs. PMID:25978379

  12. 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

  13. Leishmania major MAP kinase 10 is protective against experimental L. major infection.

    PubMed

    Kumari, Sangeeta; Singh, Sushma; Saha, Bhaskar; Paliwal, Piyush Kumar

    2011-11-01

    Leishmania, a protozoan parasite that resides and replicates obligatorily within macrophages, inflicts a complex of severe diseases known as leishmaniasis. The diseases have significant socio-economic impact through gross disfiguration, morbidity and mortality worldwide. Despite these problems, an effective anti-leishmanial vaccine remains elusive. Herein, we have analyzed the immunogenicity and protective efficacy of L. major MAP kinase 10 (LmjMAPK10) against the challenge infection with the parasite. We observe significant protection against the infection by LmjMAPK10 priming of BALB/c mouse strain, a susceptible host. The resistance to the infection is generally associated with mixed Th1/Th2 responses to the infection following immunization with LmjMAPK10 DNA or protein or a combination of both DNA and protein. Therefore, LmjMAPK10 is a probable vaccine candidate against the infection. PMID:21527301

  14. 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. PMID:23467583

  15. The Route of Leishmania tropica Infection Determines Disease Outcome and Protection against Leishmania major in BALB/c Mice

    PubMed Central

    Khalili, Ghader; Abrishami, Firoozeh; Najafy, Ali; Khaze, Vahid

    2013-01-01

    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. PMID:23467583

  16. Antileishmanial activity of licochalcone A in mice infected with Leishmania major and in hamsters infected with Leishmania donovani.

    PubMed Central

    Chen, M; Christensen, S B; Theander, T G; Kharazmi, A

    1994-01-01

    This study was designed to examine the antileishmanial activity of the oxygenated chalcone licochalcone A in mice and hamsters infected with Leishmania parasites. Intraperitoneal administration of licochalcone A at doses of 2.5 and 5 mg/kg of body weight per day completely prevented lesion development in BALB/c mice infected with Leishmania major. Treatment of hamsters infected with L. donovani with intraperitoneal administration of licochalcone A at a dose of 20 mg/kg of body weight per day for 6 consecutive days resulted in a > 96% reduction of parasite load in the liver and the spleen compared with values for untreated control animals. The [3H]thymidine uptake by the parasites isolated from the treated hamsters was only about 1% of that observed in parasites isolated from the controls. Oral administration of licochalcone A at concentrations of 5 to 150 mg/kg of body weight per day for 6 consecutive days resulted in > 65 and 85% reductions of L. donovani parasite loads in the liver and the spleen, respectively, compared with those of untreated control hamsters. These data clearly demonstrate that licochalcone A is a promising lead for the development of a new drug against leishmaniases. PMID:8092835

  17. The development of Leishmania turanica in sand flies and competition with L. major

    PubMed Central

    2012-01-01

    Background In Central Asian foci of zoonotic cutaneous leishmaniases, mixed infections of Leishmania turanica and L. major have been found in a reservoir host (the great gerbil, Rhombomys opimus) as well as in the sand fly vector Phlebotomus papatasi, but hybrids between these two Leishmania species have never been reported. In addition, the role of sand fly species other than P. papatasi in L. turanica circulation is not clear. Methods In this work we compared the development of L. turanica in three sand fly species belonging to different subgenera. In addition, we studied experimental co-infections of sand flies by both Leishmania species using GFP transfected L. turanica (MRHO/MN/08/BZ18(GFP+)) and RFP transfected L. major (WHOM/IR/-/173-DsRED(RFP+)). The possibility of Leishmania genetic exchange during the vectorial part of the life cycle was studied using flow cytometry combined with immunofluorescent microscopy. Results Late-stage infections of L. turanica with frequent colonization of the stomodeal valve were observed in the specific vector P. (Phlebotomus) papatasi and in the permissive vector P. (Adlerius) arabicus. On the other hand, in P. sergenti (the specific vector of L. tropica), L. turanica promatigotes were present only until the defecation of bloodmeal remnants. In their natural vector P. papatasi, L. turanica and L. major developed similarly, and the spatiotemporal dynamics of localization in the sand fly gut was the same for both leishmania species. Fluorescence microscopy in combination with FACS analyses did not detect any L. major / L. turanica hybrids in the experimental co-infection of P. papatasi and P. duboscqi. Conclusion Our data provide new insight into the development of different leishmania parasite species during a mixed infection in the sand fly gut. Despite the fact that both Leishmania species developed well in P. papatasi and P. duboscqi and did not outcompete each other, no genetic exchange was found. However, the ability of L

  18. Immune responses in DNA vaccine formulated with PMMA following immunization and after challenge with Leishmania major.

    PubMed

    Zarrati, Somayeh; Mahdavi, Mehdi; Tabatabaie, Fatemeh

    2016-06-01

    Leishmaniasis is a major infectious disease caused by protozoan parasites of the genus Leishmania. Despite of many efforts toward vaccine against Leishmania no effective vaccine has been approved yet. DNA vaccines can generate more powerful and broad immune responses than conventional vaccines. In order to increase immunity, the DNA vaccine has been supplemented with adjuvant. In this study a new nano-vaccine containing TSA recombinant plasmid and poly(methylmethacrylate) nanoparticles (act as adjuvant) was designed and its immunogenicity tested on BALB/c mouse. After three intramuscular injection of nano-vaccine (100 μg), the recombinant TSA protein (20 μg) was injected subcutaneously. Finally as a challenge animals were infected by Leishmania major. After the last injection of nano-vaccine, after protein booster injection, and also after challenge, cellular immune and antibody responses were evaluated by ELISA method. The findings of this study showed the new nano-vaccine was capable of induction both cytokines secretion and specific antibody responses, but predominant Th1 immune response characterized by IFN-γ production compared to control groups. Moreover, results revealed that nano-vaccine was effective in reducing parasite burden in the spleen of Leishmania major-infected BALB/c mice. Base on results, current candidate vaccine has potency for further studies. PMID:27413316

  19. 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

  20. 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. PMID:26871576

  1. First Detection of Leishmania major DNA in Sergentomyia (Spelaeomyia) darlingi from Cutaneous Leishmaniasis Foci in Mali

    PubMed Central

    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

    Background 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. Methodology/Principal Findings 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. Conclusion Our data suggest the possible involvement of P. duboscqi and potentially S. darlingi in the transmission of ZCL in Mali. PMID:22276095

  2. Leishmania major Phosphoglycans Influence the Host Early Immune Response by Modulating Dendritic Cell Functions▿

    PubMed Central

    Liu, Dong; Kebaier, Chahnaz; Pakpour, Nazzy; Capul, Althea A.; Beverley, Stephen M.; Scott, Phillip; Uzonna, Jude E.

    2009-01-01

    The precise role of Leishmania glycoconjugate molecules including phosphoglycans (PGs) and lipophosphoglycan (LPG) on host cellular responses is still poorly defined. Here, we investigated the interaction of Leishmania major LPG2 null mutant (lpg2−), which lacks both PGs and LPG, with dendritic cells (DCs) and the subsequent early immune response in infected mice. Surprisingly, the absence of phosphoglycans did not influence expression pattern of major histocompatibility complex class II (MHC II), CD40, CD80, and CD86 on DCs in vitro and in vivo. However, lpg2− L. major induced significantly higher production of interleukin-12p40 (IL-12p40) by infected bone marrow-derived DCs (BMDCs) than wild-type (WT) parasites in vitro. Furthermore, the production of IL-12p40 by draining lymph node cells from lpg2− mutant-infected mice was higher than those from WT L. major-infected mice. In model antigen presentation experiments, DCs from lpg2− mutant-infected mice induced more gamma interferon (IFN-γ) and IL-2 production by Leishmania-specific T cells than those from WT-infected mice. Lymphocytes isolated from mice infected for 3 days with lpg2− parasites produce similar levels of IFN-γ, but significantly less IL-4 and IL-10 than WT controls. Decreased IL-4 production was also seen in another general PG-deficient mutant lacking the Golgi UDP-galactose transporters (lpg5A− lpg5B−), but not with the lpg1− mutant lacking only LPG, thereby implicating PGs generally in the reduction of IL-4 production. Thus, Leishmania PGs influence host early immune response by modulating DC functions in a way that inhibits antigen presentation and promotes early IL-4 response, and their absence may impact the balance between Th1 and Th2 responses. PMID:19487470

  3. Effect of Aqueous Allium cepa and Ixora brachiata Root Extract on Leishmania major Promastigotes

    PubMed Central

    Sadeghi-Nejad, Batool; Saki, Jasem

    2014-01-01

    Background: Leishmaniasis is a major worldwide public health problem with about two to three million humans threatened by this disease annually. Allium cepa (onion) is an important dietary vegetable and was used as a herbal medicine for centuries. The root of Ixora brachiata is medicinally important. Objectives: The aim of this study was to investigate the anti-Leishmania effect of the ethanolic and methanolic extracts of Ixora brachiata root and aqueous onion extracts on Leishmania major promastigotes. Patients and Methods: The parasites isolated from cutaneous leishmaniasis were exposed with different concentrations of selected plant extracts and their inhibitory effects on the promastigotes were evaluated after 24 and 48 hours. Results: Among tested plant extracts, Ixora brachiata root extracts revealed the best activity against Leishmania major promastigotes with IC100 value of 2.5 mg/mL and IC50 value of 0.078 mg/mL. Conclusions: This study showed that aqueous Allium cepa and Ixora brachiata root extracts as natural products could be used as alternative drugs in treatment of leishmaniasis. PMID:24872942

  4. Two different dihydroorotate dehydrogenases in Lactococcus lactis.

    PubMed Central

    Andersen, P S; Jansen, P J; Hammer, K

    1994-01-01

    The pyrimidine de novo biosynthesis pathway has been characterized for a number of organisms. The general pathway consists of six enzymatic steps. In the characterization of the pyrimidine pathway of Lactococcus lactis, two different pyrD genes encoding dihydroorotate dehydrogenase were isolated. The nucleotide sequences of the two genes, pyrDa and pyrDb, have been determined. One of the deduced amino acid sequences has a high degree of homology to the Saccharomyces cerevisiae dihydroorotate dehydrogenase, and the other resembles the dihydroorotate dehydrogenase from Bacillus subtilis. It is possible to distinguish between the two enzymes in crude extracts by using different electron acceptors. We constructed mutants containing a mutated form of either one or the other or both of the pyrD genes. Only the double mutant is pyrimidine auxotrophic. Images PMID:8021180

  5. Leishmania major MPK7 Protein Kinase Activity Inhibits Intracellular Growth of the Pathogenic Amastigote Stage ▿

    PubMed Central

    Morales, Miguel A.; Pescher, Pascale; Späth, Gerald F.

    2010-01-01

    During the infectious cycle, protozoan parasites of the genus Leishmania undergo several adaptive differentiation steps that are induced by environmental factors and crucial for parasite infectivity. Genetic analyses of signaling proteins underlying Leishmania stage differentiation are often rendered difficult due to lethal null mutant phenotypes. Here we used a transgenic strategy to gain insight into the functions of the mitogen-activated Leishmania major protein kinases LmaMPK7 and LmaMPK10 in parasite virulence. We established L. major and Leishmania donovani lines expressing episomal green fluorescent protein (GFP)-LmaMPK7 and GFP-LmaMPK10 fusion proteins. The transgenic lines were normal in promastigote morphology, growth, and the ability to differentiate into metacyclic and amastigote stages. While parasites expressing GFP-LmaMPK10 showed normal infectivity by mouse footpad analysis and macrophage infection assays, GFP-LmaMPK7 transgenic parasites displayed a strong delay in lesion formation and reduced intracellular parasite growth. Significantly, the effects of GFP-LmaMPK7 on virulence and proliferation were due exclusively to protein kinase activity, as the overexpression of two kinase-dead mutants had no effect on parasite infectivity. GFP-LmaMPK7 transgenic L. donovani cells revealed a reversible, stage-specific growth defect in axenic amastigotes that was independent of cell death but linked to nonsynchronous growth arrest and a significant reduction of de novo protein biosynthesis. Our data suggest that LmaMPK7 protein kinase activity may be implicated in parasite growth control and thus relevant for the development of nonproliferating stages during the infectious cycle. PMID:19801421

  6. THE MAJOR SURFACE PROTEASE (MSP OR GP63) IN THE INTRACELLULAR AMASTIGOTE STAGE OF LEISHMANIA CHAGASI

    PubMed Central

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

    2009-01-01

    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 and MSPL RNAs, and two forms of MSPC RNA, 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 amastigote and promastigotes both express multiple MSP isoforms, but these MSPs differ biochemically and localize differently between the parasite stages. We hypothesize that MSP plays different roles in the extracellular versus intracellular forms of Leishmania spp. PMID:18067978

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

    PubMed Central

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

    2015-01-01

    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. PMID

  8. FISH analysis reveals aneuploidy and continual generation of chromosomal mosaicism in Leishmania major.

    PubMed

    Sterkers, Yvon; Lachaud, Laurence; Crobu, Lucien; Bastien, Patrick; Pagès, Michel

    2011-02-01

    The protozoan parasite Leishmania is generally considered to be diploid, although a few chromosomes have been described as aneuploid. Using fluorescence in situ hybridization (FISH), we determined the number of homologous chromosomes per individual cell in L. major (i) during interphase and (ii) during mitosis. We show that, in Leishmania, aneuploidy appears to be the rule, as it affects all the chromosomes that we studied. Moreover, every chromosome was observed in at least two ploidy states, among monosomic, disomic or trisomic, in the cell population. This variable chromosomal ploidy among individual cells generates intra-strain heterogeneity, here precisely chromosomal mosaicism. We also show that this mosaicism, hence chromosome ploidy distribution, is variable among clones and strains. Finally, when we examined dividing nuclei, we found a surprisingly high rate of asymmetric chromosome allotments, showing that the transmission of genetic material during mitosis is highly unstable in this 'divergent' eukaryote: this leads to continual generation of chromosomal mosaicism. Using these results, we propose a model for the occurrence and persistence of this mosaicism. We discuss the implications of this additional unique feature of Leishmania for its biology and genetics, in particular as a novel genetic mechanism to generate phenotypic variability from genomic plasticity. PMID:20964798

  9. 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. PMID:24699087

  10. 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. PMID:26542948

  11. Genome-wide gene expression profiling analysis of Leishmania major and Leishmania infantum developmental stages reveals substantial differences between the two species

    PubMed Central

    Rochette, Annie; Raymond, Frédéric; Ubeda, Jean-Michel; Smith, Martin; Messier, Nadine; Boisvert, Sébastien; Rigault, Philippe; Corbeil, Jacques; Ouellette, Marc; Papadopoulou, Barbara

    2008-01-01

    Background Leishmania parasites cause a diverse spectrum of diseases in humans ranging from spontaneously healing skin lesions (e.g., L. major) to life-threatening visceral diseases (e.g., L. infantum). The high conservation in gene content and genome organization between Leishmania major and Leishmania infantum contrasts their distinct pathophysiologies, suggesting that highly regulated hierarchical and temporal changes in gene expression may be involved. Results We used a multispecies DNA oligonucleotide microarray to compare whole-genome expression patterns of promastigote (sandfly vector) and amastigote (mammalian macrophages) developmental stages between L. major and L. infantum. Seven per cent of the total L. infantum genome and 9.3% of the L. major genome were differentially expressed at the RNA level throughout development. The main variations were found in genes involved in metabolism, cellular organization and biogenesis, transport and genes encoding unknown function. Remarkably, this comparative global interspecies analysis demonstrated that only 10–12% of the differentially expressed genes were common to L. major and L. infantum. Differentially expressed genes are randomly distributed across chromosomes further supporting a posttranscriptional control, which is likely to involve a variety of 3'UTR elements. Conclusion This study highlighted substantial differences in gene expression patterns between L. major and L. infantum. These important species-specific differences in stage-regulated gene expression may contribute to the disease tropism that distinguishes L. major from L. infantum. PMID:18510761

  12. In vitro activity of sulfonamides and sulfones against Leishmania major promastigotes.

    PubMed Central

    Peixoto, M P; Beverley, S M

    1987-01-01

    We examined the susceptibility of promastigotes of Leishmania major to sulfonamides and sulfones in vitro. In a completely defined medium only sulfamoxole, sulfaquinoxaline, and dapsone were inhibitory; the concentrations required for 50% inhibition of the rate of growth were 150, 600, and 600 microM, respectively. Eleven other sulfa drugs were ineffective at concentrations up to 2 mM. The growth inhibition was similar to that observed in procaryotes: the cells continued logarithmic growth for several cell doublings before inhibition was observed. Surprisingly, the addition of p-aminobenzoate or folate did not reverse the effects of the active sulfa drugs, the effects of sulfamoxole and methotrexate were additive rather than synergistic, and the addition of thymidine reversed methotrexate but not sulfa-drug inhibition. These results suggest that the mode of action of sulfa drugs on L. major is not by the classical route of inhibition of de novo folate synthesis. Promastigotes could be propagated for more than 40 passages in a completely defined medium in which the only added pterin was biopterin. The folate concentration in this medium was less than 10(-10) to 10(-11) M, as determined by a Leishmania bioassay. Although these data suggest that L. major may be capable of de novo synthesis of folate, the nonclassical mode of action of sulfa drugs, as well as other studies, favors the view that L. major is auxotrophic for folate. PMID:3435106

  13. 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

  14. Overexpression of a single Leishmania major gene enhances parasite infectivity in vivo and in vitro

    PubMed Central

    Reiling, Linda; Chrobak, Mareike; Schmetz, Christel; Clos, Joachim

    2010-01-01

    We identified a Leishmania major-specific gene that can partly compensate for the loss of virulence observed for L. major HSP100 null mutants. The gene, encoding a 46 kD protein of unknown function and lineage, also enhances the virulence of wild type L. major upon overexpression. Surprisingly, the approximately sixfold overexpression of this protein also extends the host range of L. major to normally resistant C57BL/6 mice, causing persisting lesions in this strain, even while eliciting a strong cellular immune response. This enhanced virulence in vivo is mirrored in vitro by increased parasite burden inside bone marrow-derived macrophages. The localization of the protein in the macrophage cytoplasm suggests that it may modulate the macrophage effector mechanisms. In summary, our data show that even minor changes of gene expression in L. major may alter the outcome of an infection, regardless of the host's genetic predisposition. PMID:20345655

  15. Imported cutaneous leishmaniasis caused by Leishmania major in a Chinese laborer who worked in Saudi Arabia*

    PubMed Central

    Zhang, Min; Liu, Fang; Liu, Haibo; Hu, Wenxing; Sang, Hong

    2016-01-01

    We report an imported case of cutaneous leishmaniasis in a 37-year-old man from Saudi Arabia caused by Leishmania major. He presented with non-healing nodulo-ulcerative lesions with a "volcanic crater" on the lower limbs. It was clearly cutaneous leishmaniasis - a rare disease in China - as reflected by the patient's clinical history, the lesions' morphology, histopathological examination, culture and PCR analysis of the lesions. The patient was completely cured after two cycles of sodium stibogluconate treatment. This case report demonstrates that dermatologists should be aware of sporadic cutaneous leishmaniasis cases in non-endemic areas. PMID:27438208

  16. Imported cutaneous leishmaniasis caused by Leishmania major in a Chinese laborer who worked in Saudi Arabia.

    PubMed

    Zhang, Min; Liu, Fang; Liu, Haibo; Hu, Wenxing; Sang, Hong

    2016-01-01

    We report an imported case of cutaneous leishmaniasis in a 37-year-old man from Saudi Arabia caused by Leishmania major. He presented with non-healing nodulo-ulcerative lesions with a "volcanic crater" on the lower limbs. It was clearly cutaneous leishmaniasis - a rare disease in China - as reflected by the patient's clinical history, the lesions' morphology, histopathological examination, culture and PCR analysis of the lesions. The patient was completely cured after two cycles of sodium stibogluconate treatment. This case report demonstrates that dermatologists should be aware of sporadic cutaneous leishmaniasis cases in non-endemic areas. PMID:27438208

  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. Leishmania tarentolae secreting the sand fly salivary antigen PpSP15 confers protection against Leishmania major infection in a susceptible BALB/c mice model.

    PubMed

    Katebi, A; Gholami, E; Taheri, T; Zahedifard, F; Habibzadeh, S; Taslimi, Y; Shokri, F; Papadopoulou, B; Kamhawi, S; Valenzuela, J G; Rafati, S

    2015-10-01

    Cutaneous leishmaniasis is a zoonotic, vector-borne disease causing a major health problem in several countries. No vaccine is available and there are limitations associated with the current therapeutic regimens. Immune responses to sand fly saliva have been shown to protect against Leishmania infection. A cellular immune response to PpSP15, a protein from the sand fly Phlebotomus papatasi, was sufficient to control Leishmania major infection in mice. This work presents data supporting the vaccine potency of recombinant live non-pathogenic Leishmania (L.) tarentolae secreting PpSP15 in mice and its potential as a new vaccine strategy against L. major. We generated a recombinant L. tarentolae-PpSP15 strain delivered in the presence of CpG ODN and evaluated its immunogenicity and protective immunity against L. major infection in BALB/c mice. In parallel, different vaccination modalities using PpSP15 as the target antigen were compared. Humoral and cellular immune responses were evaluated before and at three and eight weeks after challenge. Footpad swelling and parasite load were assessed at eight and eleven weeks post-challenge. Our results show that vaccination with L. tarentolae-PpSP15 in combination with CpG as a prime-boost modality confers strong protection against L. major infection that was superior to other vaccination modalities used in this study. This approach represents a novel and promising vaccination strategy against Old World cutaneous leishmaniasis. PMID:26298575

  19. Regulated expression of the Leishmania major surface virulence factor lipophosphoglycan using conditionally destabilized fusion proteins

    PubMed Central

    Madeira da Silva, Luciana; Owens, Katherine L.; Murta, Silvane M. F.; Beverley, Stephen M.

    2009-01-01

    Surface glycoconjugates play important roles in the infectious cycle of Leishmania major, including the abundant lipophosphoglycan (LPG) implicated in parasite survival in the sand fly vector and the initial stages of establishment in the mammalian host macrophage. We describe a system for inducible expression of LPG, applying a novel protein-based system that allows controlled degradation of a key LPG biosynthetic enzyme, UDP-galactopyranose mutase (UGM). This methodology relies on a mutated FK506-binding protein (FKBP) destabilizing domain (dd) fused to the protein of interest; in the absence of rapamycin analogs, such as Shld1, the dd domain is destabilized, leading to proteasomal degradation, whereas drug treatment confers stabilization. Tests in L. major using dd fusions to a panel of reporters and cellular proteins confirmed its functionality, with a high degree of regulation and low background, and we established the kinetics of protein activation and/or loss. Two inexpensive and widely available ligands, FK506 and rapamycin, functioned similarly to Shld1, without effect on Leishmania growth or differentiation. We generated parasites lacking UGM through deletion of the GLF gene and substitution with a ddGLF fusion construct, either as chromosomal knockins or through episomal complementation; these showed little or no LPG expression in the absence of inducer, whereas in its presence, high levels of LPG were attained rapidly. Complement lysis tests confirmed the correct integrity of the Leishmania LPG coat. These data suggest that the dd approach has great promise in the study of LPG and other pathways relevant to parasite survival and virulence. PMID:19383793

  20. Rhesus monkey model for Leishmania major transmitted by Phlebotomus papatasi sandfly bites.

    PubMed

    Probst, R J; Wellde, B T; Lawyer, P G; Stiteler, J S; Rowton, E D

    2001-03-01

    Leishmaniasis research needs a near-human model for investigations of natural infection processes, immunological responses and evaluation of treatments. Therefore, we developed a reproducible system using Leishmania major Yakimoff & Schokhor (Trypanosomatidae: Kinetoplastida), the cause of Old World zoonotic cutaneous leishmaniasis (ZCL), transmitted to rhesus monkeys Macaca mulatta (Zimmerman) (Primates: Cercopithecidae) by sandfly bites of experimentally infected Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae). Eight monkeys of presumed Indian origin (Leishmania naive) were exposed to bites of female sandflies that had been infected with L. major by membrane-feeding on human blood seeded with amastigotes isolated from hamster footpad lesions. Infection rates of membrane-fed sandflies averaged > 85% seven days after the infective feed, with uniformly high numbers of promastigotes in the stomodaeal valve region of the sandfly gut. Nodules and ulcerating dermal lesions developed on 7/8 monkeys 2-4 weeks post-bite and persisted for 3-7 months. Monkeys also developed satellite lesions beyond the area of sandfly bites on the head, but not on the chest. Three re-challenged monkeys developed lesions that healed faster than lesions from their primary challenges. After infection, monkeys developed delayed type hypersensitivity (DTH) responses to a panel of Leishmania skin test antigens (LSTA) and, when tested by ELISA and IFA, showed significant post-infection antibody titres which typically rose for approximately 170 days and then gradually receded during the next 100 days following the first challenge. After the second challenge, antibody titres spiked higher within approximately 50 days and receded more rapidly. In contrast, four rhesus macaques of Chinese origin developed no lesions following infected sandfly bites, although they raised antibodies and LSTA reactions, indicating subclinical infection. PMID:11297097

  1. 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

  2. Hypoxia in Leishmania major skin lesions impairs the NO-dependent leishmanicidal activity of macrophages.

    PubMed

    Mahnke, Alexander; Meier, Robert J; Schatz, Valentin; Hofmann, Julian; Castiglione, Kirstin; Schleicher, Ulrike; Wolfbeis, Otto S; Bogdan, Christian; Jantsch, Jonathan

    2014-09-01

    Cure of infections with Leishmania major is critically dependent on the ability of macrophages to induce the type 2 nitic oxide (NO) synthase (NOS2) that produces high levels of NO in the presence of ample oxygen. Therefore, we analyzed the oxygen levels found in leishmanial skin lesions and their effect on the NOS2-dependent leishmanicidal activity of macrophages (MΦ). When L. major skin lesions of self-healing C57BL/6 mice reached their maximum size, the infected tissue displayed low oxygen levels (pO2∼21 Torr). MΦ activated under these oxygen tensions failed to produce sufficient amounts of NO to clear L. major. Nos2-deficient and hypoxic wild-type macrophages displayed a similar phenotype. Killing was restored when MΦ were reoxygenated or exposed to a NO donor. The resolution of the lesion in C57BL/6 mice was paralleled by an increase of lesional pO2. When mice were kept under normobaric hypoxia, this caused a persistent suppression of the lesional pO2 and a concurrent increase of the parasite load. In Nos2-deficient mice, there was no effect of atmospheric hypoxia. Low oxygen levels found at leishmanial skin lesions impaired the NOS2-dependent leishmanicidal activity of MΦ. Hence, tissue oxygenation represents an underestimated local milieu factor that participates in the persistence of Leishmania. PMID:24583949

  3. Targeting Leishmania major parasite with peptides derived from a combinatorial phage display library.

    PubMed

    Rhaiem, Rafik Ben; Houimel, Mehdi

    2016-07-01

    Cutaneous leishmaniasis (CL) is a global problem caused by intracellular protozoan pathogens of the genus Leishmania for which there are no suitable vaccine or chemotherapy options. Thus, de novo identification of small molecules binding to the Leishmania parasites by direct screening is a promising and appropriate alternative strategy for the development of new drugs. In this study, we used a random linear hexapeptide library fused to the gene III protein of M13 filamentous bacteriophage to select binding peptides to metacyclic promastigotes from a highly virulent strain of Leishmania major (Zymodeme MON-25; MHOM/TN/94/GLC94). After four rounds of stringent selection and amplification, polyclonal and monoclonal phage-peptides directed against L. major metacyclic promastigotes were assessed by ELISA, and the optimal phage-peptides were grown individually and characterized for binding to L. major by monoclonal phage ELISA. The DNA of 42 phage-peptides clones was amplified by PCR, sequenced, and their amino acid sequences deduced. Six different peptide sequences were obtained with frequencies of occurrence ranging from 2.3% to 85.7%. The biological effect of the peptides was assessed in vitro on human monocytes infected with L. major metacyclic promastigotes, and in vivo on susceptible parasite-infected BALB/c mice. The development of cutaneous lesions in the right hind footpads of infected mice after 13 weeks post-infection showed a protection rate of 81.94% with the injected peptide P2. Moreover, Western blots revealed that the P2 peptide interacted with the major surface protease gp63, a protein of 63kDa molecular weight. Moreover, bioinformatics were used to predict the interaction between peptides and the major surface molecule of the L. major. The molecular docking showed that the P2 peptide has the minimum interaction energy and maximum shape complimentarity with the L. major gp63 active site. Our study demonstrated that the P2 peptide occurs at high frequency

  4. Genetic micro-heterogeneity of Leishmania major in emerging foci of zoonotic cutaneous leishmaniasis in Tunisia.

    PubMed

    Attia, Hanene; Sghaier, Rabiaa M; Gelanew, Tesfaye; Bali, Aymen; Schweynoch, Carola; Guerfali, Fatma Z; Mkannez, Ghada; Chlif, Sadok; Belhaj-Hamida, Nabil; Dellagi, Koussay; Schönian, Gabriele; Laouini, Dhafer

    2016-09-01

    Tunisia is endemic for zoonotic cutaneous leishmaniasis (ZCL), a parasitic disease caused by Leishmania (L.) major. ZCL displays a wide clinical polymorphism, with severe forms present more frequently in emerging foci where naive populations are dominant. In this study, we applied the multi-locus microsatellite typing (MLMT) using ten highly informative and discriminative markers to investigate the genetic structure of 35 Tunisian Leishmania (L.) major isolates collected from patients living in five different foci of Central Tunisia (two old and three emerging foci). Phylogenetic reconstructions based on genetic distances showed that nine of the ten tested loci were homogeneous in all isolates with homozygous alleles, whereas one locus (71AT) had a 58/64-bp bi-allelic profile with an allele linked to emerging foci. Promastigote-stage parasites with the 58-bp allele tend to be more resistant to in vitro complement lysis. These results, which stress the geographical dependence of the genetic micro-heterogeneity, may improve our understanding of the ZCL epidemiology and clinical outcome. PMID:27137082

  5. 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.

  6. 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. PMID:21225450

  7. 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

  8. Identification of a macrophage-binding determinant on lipophosphoglycan from Leishmania major promastigotes.

    PubMed

    Kelleher, M; Bacic, A; Handman, E

    1992-01-01

    Leishmania are obligatory intracellular parasites in mammalian macrophages that gain entry by receptor-mediated phagocytosis. Their major cell surface glycoconjugate, lipophosphoglycan (LPG), has been implicated in this process. A monoclonal antibody specific for Leishmania major LPG (WIC 79.3), which has been shown to block promastigote attachment to macrophages, was used to identify a macrophage-binding determinant of LPG. WIC 79.3 bound exclusively to the phosphorylated repeats of LPG and not to the saccharide core or lipid anchor. Furthermore, the epitope recognized by WIC 79.3 mapped to the phosphorylated oligosaccharide P5b, PO4-6[Gal(beta 1-3)Gal(beta 1-3)Gal(beta 1-3)]Gal(beta 1-4)Man(alpha 1-, which is unique to the LPG of promastigotes of L.major. Phosphorylated oligosaccharides P3, PO4-6[Gal(beta 1-3)[Gal(beta 1-4) Man(alpha 1-, and P4b, PO4-6[Gal(beta 1-3)Gal(beta 1-3)] Gal(beta 1-4)Man(alpha 1-, were also recognized by WIC 79.3 but with considerably lower (approximately 100-fold) affinities. The phosphorylated oligosaccharide P5b inhibited attachment of promastigotes of L. major to the macrophage cell line J774 to the same degree as phosphoglycan (derived from LPG) and Fab fragments of WIC 79.3, suggesting that P5b is a site of L. major LPG that is recognized by macrophage receptor(s) and is an important determinant in the attachment of promastigotes to host macrophages and initiation of infection. PMID:1370357

  9. Transcriptionally Driven DNA Replication Program of the Human Parasite Leishmania major.

    PubMed

    Lombraña, Rodrigo; Álvarez, Alba; Fernández-Justel, José Miguel; Almeida, Ricardo; Poza-Carrión, César; Gomes, Fábia; Calzada, Arturo; Requena, José María; Gómez, María

    2016-08-01

    Faithful inheritance of eukaryotic genomes requires the orchestrated activation of multiple DNA replication origins (ORIs). Although origin firing is mechanistically conserved, how origins are specified and selected for activation varies across different model systems. Here, we provide a complete analysis of the nucleosomal landscape and replication program of the human parasite Leishmania major, building on a better evolutionary understanding of replication organization in Eukarya. We found that active transcription is a driving force for the nucleosomal organization of the L. major genome and that both the spatial and the temporal program of DNA replication can be explained as associated to RNA polymerase kinetics. This simple scenario likely provides flexibility and robustness to deal with the environmental changes that impose alterations in the genetic programs during parasitic life cycle stages. Our findings also suggest that coupling replication initiation to transcription elongation could be an ancient solution used by eukaryotic cells for origin maintenance. PMID:27477279

  10. Leishmania major Telomerase TERT Protein Has a Nuclear/Mitochondrial Eclipsed Distribution That Is Affected by Oxidative Stress

    PubMed Central

    Campelo, Riward; Díaz Lozano, Isabel; Figarella, Katherine; Osuna, Antonio

    2014-01-01

    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. PMID:25312950

  11. Antigenic specificity of the 72-kilodalton major surface glycoprotein of Leishmania braziliensis braziliensis.

    PubMed Central

    Kutner, S; Pellerin, P; Breniere, S F; Desjeux, P; Dedet, J P

    1991-01-01

    We examined the expression and the antigenicity of the major surface polypeptides of Leishmania braziliensis braziliensis and Leishmania donovani chagasi, parasites which commonly coexist in the same endemic areas of Bolivia. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles from surface-iodinated promastigotes showed the presence of a unique iodinatable polypeptide of 72 kDa on the L. b. braziliensis surface and of two major components of 65 and 50 kDa exposed at the surface of L. d. chagasi. Comparison of the peptide digestion profiles of the major iodinated polypeptides of both strains showed no similarity between the maps of the 72- and the 65-kDa polypeptides of L. b. braziliensis and L. d. chagasi, respectively. Immunoprecipitation of surface-labeled L. b. braziliensis Nonidet P-40 extracts with 35 serum specimens obtained from Bolivian patients with cutaneous and mucocutaneous leishmaniasis showed that all serum specimens recognized predominantly the 72-kDa antigen and high-molecular-mass proteins in some cases. The recognition patterns were independent of the geographical origin of the patient, the type of lesion, and the serum antibody titer. Serum specimens from children with visceral leishmaniasis did not precipitate the L. b. braziliensis 72-kDa antigen. Hamster hyperimmune serum against L. b. braziliensis also recognized the 72-kDa surface antigen. However, this recognition was inhibited in the presence of the homologous nonlabeled antigen but not in the presence of heterologous (L. d. chagasi and Trypanosoma cruzi) antigens. The specific recognition of 72-kDa surface antigen in both natural and experimental L. b. braziliensis infections suggests that this antigen could be a good candidate for use in the differential immunodiagnosis and prognosis of the disease. Images PMID:2037677

  12. Differences in Lsh gene control over systemic Leishmania major and Leishmania donovani or Leishmania mexicana mexicana infections are caused by differential targeting to infiltrating and resident liver macrophage populations.

    PubMed Central

    Davies, E V; Singleton, A M; Blackwell, J M

    1988-01-01

    Earlier studies had shown that the viscerotropic NIH 173 strain of cutaneous Leishmania major fails to come under Lsh gene control. Visceral Leishmania donovani LV9 and another viscerotropic cutaneous strain, Leishmania mexicana mexicana LV4, are controlled by Lsh. The results of double-infection experiments presented here show that expression of Lsh resistance against L. mexicana mexicana was enhanced in the presence of L. donovani, whereas L. major still failed to come under Lsh gene control, even in the presence of L. donovani. Prior irradiation (850 rads) of mice showed that in the absence of infiltrating monocytes, Lsh did exert some influence over L. major. The presence of a higher infiltrate of fresh monocytes after L. major infection was confirmed in liver macrophage populations isolated from mice after infection in vivo and in liver cryosections immunostained with monoclonal antibody M1/70 directed against the type 3 complement receptor CR3. The results support the hypothesis that Lsh is expressed maximally in the resident tissue macrophages and poorly in the immature macrophages preferentially infected by L. major amastigotes. Images PMID:3356462

  13. Leishmania major possesses a unique HemG-type protoporphyrinogen IX oxidase

    PubMed Central

    Zwerschke, Dagmar; Karrie, Simone; Jahn, Dieter; Jahn, Martina

    2014-01-01

    Leishmania major was proposed to either utilize haem from its host or partially synthesize the tetrapyrrole from host provided precursors. However, only indirect evidence was available for this partial late haem biosynthetic pathway. Here, we demonstrate that the LMJF_06_1280 gene of L. major encodes a HemG-type PPO (protoporphyrinogen IX oxidase) catalysing the oxidation of protoporphyrinogen IX to protoporphyrin IX. Interestingly, trypanosomatids are currently the only known eukaryotes possessing HemG-type enzymes. The LMJF_06_1280 gene forms a potential transcriptional unit with LMJF_06_1270 encoding CPO (coproporphyrinogen III oxidase) and with LMJF_06_1290 for a cytochrome b5. In vivo function of the L. major hemG gene was shown by the functional complementation of the Escherichia coli ΔhemG strain LG285. Restored haem formation in E. coli was observed using HPLC analyses. Purified recombinant L. major HemG revealed PPO activity in vitro using different ubiquinones and triphenyltetrazolium as electron acceptors. FMN was identified as the L. major HemG cofactor. Active site residues were found to be essential for HemG catalysis. These data in combination with the solved crystal structures of L. major CPO and the physiological proof of a ferrochelatase activity provide clear-cut evidence for a partial haem biosynthetic pathway in L. major. PMID:24962471

  14. 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.

  15. 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.

  16. 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

  17. Study of Leishmania major-infected macrophages by use of lipophosphoglycan-specific monoclonal antibodies.

    PubMed

    Handman, E

    1990-07-01

    Leishmania major infection of macrophages is followed by a time-dependent appearance of lipophosphoglycan (LPG) that can be detected on the surface of infected cells by monoclonal antibodies. The origin of these LPG epitopes is probably the intracellular amastigote. LPG epitopes could be detected on the amastigote and the infected macrophage by a number of monoclonal antibodies directed to several distinct determinants on the phosphoglycan moiety. The macrophage-expressed LPG may be modified because, unlike the parasite LPG as expressed on promastigotes or amastigotes, it could not be radiolabeled by galactose oxidase or periodate treatment of infected cells followed by reduction with 3H-labeled sodium borohydride. Some LPG epitopes displayed on the macrophage may be anchored with glycosylphosphatidylinositol, and some may be in the water-soluble phosphoglycan form bound to macrophage integrins involved in its specific recognition. The water-soluble population could be released from the infected macrophage by gentle protease treatment. PMID:1694823

  18. Two functionally divergent UDP-Gal nucleotide sugar transporters participate in phosphoglycan synthesis in Leishmania major.

    PubMed

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

    2007-05-11

    In the protozoan parasite Leishmania, abundant surface and secreted molecules, such as lipophosphoglycan (LPG) and proteophosphoglycans (PPGs), contain extensive galactose in the form of phosphoglycans (PGs) based on (Gal-Man-PO(4)) 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. Data base mining revealed 12 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 knock-out studies established that two genes (LPG5A and LPG5B) encoded UDP-Gal NSTs. Although 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, since the lpg5A(-) mutant showed a partial defect in LPG but not PPG phosphoglycosylation, whereas 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

  19. 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

  20. Blockade of CD86 ameliorates Leishmania major infection by down-regulating the Th2 response.

    PubMed

    Brown, J A; Titus, R G; Nabavi, N; Glimcher, L H

    1996-12-01

    The costimulatory molecules CD80 and CD86 affect the differentiation of Th1 and Th2 subsets in experimental allergic encephalomyelitis, an autoimmune disorder. It is reported that the CD86 costimulator significantly affects disease outcome in Leishmania major infection, a classic model of Th subset polarization. Treatment of both L. major-resistant (C57BL/6) and susceptible (BALB/c) strains of mice with anti-CD86 substantially decreased parasite burden. This was accompanied, in BALB/c mice, by a decrease in Th2 cytokines. In contrast, anti-CD80 treatment did not affect parasite burden or cytokine levels in either strain. These data illustrate that in L. major infection, anti-CD86 can abrogate Th2 differentiation in a Th2-dominated susceptible mouse and can ameliorate disease in a Th1-dominated resistant strain, although the mechanism involved in the latter is not clear. It is concluded that in L. major infection, Th2 subset differentiation is critically dependent on interaction with the CD86 costimulatory molecule. PMID:8940222

  1. Identification of the defect in lipophosphoglycan biosynthesis in a non-pathogenic strain of Leishmania major.

    PubMed

    McConville, M J; Homans, S W

    1992-03-25

    The major macromolecule on the surface of the protozoan parasite, Leishmania major, is a complex lipophosphoglycan (LPG), which is anchored to the plasma membrane by an inositol-containing phospholipid. A defect in LPG biosynthesis is thought to be responsible for the avirulence of the L. major strain LRC L119 in mice. In order to identify the nature of this defect we have characterized two truncated forms of LPG, which are accumulated in this strain, by one- and two-dimensional 500-MHz 1H NMR spectroscopy, two-dimensional heteronuclear 1H-31P NMR spectroscopy, methylation analysis, and exoglycosidase digestions. The structures of these glycoinositolphospholipids, termed GIPL-4 and -6, are as follows: [formula: see text] The glycan moieties of GIPL-4 and -6 are identical to the anchor region of LPG, which is also substituted with a Glc-1-PO4 residue in approximately 60% of the structures. However, instead of being capped with chains of phosphorylated oligosaccharide repeat units, both glycan moieties terminate in Man alpha 1-PO4, suggesting that the defect in LPG biosynthesis is in the transfer of galactose to this residue to form the disaccharide backbone of the first repeat unit. These results indicate that the phosphoglycan moiety of LPG is essential for intracellular survival of the parasite and have implications for LPG biosynthesis. PMID:1532574

  2. Metabolic, immune, and gut microbial signals mount a systems response to Leishmania major infection.

    PubMed

    Lamour, Sabrina D; Veselkov, Kirill A; Posma, Joram M; Giraud, Emilie; Rogers, Matthew E; Croft, Simon; Marchesi, Julian R; Holmes, Elaine; Seifert, Karin; Saric, Jasmina

    2015-01-01

    Parasitic infections such as leishmaniasis induce a cascade of host physiological responses, including metabolic and immunological changes. Infection with Leishmania major parasites causes cutaneous leishmaniasis in humans, a neglected tropical disease that is difficult to manage. To understand the determinants of pathology, we studied L. major infection in two mouse models: the self-healing C57BL/6 strain and the nonhealing BALB/c strain. Metabolic profiling of urine, plasma, and feces via proton NMR spectroscopy was performed to discover parasite-specific imprints on global host metabolism. Plasma cytokine status and fecal microbiome were also characterized as additional metrics of the host response to infection. Results demonstrated differences in glucose and lipid metabolism, distinctive immunological phenotypes, and shifts in microbial composition between the two models. We present a novel approach to integrate such metrics using correlation network analyses, whereby self-healing mice demonstrated an orchestrated interaction between the biological measures shortly after infection. In contrast, the response observed in nonhealing mice was delayed and fragmented. Our study suggests that trans-system communication across host metabolism, the innate immune system, and gut microbiome is key for a successful host response to L. major and provides a new concept, potentially translatable to other diseases. PMID:25369177

  3. Intravenous injection of irradiated Leishmania major into susceptible BALB/c mice: immunization or protective tolerance.

    PubMed

    Aebischer, T; Morris, L; Handman, E

    1994-10-01

    It is well established that BALB/c mice can be protected from fatal infection with Leishmania major by prophylactic intravenous (i.v.) immunization with irradiated parasites. Protection is critically dependent on the route of injection with i.v. injection being protective and subcutaneous injection not protective. We used this BALB/c-L. major model system to investigate this phenomenon. We analyzed quantitatively the parasite-specific, CD4+ T cell mediated immune responses by limiting dilution. Subcutaneous vaccination resulted in priming of CD4+ precursor T cells, whereas i.v. vaccination was ineffectual. Moreover, i.v. injection prevented the increase in the number of specific precursor cells induced by infection of normal mice during the first weeks post-challenge with virulent parasites. We show here that this was not due to the elimination of the virulent challenge parasites as a result of immunity nor to inefficient antigen presentation of the irradiated organisms after i.v. injection. The data presented here suggest that i.v. injection results in tolerization rather than immunization. Tolerization as a mechanism of host protection is consistent with earlier observations that transient immunosuppression results in cure of L. major infection in BALB/c mice. Transfer of antigen presenting cells (APC) isolated from spleens of mice injected previously with irradiated parasites mimicked to some extent the effect of i.v. immunization with irradiated parasites. The possible involvement of these APC in decreasing the parasite-specific T cell response is discussed. PMID:7826944

  4. 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

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

    PubMed

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

    2016-06-01

    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

  6. Novel arylalkylamine compounds exhibits potent selective antiparasitic activity against Leishmania major

    PubMed Central

    Iniguez, Eva A.; Perez, Andrea; Maldonado, Rosa A.; Skouta, Rachid

    2015-01-01

    Leishmania major (L. major) is a protozoan parasite causal agent of Leishmaniasis. It is estimated that 12 million people are currently infected and around 2 million infections occur each year. Current treatments suffer of high toxicity for the patient, low efficacy toward the parasite, high cost, and are losing effectiveness due to parasite resistance. Discovering novel small molecule with high specificity/selectivity and drug-like properties for anti-leishmanial activity remains a significant challenge. The purpose of this study is to communicate the design and synthesis strategies of novel chemical compounds based of the arylalkylamine scaffold with selective toxicity towards L. major and less toxicity to human cells in vitro. Here, we have developed a structure activity relationship (SAR) study of arylalkylamine AA1 in order to study their anti-parasitic effect in L. major. Overall, 27 arylalkylamine compounds derived from AA1 were synthesized and purified by silica gel column chromatography. The purity of each analog was confirmed by spectroscopic methods (1H, 13C NMR and LC/MS). Among these analogs, the compound AA9 showed the best toxic activity on L. major (LD50 = 3.34 μM), which represents a 9 fold higher lethality as compared with its parental AA1 (Fer-1) compound (LD50 = 28.75 μM). In addition, AA9 showed no significant toxicity at 80 μM on U20S Human Osteoblasts, Raw 264.7 Macrophages or intraperitoneal macrophages. In summary, our combined SAR study and biological evaluation data of AA1-AA27 compounds allow the identification of novel arylalkylamine compound AA9 that exhibits potent cytotoxicity against L. major promastigote with minimum toxic effect on human cells. PMID:26410073

  7. 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

  8. Expression of a stage-specific lipophosphoglycan in Leishmania major amastigotes.

    PubMed

    Turco, S J; Sacks, D L

    1991-03-01

    Amastigotes of Leishmania major were isolated from infected mice and radiolabeled for 2 h with [3H]galactose. An acidic [3H]glycoconjugate was extracted from a dilipidated residue fraction with the solvent water/ethanol/diethylether/pyridine/NH4OH (15:15:5:1:0.017). The radioactivity labeled glycoconjugate was found to possess the following characteristics that were similar to the lipophosphoglycan extractable from promastigotes: (i) migrated as a broad band upon electrophoresis on SDS polyacrylamide gels; (ii) deaminated with nitrous acid; and (iii) hydrolyzed with phosphatidylinositol-specific phospholipase C. Furthermore, analysis of the aqueous soluble material released by the latter enzyme revealed a negatively-charged [3H]polysaccharide intermediate in size compared to the analogous portions of LPG isolated from non-infective and metacyclic promastigotes. Most importantly, the [3H]polysaccharide was found to contain phosphate and was susceptible to mild acid hydrolysis, establishing that the intact molecule is a lipophosphoglycan. A structural difference, however, was found in the major, mild acid-generated fragment of the amastigote phosphoglycan, which was larger in size and not as anionic as the analogous fragment from the promastigote phosphoglycans. These results indicate that the amastigotes do express a lipophosphoglycan, but that it is structurally distinct from its promastigote counterparts. PMID:1646960

  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. Parameter estimation and sensitivity analysis in an agent-based model of Leishmania major infection

    PubMed Central

    Jones, Douglas E.; Dorman, Karin S.

    2009-01-01

    Computer models of disease take a systems biology approach toward understanding host-pathogen interactions. In particular, data driven computer model calibration is the basis for inference of immunological and pathogen parameters, assessment of model validity, and comparison between alternative models of immune or pathogen behavior. In this paper we describe the calibration and analysis of an agent-based model of Leishmania major infection. A model of macrophage loss following uptake of necrotic tissue is proposed to explain macrophage depletion following peak infection. Using Gaussian processes to approximate the computer code, we perform a sensitivity analysis to identify important parameters and to characterize their influence on the simulated infection. The analysis indicates that increasing growth rate can favor or suppress pathogen loads, depending on the infection stage and the pathogen’s ability to avoid detection. Subsequent calibration of the model against previously published biological observations suggests that L. major has a relatively slow growth rate and can replicate for an extended period of time before damaging the host cell. PMID:19837088

  11. 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

  12. Deletion of the Aryl Hydrocarbon Receptor Enhances the Inflammatory Response to Leishmania major Infection

    PubMed Central

    Elizondo, Guillermo; Rodríguez-Sosa, Miriam; Estrada-Muñiz, Elizabet; Gonzalez, Frank J.; Vega, Libia

    2011-01-01

    The aryl hydrocarbon receptor (AhR) is a ligand-activated receptor that mediates the toxicity of environmental pollutants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Recently, it has been shown that the AhR plays a role in immune and inflammatory regulation. However, most of these studies are based on the activation of AhR by exogenous ligands. Therefore, in the present study, we addressed the role of this transcription factor, in the absent of exogenous ligand, on the immune response to Leishmania major infection. Our results indicate that inactivation of the AhR results in an alteration of the levels of several cytokines. Lymph node cells from infected Ahr-null animals displayed an increase in IFNγ and IL-12 levels, together with a decrease in IL-4 and IL-10 levels compared to wild-type (wt) mice. Ahr-null mice also presented higher serum levels of the pro-inflammatory cytokine TNF-α prior to parasite inoculation and during infection compared to wt mice. Moreover, a 30% decrease in the population of Treg cells was observed in Ahr-null mice. This decrease was associated with a reduction in Foxp3 mRNA levels. Finally, the alteration in the cytokine profile results in a better resolution of the L. major infection. PMID:22110376

  13. An Essential Type I Nitroreductase from Leishmania major Can Be Used to Activate Leishmanicidal Prodrugs

    PubMed Central

    Voak, Andrew A.; Gobalakrishnapillai, Vithurshaa; Seifert, Karin; Balczo, Edina; Hu, Longqin; Hall, Belinda S.; Wilkinson, Shane R.

    2013-01-01

    Nitroaromatic prodrugs are used to treat a range of microbial infections with selectivity achieved by specific activation reactions. For trypanosomatid parasites, this is mediated by type I nitroreductases. Here, we demonstrate that the causative agent of leishmaniasis, Leishmania major, expresses an FMN-containing nitroreductase (LmNTR) that metabolizes a wide range of substrates, and based on electron donor and acceptor preferences, it may function as an NADH:quinone oxidoreductase. Using gene deletion approaches, we demonstrate that this activity is essential to L. major promastigotes, the parasite forms found in the insect vector. Intriguingly, LmNTR+/− heterozygote promastigote parasites could readily differentiate into infectious metacyclic cells but these were unable to establish infections in cultured mammalian cells and caused delayed pathology in mice. Furthermore, we exploit the LmNTR activity evaluating a library of nitrobenzylphosphoramide mustards using biochemical and phenotypic screens. We identify a subset of compounds that display significant growth inhibitory properties against the intracellular parasite form found in the mammalian hosts. The leishmanicidal activity was shown to be LmNTR-specific as the LmNTR+/− heterozygote promastigotes displayed resistance to the most potent mustards. We conclude that LmNTR can be targeted for drug development by exploiting its prodrug activating property or by designing specific inhibitors to block its endogenous function. PMID:23946481

  14. 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. PMID:19635872

  15. Genome-Wide Prediction of Vaccine Candidates for Leishmania major: An Integrated Approach

    PubMed Central

    Singh, Satarudra Prakash; Roopendra, Kriti; Mishra, Bhartendu Nath

    2015-01-01

    Despite the wealth of information regarding genetics of the causative parasite and experimental immunology of the cutaneous leishmaniasis, there is currently no licensed vaccine against it. In the current study, a two-level data mining strategy was employed, to screen the Leishmania major genome for promising vaccine candidates. First, we screened a set of 25 potential antigens from 8312 protein coding sequences, based on presence of signal peptides, GPI anchors, and consensus antigenicity predictions. Second, we conducted a comprehensive immunogenic analysis of the 25 antigens based on epitopes predicted by NetCTL tool. Interestingly, results revealed that candidate antigen number 1 (LmjF.03.0550) had greater number of potential T cell epitopes, as compared to five well-characterized control antigens (CSP-Plasmodium falciparum, M1 and NP-Influenza A virus, core protein-Hepatitis B virus, and PSTA1-Mycobacterium tuberculosis). In order to determine an optimal set of epitopes among the highest scoring predicted epitopes, the OptiTope tool was employed for populations susceptible to cutaneous leishmaniasis. The epitope (127SLWSLLAGV) from antigen number 1, found to bind with the most prevalent allele HLA-A⁎0201 (25% frequency in Southwest Asia), was predicted as most immunogenic for all the target populations. Thus, our study reasserts the potential of genome-wide screening of pathogen antigens and epitopes, for identification of promising vaccine candidates. PMID:26681959

  16. 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

  17. 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

  18. 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

  19. Leishmania tropica major in mice: vaccination against cutaneous leishmaniasis in mice of high genetic susceptibility.

    PubMed

    Mitchell, G F; Handman, E

    1983-02-01

    BALB/c and BALB/c.H-2b mice are genetically susceptible to development of persistent and severe disease following cutaneous injection of promastigotes of the protozoan parasite, Leishmania tropica major, whereas C57BL/6 are relatively resistant. Resistance in C57BL/6 can be further increased by intraperitoneal injection of living, but not killed, promastigotes prior to cutaneous challenge. Severely diseased BALB/c mice can show resistance to development of a second cutaneous lesion but apparently only in the advanced stages of systemic life-threatening disease. A striking level of resistance to persistent disease has been demonstrated in BALB/c.H-2b mice pre-injected with frozen and thawed L. t. major-infected macrophages of the continuous macrophage cell line IC-21 (H-2b) together with Corynebacterium parvum. No resistance is seen in recipients of either C. parvum or the crude antigen mixture alone. Protection is afforded by intraperitoneal and not subcutaneous injection of crude antigen plus adjuvant. In these vaccination studies all evidence points to the infected macrophage as most appropriate source of 'host-protective' antigens as well as being the most likely target of host-protective immunity. Resistance is expressed in vaccinated mice as minimal signs of cutaneous disease and rapid resolution of any small lesions which do develop. Frozen and thawed promastigotes plus C. parvum will not induce resistance to persistent disease in BALB/c.H-2b mice and preincubation of promastigotes with sera from resistant vaccinated mice does not influence their capacity to cause cutaneous disease. The results provide baseline data for vaccination attempts in genetically susceptible hosts using isolated L. t. major antigens (and, in particular, infected macrophage antigens) and highlight the utility of the intraperitoneal route of injection and the use of the therapeutic biological, C. parvum, as an adjuvant in such studies. PMID:6870673

  20. Evaluation of recombinant Leishmania poly-protein plus GLA-SE vaccines against sand fly-transmitted Leishmania major in C57Bl/6 mice1

    PubMed Central

    Peters, Nathan C.; Bertholet, Sylvie; Lawyer, Phillip G.; Charmoy, Melanie; Romano, Audrey; Ribeiro-Gomes, Flavia L.; Stamper, Lisa W.; Sacks, David L.

    2012-01-01

    Numerous experimental Leishmania vaccines have been developed to prevent the visceral and cutaneous forms of Leishmaniasis, which occur after exposure to the bite of an infected sand fly, yet only one is under evaluation in humans. KSAC and L110f, recombinant Leishmania poly-proteins delivered in a stable emulsion (SE) with the TLR 4 agonists monophosphoryl lipid A (MPL) or glucopyranosyl lipid A (GLA) have shown protection in animal models. KSAC+GLA-SE protected against cutaneous disease following sand fly transmission of L. major in susceptible BALB/c mice. Similar poly-protein adjuvant combinations are the vaccine candidates most likely to see clinical evaluation. We assessed immunity generated by KSAC or L110f vaccination with GLA-SE following challenge with L. major by needle or infected sand fly bite in resistant C57BL/6 mice. Poly-protein vaccinated mice had a 60-fold increase in CD4+IFN-γ+ T cell numbers versus control animals at 2 weeks post needle inoculation of L. major and this correlated with a 100-fold reduction in parasite load. Immunity did not, however, reach levels observed in mice with a healed primary infection. Following challenge by infected sand fly bite, poly-protein vaccinated animals had comparable parasite loads, greater numbers of neutrophils at the challenge site, and reduced CD4+ IFN-γ+:IL-17+ ratios versus non-vaccinated controls. In contrast, healed animals had significantly reduced parasite loads and higher CD4+ IFN-γ+:IL-17+ ratios. These observations demonstrate that vaccine-induced protection against needle challenge does not necessarily translate to protection following challenge by infected sand fly bite. PMID:23045616

  1. Developmental modification of lipophosphoglycan during the differentiation of Leishmania major promastigotes to an infectious stage.

    PubMed

    McConville, M J; Turco, S J; Ferguson, M A; Sacks, D L

    1992-10-01

    Protozoan parasites of the genus Leishmania produce the novel surface glycoconjugate, lipophosphoglycan (LPG), which is required for parasite infectivity. In this study we show that LPG structure is modified during the differentiation of L. major promastigotes from a less infectious form in logarithmic growth phase to a highly infectious 'metacyclic' form during stationary growth phase. In both stages, the LPGs comprise linear chains of phosphorylated oligosaccharide repeat units which are anchored to the membrane via a glycosyl-phosphatidylinositol glycolipid anchor. During metacyclogenesis there is (i) an approximate doubling in the average number of repeat units per molecule from 14 to 30, (ii) a pronounced decrease in the relative abundance of repeat units with side chains of beta Gal or Gal beta 1-3Gal beta 1-, and a corresponding increase in repeat units with either no side chains or with side chains of Arap alpha 1-2 Gal beta 1- and (iii) a decrease in the frequency with which the glycolipid anchor is substituted with a single glucose alpha 1-phosphate residue. While the majority of the LPG phosphoglycan chains are capped with the neutral disaccharide, Man alpha 1-2Man, a significant minority of the chains appeared to terminate in non-phosphorylated repeat units and may represent incompletely capped species. We suggest that the developmental modification of LPG may be important in modulating the binding of promastigotes to receptors in the sandfly midgut and on human macrophages and in increasing the resistance of metacyclic promastigotes to complement-mediated lysis. PMID:1396559

  2. Mortality of Leishmania major in Phlebotomus papatasi caused by plant feeding of the sand flies.

    PubMed

    Schlein, Y; Jacobson, R L

    1994-01-01

    The plant feeding of Phlebotomus papatasi and the effects of plant diets on the Leishmania major infections were investigated. Plant-fed flies had small free particles and membranous shreds in their gut that were stained by calcofluor as cellulosic plant tissues. They were found in 34.0% of the female and 14.3% of the male sand flies following feeding on the caper plant (Capparis spinosa). No plant residues were found in 54 females that had been fed on plant-derived honeydew secretions of Aphis craccivora offered on a branch of the host plant. Calcofluor-stained particles were also absent from the gut lumen of unfed flies. The proportion of sugar feeding, regardless of the intake of plant tissue, in the series that had been offered caper plant or honeydew was estimated by testing for the presence of fructose in the gut. The proportion of fructose-positive flies in each series, among both males and females, was 45%. Plant feeding in the field was demonstrated by finding tissue residues in the gut of 32.8% of female and 10.3% of male P. papatasi from the Jordan Valley. Feeding on specific plants was demonstrated using baits of branches suffused with food dye and finding the dye marker in wild-caught P. papatasi. The influence of plant diets on L. major infections in P. papatasi was as follows: Malva nicaeensis and the honeydew of Icerya purchasi produced thriving parasitemias; however, feeding on Ricinus communis, Capparis spinosa, and Solanum luteum caused > 50% mortality and deformation of parasites in 88%, 55%, and 46% of the infections, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8304569

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

    PubMed

    Masic, Anita; Valencia Hernandez, Ana Maria; 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

  4. 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

  5. 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

  6. The role of alanine 163 in solute permeability of Leishmania major aquaglyceroporin LmAQP1.

    PubMed

    Mukhopadhyay, Rita; Mandal, Goutam; Atluri, Venkata Subba Rao; Figarella, Katherine; Uzcategui, Nestor L; Zhou, Yao; Beitz, Eric; Ajees, A Abdul; Bhattacharjee, Hiranmoy

    2011-01-01

    Leishmania major aquaglyceroporin LmAQP1 allows adventitious passage of antimonite, an activated form of the drug Pentostam, which is used as the first line treatment for leishmaniasis. The extracellular C-loop of an aquaglyceroporin confers substrate specificity. Alteration of Glu125 to serine in the Plasmodium falciparum aquaglyceroporin PfAQP has been shown to selectively affect water but not glycerol permeability. The C-loop of LmAQP1 is twelve residues longer than PfAQP, and Ala163 is at an equivalent position as Glu125 of PfAQP. The role of Ala163 in LmAQP1 solute permeability was investigated. Alteration of Ala163 to serine or threonine did not significantly affect conduction of solutes. However, alteration to aspartate, glutamate, and glutamine blocked passage of water, glycerol, and other organic solutes. While LmAQP1 is a mercurial insensitive water channel, mutation of the adjacent threonine (Thr164) to cysteine led to inhibition of water passage by Hg(2+). This inhibition could be reversed upon addition of β-mercaptoethanol. These data suggest that, unlike Glu125 (PfAQP), Ala163 is not involved in stabilization of the C-loop and selective solute permeability. Ala163 is located near the pore mouth of the channel, and replacement of Ala163 by bulkier residue sterically hinders the passage of solutes. Alteration of Ala163 to serine or threonine affected metalloid uptake in the order, wild-type>A163S>A163T. Metalloid conduction was near completely blocked when Ala163 was mutagenized to aspartate, glutamate, or glutamine. Mutations such as A163S and A163T that reduced the permeability to antimonite, without a significant loss in water or solute conductivity raises the possibility that, subtle changes in the side chain of the amino acid residue in position 163 of LmAQP1 may play a role in drug resistance. PMID:20888371

  7. Deletion of UDP-glucose pyrophosphorylase reveals a UDP-glucose independent UDP-galactose salvage pathway in Leishmania major

    PubMed Central

    Lamerz, Anne-Christin; Damerow, Sebastian; Kleczka, Barbara; Wiese, Martin; van Zandbergen, Ger; Lamerz, Jens; Wenzel, Alexander; Hsu, Fong-Fu; Turk, John; Beverley, Stephen M.; Routier, Françoise H.

    2010-01-01

    The nucleotide sugar UDP-galactose (UDP-Gal) is essential for the biosynthesis of several abundant glycoconjugates forming the surface glycocalyx of the protozoan parasite Leishmania major. Current data suggest that UDP-Gal could arise de novo by epimerization of UDP-glucose (UDP-Glc) or by a salvage pathway involving phosphorylation of Gal and the action of UDP-glucose:α-d-galactose-1-phosphate uridylyltransferase as described by Leloir. Since both pathways require UDP-Glc, inactivation of the UDP-glucose pyrophosphorylase (UGP) catalyzing activation of glucose-1 phosphate to UDP-Glc was expected to deprive parasites of UDP-Gal required for Leishmania glycocalyx formation. Targeted deletion of the gene encoding UGP, however, only partially affected the synthesis of the Gal-rich phosphoglycans. Moreover, no alteration in the abundant Gal-containing glycoinositolphospholipids was found in the deletion mutant. Consistent with these findings, the virulence of the UGP-deficient mutant was only modestly affected. These data suggest that Leishmania elaborates a UDP-Glc independent salvage pathway for UDP-Gal biosynthesis. PMID:20335578

  8. The role(s) of lipophosphoglycan (LPG) in the establishment of Leishmania major infections in mammalian hosts

    PubMed Central

    Späth, Gerald F.; Garraway, L. A.; Turco, Salvatore J.; Beverley, Stephen M.

    2003-01-01

    The abundant cell surface glycolipid lipophosphoglycan (LPG) was implicated in many steps of the Leishmania infectious cycle by biochemical tests. The presence of other abundant surface or secreted glycoconjugates sharing LPG domains, however, has led to uncertainty about the relative contribution of LPG in vivo. Here we used an Leishmania major lpg1- mutant, which lacks LPG alone and shows attenuated virulence, to dissect the role of LPG in the establishment of macrophage infections in vivo. lpg1- was highly susceptible to human complement, had lost the ability to inhibit phagolysosomal fusion transiently, and was oxidant sensitive. Studies of mouse mutants defective in relevant defense mechanisms confirmed the role of LPG in oxidant resistance but called into question the importance of transient inhibition of phagolysosomal fusion for Leishmania macrophage survival. Moreover, the limited lytic activity of mouse complement appears to be an ineffective pathogen defense mechanism in vitro and in vivo, unlike human hosts. In contrast, lpg1- parasites bound C3b and resisted low pH and proteases normally, entered macrophages efficiently and silently, and continued to inhibit host-signaling pathways. These studies illustrate the value of mechanistic approaches focusing on both parasite and host defense pathways in dissecting the specific biological roles of complex virulence factors such as LPG. PMID:12869694

  9. 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

  10. Localization of major histocompatibility complex class II molecules in phagolysosomes of murine macrophages infected with Leishmania amazonensis.

    PubMed Central

    Antoine, J C; Jouanne, C; Lang, T; Prina, E; de Chastellier, C; Frehel, C

    1991-01-01

    Leishmania-infected macrophages are potential antigen-presenting cells for CD4+ T lymphocytes, which recognize parasite antigens bound to major histocompatibility complex class II molecules (Ia). However, the intracellular sites where Ia and antigens may interact are far from clear, since parasites grow within the modified lysosomal compartment of the host cell, whereas Ia molecules seem to be targeted to endosomes. To address this question, the expression and fate of Ia molecules were studied by immunocytochemistry in Leishmania amazonensis-infected murine macrophages stimulated with gamma interferon. In uninfected macrophages, Ia molecules were localized on the plasma membrane and in perinuclear vesicles, but they underwent a dramatic redistribution after infection, since most of the intracellular staining was then associated with the periphery of the parasitophorous vacuoles (p.v.) and quite often polarized towards amastigote-binding sites. The Ii invariant chain, which is transiently associated with Ia during their intracellular transport, although well expressed in infected macrophages, apparently did not reach the p.v. Similar findings were observed with macrophages from mice either resistant or highly susceptible to Leishmania infection. In order to determine the origin of p.v.-associated Ia, the fate of plasma membrane, endosomal, and lysosomal markers, detected with specific antibodies, was determined after infection. At 48 h after infection, p.v. was found to exhibit a membrane composition typical of mature lysosomes. Overall, these data suggest that (i) Ia located in p.v. originate from secondary lysosomes involved in the biogenesis of this compartment or circulate in several endocytic organelles, including lysosomes and (ii) p.v. could play a role in antigen processing and presentation. Alternatively, the presence of high amounts of Ia in p.v. could be due to a Leishmania-induced mechanism by means of which this organism may evade the immune response

  11. Skin-resident memory CD4+ T cells enhance protection against Leishmania major infection

    PubMed Central

    Glennie, Nelson D.; Yeramilli, Venkata A.; Beiting, Daniel P.; Volk, Susan W.; Weaver, Casey T.

    2015-01-01

    Leishmaniasis causes a significant disease burden worldwide. Although Leishmania-infected patients become refractory to reinfection after disease resolution, effective immune protection has not yet been achieved by human vaccines. Although circulating Leishmania-specific T cells are known to play a critical role in immunity, the role of memory T cells present in peripheral tissues has not been explored. Here, we identify a population of skin-resident Leishmania-specific memory CD4+ T cells. These cells produce IFN-γ and remain resident in the skin when transplanted by skin graft onto naive mice. They function to recruit circulating T cells to the skin in a CXCR3-dependent manner, resulting in better control of the parasites. Our findings are the first to demonstrate that CD4+ TRM cells form in response to a parasitic infection, and indicate that optimal protective immunity to Leishmania, and thus the success of a vaccine, may depend on generating both circulating and skin-resident memory T cells. PMID:26216123

  12. 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

  13. 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

  14. Leishmania major Survival in Selective Phlebotomus papatasi Sand Fly Vector Requires a Specific SCG-Encoded Lipophosphoglycan Galactosylation Pattern

    PubMed Central

    Dobson, Deborah E.; Kamhawi, Shaden; Lawyer, Phillip; Turco, Salvatore J.; Beverley, Stephen M.; Sacks, David L.

    2010-01-01

    Phlebotomine sand flies that transmit the protozoan parasite Leishmania differ greatly in their ability to support different parasite species or strains in the laboratory: while some show considerable selectivity, others are more permissive. In “selective” sand flies, Leishmania binding and survival in the fly midgut typically depends upon the abundant promastigote surface adhesin lipophosphoglycan (LPG), which exhibits species- and strain-specific modifications of the dominant phosphoglycan (PG) repeat units. For the “selective” fly Phlebotomus papatasi PpapJ, side chain galactosyl-modifications (scGal) of PG repeats play key roles in parasite binding. We probed the specificity and properties of this scGal-LPG PAMP (Pathogen Associated Molecular Pattern) through studies of natural isolates exhibiting a wide range of galactosylation patterns, and of a panel of isogenic L. major engineered to express similar scGal-LPG diversity by transfection of SCG-encoded β1,3-galactosyltransferases with different activities. Surprisingly, both ‘poly-scGal’ and ‘null-scGal’ lines survived poorly relative to PpapJ-sympatric L. major FV1 and other ‘mono-scGal’ lines. However, survival of all lines was equivalent in P. duboscqi, which naturally transmit L. major strains bearing ‘null-scGal’-LPG PAMPs. We then asked whether scGal-LPG-mediated interactions were sufficient for PpapJ midgut survival by engineering Leishmania donovani, which normally express unsubstituted LPG, to express a ‘PpapJ-optimal’ scGal-LPG PAMP. Unexpectedly, these “L. major FV1-cloaked” L. donovani-SCG lines remained unable to survive within PpapJ flies. These studies establish that midgut survival of L. major in PpapJ flies is exquisitely sensitive to the scGal-LPG PAMP, requiring a specific ‘mono-scGal’ pattern. However, failure of ‘mono-scGal’ L. donovani-SCG lines to survive in selective PpapJ flies suggests a requirement for an additional, as yet unidentified L

  15. Leishmania major survival in selective Phlebotomus papatasi sand fly vector requires a specific SCG-encoded lipophosphoglycan galactosylation pattern.

    PubMed

    Dobson, Deborah E; Kamhawi, Shaden; Lawyer, Phillip; Turco, Salvatore J; Beverley, Stephen M; Sacks, David L

    2010-01-01

    Phlebotomine sand flies that transmit the protozoan parasite Leishmania differ greatly in their ability to support different parasite species or strains in the laboratory: while some show considerable selectivity, others are more permissive. In "selective" sand flies, Leishmania binding and survival in the fly midgut typically depends upon the abundant promastigote surface adhesin lipophosphoglycan (LPG), which exhibits species- and strain-specific modifications of the dominant phosphoglycan (PG) repeat units. For the "selective" fly Phlebotomus papatasi PpapJ, side chain galactosyl-modifications (scGal) of PG repeats play key roles in parasite binding. We probed the specificity and properties of this scGal-LPG PAMP (Pathogen Associated Molecular Pattern) through studies of natural isolates exhibiting a wide range of galactosylation patterns, and of a panel of isogenic L. major engineered to express similar scGal-LPG diversity by transfection of SCG-encoded β1,3-galactosyltransferases with different activities. Surprisingly, both 'poly-scGal' and 'null-scGal' lines survived poorly relative to PpapJ-sympatric L. major FV1 and other 'mono-scGal' lines. However, survival of all lines was equivalent in P. duboscqi, which naturally transmit L. major strains bearing 'null-scGal'-LPG PAMPs. We then asked whether scGal-LPG-mediated interactions were sufficient for PpapJ midgut survival by engineering Leishmania donovani, which normally express unsubstituted LPG, to express a 'PpapJ-optimal' scGal-LPG PAMP. Unexpectedly, these "L. major FV1-cloaked" L. donovani-SCG lines remained unable to survive within PpapJ flies. These studies establish that midgut survival of L. major in PpapJ flies is exquisitely sensitive to the scGal-LPG PAMP, requiring a specific 'mono-scGal' pattern. However, failure of 'mono-scGal' L. donovani-SCG lines to survive in selective PpapJ flies suggests a requirement for an additional, as yet unidentified L. major-specific parasite factor(s). The

  16. 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

  17. Characterization of the RNA polymerase II and III complexes in Leishmania major.

    PubMed

    Martínez-Calvillo, Santiago; Saxena, Alka; Green, Amanda; Leland, Aaron; Myler, Peter J

    2007-04-01

    Transcription of protein-coding genes in Leishmania major and other trypanosomatids differs from that in most eukaryotes and bioinformatic analyses have failed to identify several components of the RNA polymerase (RNAP) complexes. To increase our knowledge about this basic cellular process, we used tandem affinity purification (TAP) to identify subunits of RNAP II and III. Mass spectrometric analysis of the complexes co-purified with TAP-tagged LmRPB2 (encoded by LmjF31.0160) identified seven RNAP II subunits: RPB1, RPB2, RPB3, RPB5, RPB7, RPB10 and RPB11. With the exception of RPB10 and RPB11, and the addition of RPB8, these were also identified using TAP-tagged constructs of one (encoded by LmjF34.0890) of the two LmRPB6 orthologues. The latter experiments also identified the RNAP III subunits RPC1 (C160), RPC2 (C128), RPC3 (C82), RPC4 (C53), RPC5 (C37), RPC6 (C34), RPC9 (C17), RPAC1 (AC40) and RPAC2 (AC19). Significantly, the complexes precipitated by TAP-tagged LmRPB6 did not contain any RNAP I-specific subunits, suggesting that, unlike in other eukaryotes, LmRPB6 is not shared by all three polymerases but is restricted to RNAP II and III, while the LmRPB6z (encoded by LmjF25.0140) isoform is limited to RNAP I. Similarly, we identified peptides from only one (encoded by LmjF18.0780) of the two RPB5 orthologues and one (LmjF13.1120) of the two RPB10 orthologues, suggesting that LmRPB5z (LmjF18.0790) and LmRPB10z (LmjF13.1120) are also restricted to RNAP I. In addition to these RNAP subunits, we also identified a number of other proteins that co-purified with the RNAP II and III complexes, including a potential transcription factor, several histones, an ATPase involved in chromosome segregation, an endonuclease, four helicases, RNA splicing factor PTSR-1, at least two RNA binding proteins and several proteins of unknown function. PMID:17275824

  18. Metabolomics-Based Study of Logarithmic and Stationary Phases of Promastigotes in Leishmania major by 1H NMR Spectroscopy

    PubMed Central

    Arjmand, Mohammad; Madrakian, Azadeh; Khalili, Ghader; Najafi, Ali; Zamani, Zahra; Akbari, Ziba

    2016-01-01

    Background: Cutaneous leishmaniasis is one of the most important parasitic diseases in humans. In this disease, one of the responsible organisms is Leishmania major, which is transmitted by sandfly vector. There are specific differences in biochemical profiles and metabolite pathways in logarithmic and stationary phases of Leishmania parasites. In the present study, 1H NMR spectroscopy was used to examine the metabolites outliers in the logarithmic and stationary phases of promastigotes in L. major to enlighten more about the transmission mechanism in metacyclogenesis of L. major. Methods: Promastigote was cultured, logarithmic and stationary phases were separated by the peanut agglutinin, and cell metabolites were extracted. 1H NMR spectroscopy was applied, and outliers were analyzed using principal component analysis. Results: The most altered metabolites in stationary and logarithmic phases were limited to citraconic acid, isopropylmalic acid, L-leucine, ornithine, caprylic acid, capric acid, and acetic acid. Conclusion: 1H NMR spectroscopy could play an important role in the characterization of metabolites in biochemical pathways during a metacyclogenesis process. These metabolites and their pathways can help in exploiting a transmission mechanism in metacyclogenesis, and outcoming data might be used in the metabolic network reconstruction of L. major modeling. PMID:26592771

  19. Characterization of integral membrane proteins of Leishmania major by Triton X-114 fractionation and analysis of vaccination effects in mice.

    PubMed Central

    Murray, P J; Spithill, T W; Handman, E

    1989-01-01

    The total integral membrane proteins of promastigotes of Leishmania major were extracted by using the Triton X-114 phase separation technique and were characterized by immunoprecipitation, Western blotting (immunoblotting), and lectin chromatography. Of the 40 or more proteins which partitioned into the detergent phase, only about 10 proteins could be surface radioiodinated on live promastigotes, suggesting their surface orientation. The abundance of the gp58-63 antigen varied markedly between two strains of L. major. Sera from patients with visceral leishmaniasis caused by Leishmania donovani chagasi recognized the gp58-63 complex and an additional Mr-42,000 polypeptide shared between L. major and L. donovani chagasi. A subpopulation of six surface proteins, including the abundant gp58-63 antigen and a group of proteins of Mr 81,000 to 105,000, were glycoproteins recognized by antiserum to wheat germ agglutinin- or concanavalin A-binding proteins. The membrane proteins of the LRC-L119 isolate of L. major could successfully vaccinate genetically susceptible mice, thus opening the way for a molecularly defined subunit vaccine composed of glycolipid and membrane protein antigens. Images PMID:2731987

  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. Species- and infective stage-specific monoclonal antibodies to Leishmania major produced by an in vitro immunization method.

    PubMed

    Wu, S J; Rowton, E D; Ma, M; Andre, R G

    1990-12-01

    Monoclonal antibodies specific to the infective-stage promastigotes of Leishmania major are needed for developing rapid diagnostic assays of infected sand flies. An in vitro immunization protocol was applied for the production of monoclonal antibodies using small amounts of L. major. Infective-stage promastigotes were isolated from sand flies (Phlebotomus papatasi) 7-10 days after infection and used as antigen for immunization. Two weeks after a primary immunization, murine splenocytes were removed and immunized in vitro with antigen in murine EL-4 thymoma cell conditioned medium. Three fusions were performed using X63-Ag.653 myeloma cells as fusion partners and two fusions were performed using FOX-NY cells. Antibodies specific to promastigotes were detected using an indirect enzyme-linked immunosorbent assay (ELISA). Initially 56 monoclonal antibodies were selected, and their species and stage specificity were determined using both an ELISA and an indirect fluorescent antibody assay (IFA). Twelve monoclonal antibodies showed species specificity to L. major when tested against four sympatric species of Leishmania. Four other monoclonal antibodies showed species and infective-stage specificity to L. major promastigotes. When tested in immunoblots, all four species- and stage-specific monoclonal antibodies bound to five protein bands that were unique to the infective-stage promastigotes. PMID:2087235

  2. 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

  3. Inoculation of killed Leishmania major into immune mice rapidly disrupts immunity to a secondary challenge via IL-10-mediated process

    PubMed Central

    Okwor, Ifeoma; Liu, Dong; Beverley, Stephen M.; Uzonna, Jude E.

    2009-01-01

    Recovery from natural or experimental Leishmania major infection, the causative agent of cutaneous leishmaniasis, results in development of durable immunity in mice and humans that is manifested as rapid control of parasite replication and resolution of cutaneous lesion after secondary challenge. This form of “infection-induced” immunity is thought to occur naturally in endemic areas and is generally considered the gold standard for any effective vaccine against cutaneous leishmaniasis. To determine factors that might heighten or abrogate infection-induced immunity, we investigated the impact of inoculating dead antigen in the form of killed Leishmania parasites to healed mice. We show that inoculation of killed parasites into mice that resolved their primary virulent L. major infection results in rapid and relatively sustained loss of infection-induced immunity. This loss of immunity was not due to the inability of killed parasites to induce inflammatory responses (such as delayed type hypersensitivity), but it was related to their failure to induce robust IFN-γ response. Furthermore, inoculation of killed Leishmania parasites into healed mice led to rapid expansion of IL-10-producing CD4+CD25+Foxp3+ T cells in lymph nodes draining the primary infection site. Treatment with anti-CD25 or anti-IL-10R mAb abolished killed parasite-induced loss of immunity. Our study suggests that vaccination with killed parasites could predispose naturally immune individuals to become susceptible to new infections and/or disease reactivation. This may account for the lack of efficacy of such vaccines in field trials in endemic regions. These findings have important implications for vaccine design and vaccination strategies against human cutaneous leishmaniasis. PMID:19666482

  4. Leishmania major lacking arginase are auxotrophic for polyamines but retain infectivity to susceptible BALB/c mice

    PubMed Central

    Reguera, Rosa M.; Balaña-Fouce, Rafael; Showalter, Melissa; Hickerson, Suzanne; Beverley, Stephen M.

    2009-01-01

    Polyamines are essential metabolites in eukaryotes participating in a variety of proliferative processes, and in trypanosomatid protozoa play an additional role in the synthesis of the critical thiol trypanothione. Whereas the polyamine biosynthesis arising from L-ornithine has been well studied in protozoa, the metabolic origin(s) of L-ornithine have received less attention. Arginase (EC 3.5.3.1) catalyzes the enzymatic hydrolysis of L-arginine to L-ornithine and urea, and we tested the role of arginase in polyamine synthesis by the generation of an arg− knockout in Leishmania major by double targeted gene replacement. This mutant lacked arginase activity and required the nutritional provision of polyamines or L-ornithine for growth. A complemented line (arg−/+ARG) expressing arginase from a multicopy expression vector showed 30-fold elevation of arginase activity, similar polyamine and ornithine levels as the wild-type, and resistance to the inhibitors α-difluoromethylornithine (DFMO) and Nω-hydroxy-L-arginine (NOHA). This established that arginase is the major route of polyamine synthesis in promastigotes cultured in vitro. The arg− parasites retained the ability to differentiate normally to the infective metacyclic stage, and were able to induce progressive disease following inoculation into susceptible BALB/c mice, albeit less efficiently than WT parasites. These data suggest that the infective amastigote form of Leishmania, which normally resides within an acidified parasitophorous vacuole, can survive in vivo through salvage of host polyamines and/or other molecules, aided by the tendency of acidic compartments to concentrate basic metabolites. This may thus contribute to the relative resistance of Leishmania to ornithine decarboxylase (ODC) inhibitors. The availability of infective, viable, arginase-deficient parasites should prove useful in dissecting the role of L-arginine metabolism in both pro- and anti-parasitic responses involving host nitric

  5. Protective Response to Leishmania major in BALB/c Mice Requires Antigen Processing in the Absence of DM1

    PubMed Central

    Kamala, Tirumalai; Nanda, Navreet K.

    2009-01-01

    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-γ. 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. PMID:19342667

  6. 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

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

    PubMed

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

    2016-05-01

    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

  8. 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. PMID:25233487

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

    PubMed

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

    2010-07-01

    Leishmania synthesize abundant phosphoglycan-containing molecules made up of [Gal-Man-PO(4)] 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

  10. Identification of genes encoding arabinosyltransferases (SCA) mediating developmental modifications of lipophosphoglycan required for sand fly transmission of leishmania major.

    PubMed

    Dobson, Deborah E; Mengeling, Brenda J; Cilmi, Salvatore; Hickerson, Suzanne; Turco, Salvatore J; Beverley, Stephen M

    2003-08-01

    At key steps in the infectious cycle pathogens must adhere to target cells, but at other times detachment is required for transmission. During sand fly infections by the protozoan parasite Leishmania major, binding of replicating promastigotes is mediated by galactosyl side chain (scGal) modifications of phosphoglycan repeats of the major surface adhesin, lipophosphoglycan (LPG). Release is mediated by arabinosyl (Ara) capping of LPG scbetaGal residues upon differentiation to the infective metacyclic stage. We used intraspecific polymorphisms of LPG structure to develop a genetic strategy leading to the identification of two genes (SCA1/2) mediating scAra capping. These LPG side chain beta1,2-arabinosyltransferases (scbetaAraTs) exhibit canonical glycosyltransferase motifs, and their overexpression leads to elevated microsomal scbetaAraT activity. Although the level of scAra caps is maximal in metacyclic parasites, scbetaAraT activity is maximal in log phase cells. Because quantitative immunolocalization studies suggest this is not mediated by sequestration of SCA scbetaAraTs away from the Golgi apparatus during log phase, regulation of activated Ara precursors may control LPG arabinosylation in vivo. The SCA genes define a new family of eukaryotic betaAraTs and represent novel developmentally regulated LPG-modifying activities identified in Leishmania. PMID:12750366

  11. 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

  12. 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

  13. Choline transport in Leishmania major promastigotes and its inhibition by choline and phosphocholine analogs.

    PubMed

    Zufferey, Rachel; Mamoun, Choukri Ben

    2002-01-01

    Phosphatidylcholine is the most abundant phospholipid in the membranes of the human parasite Leishmania. The metabolic pathways leading to its biosynthesis are likely to play a critical role in parasite development and survival and may offer a good target for antileishmanial chemotherapy. Phosphatidylcholine synthesis via the CDP-choline pathway requires transport of the choline precursor from the host. Here, we report the first characterization of choline transport in this parasite, which is carrier-mediated and exhibits Michaelis-Menten kinetics with an apparent K(m) value of 2.5 microM for choline. This process is Na(+)-independent and requires an intact proton gradient to be fully functional. Choline transport into Leishmania is highly specific for choline and is inhibited by the choline carrier inhibitor hemicholinium-3, the channel blocker quinacrine, the antimalarial aminoquinolines quinine and quinidine, the antileishmanial phosphocholine analogs, miltefosine and edelfosine, and by choline analogs, most of which have antimalarial activities. Most importantly, choline analogs kill the promastigote form of the parasite in vitro in the low micromolar range. These results set the stage for the use of choline analogs in antileishmanial chemotherapy and shed new lights on the mechanism of action of the leishmanicidal phosphocholine analogs. PMID:12467980

  14. 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. PMID:26503273

  15. 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

  16. 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

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

    PubMed

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

    2016-08-01

    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

  18. Prediction of T Cell Epitopes from Leishmania major Potentially Excreted/Secreted Proteins Inducing Granzyme B Production

    PubMed Central

    Naouar, Ikbel; Boussoffara, Thouraya; Chenik, Mehdi; Gritli, Sami; Ben Ahmed, Melika; Belhadj Hmida, Nabil; Bahi-Jaber, Narges; Bardi, Rafika; Gorgi, Yousr; Ben Salah, Afif; Louzir, Hechmi

    2016-01-01

    Leishmania-specific cytotoxic T cell response is part of the acquired immune response developed against the parasite and contributes to resistance to reinfection. Herein, we have used an immune-informatic approach for the identification, among Leishmania major potentially excreted/secreted proteins previously described, those generating peptides that could be targeted by the cytotoxic immune response. Seventy-eight nonameric peptides that are predicted to be loaded by HLA-A*0201 molecule were generated and their binding capacity to HLA-A2 was evaluated. These peptides were grouped into 20 pools and their immunogenicity was evaluated by in vitro stimulation of peripheral blood mononuclear cells from HLA-A2+-immune individuals with a history of zoonotic cutaneous leishmaniasis. Six peptides were identified according to their ability to elicit production of granzyme B. Furthermore, among these peptides 3 showed highest affinity to HLA-A*0201, one derived from an elongation factor 1-alpha and two from an unknown protein. These proteins could constitute potential vaccine candidates against leishmaniasis. PMID:26771180

  19. A family of heat shock protein 70-related genes are expressed in the promastigotes of Leishmania major.

    PubMed Central

    Searle, S; Campos, A J; Coulson, R M; Spithill, T W; Smith, D F

    1989-01-01

    We describe the isolation and characterisation of two novel genes of the parasitic protozoan Leishmania major that are related by nucleotide sequence homology to eukaryotic genes encoding 70 Kd. heat shock proteins. The transcription of neither gene is heat-inducible but both are constituitively-expressed throughout the promastigote stage of the parasite life cycle. A third gene shows differential expression between non-infective and infective promastigote stages in the absence of any temperature change. These genes are related by sequence homology to the tandemly-repeated hsp70 genes of trypanosomatids, but are located on different, dispersed chromosomes within the genome of L. major. The open reading frame for translation derived from one of these sequences contains a putative mitochondrial signal peptide at its amino-terminus. Images PMID:2762121

  20. The Mating Competence of Geographically Diverse Leishmania major Strains in Their Natural and Unnatural Sand Fly Vectors

    PubMed Central

    Inbar, Ehud; Akopyants, Natalia S.; Charmoy, Melanie; Romano, Audrey; Lawyer, Phillip; Elnaiem, Dia-Eldin A.; Kauffmann, Florence; Barhoumi, Mourad; Grigg, Michael; Owens, Katherine; Fay, Michael; Dobson, Deborah E.; Shaik, Jahangheer; Beverley, Stephen M.; Sacks, David

    2013-01-01

    Invertebrate stages of Leishmania are capable of genetic exchange during their extracellular growth and development in the sand fly vector. Here we explore two variables: the ability of diverse L. major strains from across its natural range to undergo mating in pairwise tests; and the timing of the appearance of hybrids and their developmental stage associations within both natural (Phlebotomus duboscqi) and unnatural (Lutzomyia longipalpis) sand fly vectors. Following co-infection of flies with parental lines bearing independent drug markers, doubly-drug resistant hybrid progeny were selected, from which 96 clonal lines were analyzed for DNA content and genotyped for parent alleles at 4–6 unlinked nuclear loci as well as the maxicircle DNA. As seen previously, the majority of hybrids showed ‘2n’ DNA contents, but with a significant number of ‘3n’ and one ‘4n’ offspring. In the natural vector, 97% of the nuclear loci showed both parental alleles; however, 3% (4/150) showed only one parental allele. In the unnatural vector, the frequency of uniparental inheritance rose to 10% (27/275). We attribute this to loss of heterozygosity after mating, most likely arising from aneuploidy which is both common and temporally variable in Leishmania. As seen previously, only uniparental inheritance of maxicircle kDNA was observed. Hybrids were recovered at similar efficiencies in all pairwise crosses tested, suggesting that L. major lacks detectable ‘mating types’ that limit free genetic exchange. In the natural vector, comparisons of the timing of hybrid formation with the presence of developmental stages suggest nectomonads as the most likely sexually competent stage, with hybrids emerging well before the first appearance of metacyclic promastigotes. These studies provide an important perspective on the prevalence of genetic exchange in natural populations of L. major and a guide for experimental studies to understand the biology of mating. PMID:23935521

  1. 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. PMID:24623031

  2. Cathepsin B in antigen-presenting cells controls mediators of the Th1 immune response during Leishmania major infection.

    PubMed

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

    2014-09-01

    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

  3. Structure of Leishmania major Methionyl-tRNA Synthetase in Complex with Intermediate Products Methionyladenylate and Pyrophosphate

    PubMed Central

    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.

    2011-01-01

    Leishmania parasites cause two million new cases of leishmaniasis each year with several hundreds of millions 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 Mg2+ 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 E. 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-tRNAMet 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. PMID:21144880

  4. Batf3-dependent CD103+ dendritic cells are major producers of IL-12 that drive local Th1 immunity against Leishmania major infection in mice

    PubMed Central

    Martínez-López, María; Iborra, Salvador; Conde-Garrosa, Ruth; Sancho, David

    2015-01-01

    The role of different DC subsets in priming and maintenance of immunity against Leishmania major (L. major) infection is debated. The transcription factor basic leucine zipper transcription factor, ATF-like 3 (Batf3) is essential for the development of mouse CD103+ DCs and some functions of CD8α+ DCs. We found that CD103+ DCs were significantly reduced in the dermis of Batf3-deficient C57BL/6 mice. Batf3−/− mice developed exacerbated and unresolved cutaneous pathology following a low dose of intradermal L. major infection in the ear pinnae. Parasite load was increased 1000-fold locally and expanded systemically. Batf3 deficiency did not affect L. major antigen presentation to T cells, which was directly exerted by CD8α− conventional DCs (cDCs) in the skin draining LN. However, CD4+ T-cell differentiation in the LN and skin was skewed to nonprotective Treg- and Th2-cell subtypes. CD103+ DCs are major IL-12 producers during L. major infection. Local Th1 immunity was severely hindered, correlating with impaired IL-12 production and reduction in CD103+ DC numbers. Adoptive transfer of WT but not IL-12p40−/− Batf3-dependent DCs significantly improved anti-L. major response in infected Batf3−/− mice. Our results suggest that IL-12 production by Batf3-dependent CD103+ DCs is crucial for maintenance of local Th1 immunity against L. major infection. PMID:25312824

  5. 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

  6. 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

  7. Sphingolipid Degradation by Leishmania major Is Required for Its Resistance to Acidic pH in the Mammalian Host▿†

    PubMed Central

    Xu, Wei; Xin, Lijun; Soong, Lynn; Zhang, Kai

    2011-01-01

    Leishmania parasites alternate between flagellated promastigotes in sand flies and nonflagellated amastigotes in mammals, causing a spectrum of serious diseases. To survive, they must resist the harsh conditions in phagocytes (including acidic pH, elevated temperature, and increased oxidative/nitrosative stress) and evade the immune response. Recent studies have highlighted the importance of sphingolipid (SL) metabolism in Leishmania virulence. In particular, we have generated a Leishmania major iscl− mutant which is deficient in SL degradation but grows normally as promastigotes in culture. Importantly, iscl− mutants cannot induce pathology in either immunocompetent or immunodeficient mice yet are able to persist at low levels. In this study, we investigated how the degradation of SLs might contribute to Leishmania infection. First, unlike wild-type (WT) L. major, iscl− mutants do not trigger polarized T cell responses in mice. Second, like WT parasites, iscl− mutants possess the ability to downregulate macrophage activation by suppressing the production of interleukin-12 (IL-12) and nitric oxide. Third, during the stationary phase, iscl− promastigotes were extremely vulnerable to acidic pH but not to other adverse conditions, such as elevated temperature and oxidative/nitrosative stress. In addition, inhibition of phagosomal acidification significantly improved iscl− survival in murine macrophages. Together, these findings indicate that SL degradation by Leishmania is essential for its adaption to the acidic environment in phagolysosomes but is not required for the suppression of host cell activation. Finally, our studies with iscl− mutant-infected mice suggest that having viable, persistent parasites is not sufficient to provide immunity against virulent Leishmania challenge. PMID:21576322

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

    PubMed Central

    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.

    2014-01-01

    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. PMID:24072877

  9. 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. PMID:22227102

  10. 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.

  11. Spontaneous Recovery of Pathogenicity by Leishmania major hsp100−/− Alters the Immune Response in Mice

    PubMed Central

    Reiling, Linda; Jacobs, Thomas; Kroemer, Manfred; Gaworski, Iris; Graefe, Sebastian; Clos, Joachim

    2006-01-01

    By using repeated mouse infection cycles, we obtained an escape variant with restored infectivity and pathogenicity that originated from a single, noninfectious hsp100−/− gene (formerly known as ΔclpB) replacement clone of Leishmania major, the causative agent of cutaneous leishmaniasis. This isolate elicited increased infiltration of immune cells to the site of infection and altered the polarization of the immune response in BALB/c mice from a predominantly TH2 type to a TH1 type. A clonal analysis resulted in isolation of two clones with antagonistic properties. While one clone exhibited restored infectivity in isolated macrophages but caused no persistent infection in the mouse model, the second clone was unable to infect macrophages in vitro but could establish a lasting infection and form progressive lesions in BALB/c mice. Our results add to the evidence that the TH1-TH2 dichotomy of the early immune response against L. major not only depends on the genetic predisposition of the host but also depends on intrinsic properties of the parasite. PMID:17057085

  12. Targeting the Midgut Secreted PpChit1 Reduces Leishmania major Development in Its Natural Vector, the Sand Fly Phlebotomus papatasi

    PubMed Central

    Coutinho-Abreu, Iliano V.; Sharma, Narinder K.; Robles-Murguia, Maricela; Ramalho-Ortigao, Marcelo

    2010-01-01

    Background During its developmental cycle within the sand fly vector, Leishmania must survive an early proteolytic attack, escape the peritrophic matrix, and then adhere to the midgut epithelia in order to prevent excretion with remnants of the blood meal. These three steps are critical for the establishment of an infection within the vector and are linked to interactions controlling species-specific vector competence. PpChit1 is a midgut-specific chitinase from Phlebotomus papatasi presumably involved in maturation and degradation of the peritrophic matrix. Sand fly midgut chitinases, such as PpChit1, whether acting independently or in a synergistic manner with Leishmania-secreted chitinase, possibly play a role in the Leishmania escape from the endoperitrophic space. Thus, we predicted that silencing of sand fly chitinase will lead to reduction or elimination of Leishmania within the gut of the sand fly vector. Methodology/Principal Findings We used injection of dsRNA to induce knock down of PpChit1 transcripts (dsPpChit1) and assessed the effect on protein levels post blood meal (PBM) and on Leishmania major development within P. papatasi. Injection of dsPpChit1 led to a significant reduction of PpChit1 transcripts from 24 hours to 96 hours PBM. More importantly, dsPpChit1 led to a significant reduction in protein levels and in the number of Le. major present in the midgut of infected P. papatasi following a infective blood meal. Conclusion/Significance Our data supports targeting PpChit1 as a potential transmission blocking vaccine candidate against leishmaniasis. PMID:21152058

  13. Vaccination with phosphoglycan-deficient Leishmania major protects highly susceptible mice from virulent challenge without inducing a strong Th1 response.

    PubMed

    Uzonna, Jude E; Späth, Gerald F; Beverley, Stephen M; Scott, Phillip

    2004-03-15

    Long-term immunity to Leishmania may require the continued presence of parasites, but previous attempts to create attenuated parasites that persist without causing disease have had limited success. Since Leishmania major mutants that lack lipophosphoglycan and other secreted phosphoglycans, termed lpg2-, persist indefinitely in infected mice without inducing any disease, we tested their ability to provide protection to virulent L. major challenge. In response to leishmanial Ag stimulation, cells from lpg2--infected mice produced minimal levels of IL-4 and IL-10, as well as very low levels of IFN-gamma. Nevertheless, when BALB/c mice infected with lpg2- parasites were challenged with virulent L. major they were protected from disease. Thus, these findings report on attenuated parasites that may be used to induce long-term protection against leishmaniasis and indicate that the immunity induced can be maintained in the absence of a strong Th1 response. PMID:15004184

  14. DNA based vaccination with a cocktail of plasmids encoding immunodominant Leishmania (Leishmania) major antigens confers full protection in BALB/c mice.

    PubMed

    Ahmed, Sami Ben Hadj; Touihri, Leila; Chtourou, Yessine; Dellagi, Koussay; Bahloul, Chokri

    2009-01-01

    Despite the lack of effective vaccines against parasitic diseases, the prospects of developing a vaccine against leishmaniasis are still high. With this objective, we have tested four DNA based candidate vaccines encoding to immunodominant leishmania antigens (LACKp24, TSA, LmSTI1 and CPa). These candidates have been previously reported as capable of eliciting at least partial protections in the BALB/c mice model of experimental cutaneous leishmaniasis. When tested under similar experimental conditions, all of them were able to induce similar partial protective effects, but none could induce a full protection. In order to improve the level of protection we have explored the approach of DNA based vaccination with different cocktails of plasmids encoding to the different immunodominant Leishmania antigens. A substantial increase of protection was achieved when the cocktail is composed of all of the four antigens; however, no full protection was achieved when mice were challenged with a high dose of parasite in their hind footpad. The full protection was only achieved after a challenge with a low parasitic dose in the dermis of the ear. It was difficult to determine clear protection correlates, other than the mixture of immunogens induced specific Th1 immune responses against each component. Therefore, such an association of antigens increased the number of targeted epitopes by the immune system with the prospects that the responses are at least additive if not synergistic. Even though, any extrapolation of this approach when applied to other animal or human models is rather hazardous, it undoubtedly increases the hopes of developing an effective leishmania vaccine. PMID:18951941

  15. 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

  16. Effect of root bark extract of Berberis vulgaris L. on Leishmania major on BALB/c mice.

    PubMed

    Salehabadi, Alireza; Karamian, Mahdi; Farzad, Motevalli Haghi; Namaei, Mohammad Hasan

    2014-03-01

    Leishmaniasis is one of the most important diseases transmitted by arthropod. This disease is present in 88 countries. Approximately 400 million people are at risk, and 12 million are involved. We aimed to examine the application of ethanolic extract of the root bark of Berberis vulgaris L. for treatment of mice infected with cutaneous leishmaniasis. At first, 40 BALB/c mice were infected to Leishmania major promastigotes and were divided in two groups A and B. Then, each of A and B groups were divided to two subgroups. Mice from subgroup A1 were treated with 10% root bark alcoholic extract, and mice from subgroup A2 were treated with only alcohol (control). Mice from subgroup B1 were treated with 20% root bark alcoholic extract, and mice from subgroup B2 were treated with only alcohol (control). The 90% recovery was found in the mice treated with 20% root bark extract, and 55% recovery was found with 10% root bark extract, but in the control group, 0% recovery was found. The results of our study showed that the lotion of root bark extract has good suppression effects on parasites. Therefore, it might be a pro for developing new antileishmanial drugs. PMID:24337510

  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. PMID:27065601

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

    PubMed Central

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

    2014-01-01

    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 Ca2+ ions. A third inhibitor, 3-fluoro-1-(2-hydroxy-2,2-diphosphonoethyl)pyridinium (46I), was found to bind two Mg2+ 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. PMID:24598749

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

    PubMed

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

    2014-03-01

    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(2+) ions. A third inhibitor, 3-fluoro-1-(2-hydroxy-2,2-diphosphonoethyl)pyridinium (46I), was found to bind two Mg(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. PMID:24598749

  2. Vector-host-parasite inter-relationships in leishmaniasis. IV. Electrophoretic studies on proteins of four vertebrate bloods with and without Leishmania infantum or L. major.

    PubMed

    Daba, S; Mansour, N S; Youssef, F G; Shanbaky, N M; el Sawaf, B M

    1997-12-01

    Fifty five protein bands with relative mobilities of 8,954 to 245,471 kilo Daltons (kD) were electrophoretically separated from 12 feeding media of blood from 4 natural vertebrate hosts of Phlebotomus langeroni. The feeding media included human, dog (Canis familiaris), rat (Rattus rattus) and turkey (Melagris gallopava) bloods without or with Leishmania infantum or L. major promastigotes. Protein bands were identical among the feeding media of one host's blood but varied in number (24-28 bands) and relative mobilities among the various hosts' blood. Some protein fractions were common among the various hosts blood, others were only present in two or three hosts' blood and some were restricted to one host blood and were unique for each host. This study provides data which may help in understanding why blood from different natural hosts may variably influence the life cycle of Leishmania parasite in the sand fly gut. PMID:9425823

  3. Immunostimulatory responses to crude extracts of Warburgia ugandensis (sprague) subsp ugandensis (canellaceae) by Balb/c mice infected with Leishmania major

    PubMed Central

    Ngure, Peter; Ng'ang'a, Zipporah; Kimutai, Albert; Kepha, Stella; Mong'are, Samuel; Ingonga, Johnnie; Tonui, Willy

    2014-01-01

    Introduction To determine the immunostimulatory potential of crude extracts of Warburgia ugandensis subsp. ugandensis with a soluble leishmanial antigen in vaccinating BALB/c mice. Methods Seventy two female BALB/c mice were randomly assigned into six groups. The mice were vaccinated with soluble leishmania antigens (SLA) alone, hexane, ethyl acetate, and dichloromethane extract co-administered with SLA. Unvaccinated mice formed the control group. The induction of cell-mediated immunity following vaccination was determined by measuring in vitro lymphocyte proliferation and the production of interleukin (IL)-4 and gamma interferon (IFN-γ) determined by flow cytometry. Protection against L. major was determined by quantifying parasite burdens in L. major infected footpads using a limiting dilution assay and by measuring lesion sizes of the infected footpad compared to the contralateral uninfected footpad. Results On vaccination with extracts of W. ugandensis subsp. ugandensis alone or as adjuvants when used in combination with Leishmania antigens, the hexane extract and the dichloromethane extract plus SLA stimulated moderate production of IFN-γ and low levels of IL-4.These mice were partially protected from cutaneous leishmaniasis as shown by the slow development of lesions and comparatively less parasite burdens. Conclusion These data suggest that extracts of W. ugandensis subsp. ugandensis are suitable adjuvants for Leishmania vaccines. However, since W. ugandensis subsp. ugandensis has been shown to be effective against Leishmania parasites in vitro and in vivo, further studies ought to be conducted to determine its immunochemotherapeutic potential when co-administered with a soluble leishmanial antigen in vaccinating BALB/c mice. PMID:24624248

  4. Detection, identification and molecular typing of Leishmania major in Phlebotomus papatasi from a focus of zoonotic cutaneous leishmaniasis in central of Iran.

    PubMed

    Parvizi, Parviz; Baghban, Nassrin; Novin, Elnaz Alaee; Absavaran, Azad

    2010-02-01

    Leishmania major is the causative agent and Phlebotomus papatasi is the main vector of rural zoonotic cutaneous leishmaniasis (ZCL) in Iran and elsewhere. Nested PCR protocols were used to amplify a region of the ribosomal RNA amplicon of Leishmania (ITS1-5.8S rRNA gene) in female P. papatasi. In the current investigation, L. major was found in Natanz, Isfahan province in centre of Iran, in a focus of rural ZCL. Ten (1.8%) out of 549 female P. papatasi was found to be infected with L. major based on the PCR detection and sequencing of parasite ITS-rDNA. Nine (1.8%) out of 498 female P. papatasi infected with L. major came from animal shelters, inside houses and yards. And one (1.9%) out of 51 female P. papatasi infected with L. major came from gerbil borrows. Infection rates were higher for females containing red blood meals, large eggs (semi-mature and mature) than for those without either blood meals or eggs. From the 10 infections detected three different haplotypes of L. major were identified. Two haplotypes were found to be novel. The other haplotypes of L. major was found to be identical to that of isolates of L. major from Iran and in elsewhere using GenBank data. Comparisons of infection rates between habitats will be inaccurate when the proportions of blood-fed and gravid flies differ among sandfly samples. PMID:19854172

  5. Golgi-located NTPDase1 of Leishmania major is required for lipophosphoglycan elongation and normal lesion development whereas secreted NTPDase2 is dispensable for virulence.

    PubMed

    Sansom, Fiona M; Ralton, Julie E; Sernee, M Fleur; Cohen, Alice M; Hooker, David J; Hartland, Elizabeth L; Naderer, Thomas; McConville, Malcolm J

    2014-12-01

    Parasitic protozoa, such as Leishmania species, are thought to express a number of surface and secreted nucleoside triphosphate diphosphohydrolases (NTPDases) which hydrolyze a broad range of nucleoside tri- and diphosphates. However, the functional significance of NTPDases in parasite virulence is poorly defined. The Leishmania major genome was found to contain two putative NTPDases, termed LmNTPDase1 and 2, with predicted NTPDase catalytic domains and either an N-terminal signal sequence and/or transmembrane domain, respectively. Expression of both proteins as C-terminal GFP fusion proteins revealed that LmNTPDase1 was exclusively targeted to the Golgi apparatus, while LmNTPDase2 was predominantly secreted. An L. major LmNTPDase1 null mutant displayed increased sensitivity to serum complement lysis and exhibited a lag in lesion development when infections in susceptible BALB/c mice were initiated with promastigotes, but not with the obligate intracellular amastigote stage. This phenotype is characteristic of L. major strains lacking lipophosphoglycan (LPG), the major surface glycoconjugate of promastigote stages. Biochemical studies showed that the L. major NTPDase1 null mutant synthesized normal levels of LPG that was structurally identical to wild type LPG, with the exception of having shorter phosphoglycan chains. These data suggest that the Golgi-localized NTPase1 is involved in regulating the normal sugar-nucleotide dependent elongation of LPG and assembly of protective surface glycocalyx. In contrast, deletion of the gene encoding LmNTPDase2 had no measurable impact on parasite virulence in BALB/c mice. These data suggest that the Leishmania major NTPDase enzymes have potentially important roles in the insect stage, but only play a transient or non-major role in pathogenesis in the mammalian host. PMID:25521752

  6. Golgi-Located NTPDase1 of Leishmania major Is Required for Lipophosphoglycan Elongation and Normal Lesion Development whereas Secreted NTPDase2 Is Dispensable for Virulence

    PubMed Central

    Sansom, Fiona M.; Ralton, Julie E.; Sernee, M. Fleur; Cohen, Alice M.; Hooker, David J.; Hartland, Elizabeth L.; Naderer, Thomas; McConville, Malcolm J.

    2014-01-01

    Parasitic protozoa, such as Leishmania species, are thought to express a number of surface and secreted nucleoside triphosphate diphosphohydrolases (NTPDases) which hydrolyze a broad range of nucleoside tri- and diphosphates. However, the functional significance of NTPDases in parasite virulence is poorly defined. The Leishmania major genome was found to contain two putative NTPDases, termed LmNTPDase1 and 2, with predicted NTPDase catalytic domains and either an N-terminal signal sequence and/or transmembrane domain, respectively. Expression of both proteins as C-terminal GFP fusion proteins revealed that LmNTPDase1 was exclusively targeted to the Golgi apparatus, while LmNTPDase2 was predominantly secreted. An L. major LmNTPDase1 null mutant displayed increased sensitivity to serum complement lysis and exhibited a lag in lesion development when infections in susceptible BALB/c mice were initiated with promastigotes, but not with the obligate intracellular amastigote stage. This phenotype is characteristic of L. major strains lacking lipophosphoglycan (LPG), the major surface glycoconjugate of promastigote stages. Biochemical studies showed that the L. major NTPDase1 null mutant synthesized normal levels of LPG that was structurally identical to wild type LPG, with the exception of having shorter phosphoglycan chains. These data suggest that the Golgi-localized NTPase1 is involved in regulating the normal sugar-nucleotide dependent elongation of LPG and assembly of protective surface glycocalyx. In contrast, deletion of the gene encoding LmNTPDase2 had no measurable impact on parasite virulence in BALB/c mice. These data suggest that the Leishmania major NTPDase enzymes have potentially important roles in the insect stage, but only play a transient or non-major role in pathogenesis in the mammalian host. PMID:25521752

  7. Non-MHC-linked Th2 cell development induced by soluble protein administration predicts susceptibility to Leishmania major infection.

    PubMed

    Guéry, J C; Galbiati, F; Smiroldo, S; Adorini, L

    1997-09-01

    Continuous administration of soluble protein Ag followed by immunization with the same Ag in adjuvant results in the selective development of Ag-specific CD4+ Th2 cells in both normal and beta2-microglobulin-deficient BALB/c mice. In addition to chronic administration by mini-osmotic pump, single bolus i.p., but not i.v., injection of protein Ag induces Th2 cell expansion. Strong Th2 cell priming depends on a non-MHC-linked genetic polymorphism. It is observed in all congenic strains on BALB background tested, BALB/c, BALB/b, and BALB/k, but not in MHC-matched strains on disparate genetic background, B10.D2, C57BL/6, and C3H. DBA/2 mice appear to have an intermediate phenotype, as shown by their weaker capacity to mount Th2 responses as compared with BALB/c mice after soluble Ag administered by either mini-osmotic pumps or single bolus i.p. Conversely, induction of Th1 cell unresponsiveness by soluble protein is observed in any mouse strain tested, following any mode of Ag administration. These data demonstrate that non-MHC-linked genetic polymorphism controls the priming of Th2 but not the inhibition of Th1 cells induced by administration of soluble protein. The pattern of Th2 responses in these different strains is predictive of disease outcome following Leishmania major infection and supports the hypothesis that systemic Ag presentation in the absence of strong inflammatory signals may represent an important stimulus leading to selective Th2 cell development in susceptible mouse strains. PMID:9278301

  8. NF-kappa B1 is required for optimal CD4+ Th1 cell development and resistance to Leishmania major.

    PubMed

    Artis, David; Speirs, Kendra; Joyce, Karen; Goldschmidt, Michael; Caamaño, Jorge; Hunter, Christopher A; Scott, Phillip

    2003-02-15

    The NF-kappaB family of transcription factors regulates the expression of a wide range of immune response genes involved in immunity to pathogens. However, the need for individual family members in regulating innate and adaptive immune responses in vivo has yet to be clearly defined. We investigated the role of NF-kappaB1 in the induction of protective IL-12-dependent Th1 cell responses following infection with the intracellular protozoan parasite Leishmania major. Whereas wild-type C57BL/6 mice controlled parasite replication, NF-kappaB1 knockout (KO) mice were susceptible to infection, developing chronic unresolving lesions associated with persistent parasites. There was a profound defect in Ag-specific CD4(+) T cell proliferation and IFN-gamma production in infected KO mice, although innate responses-including IL-12 production and control of intracellular parasite replication by macrophages-were intact. In vitro polyclonal stimulation of purified naive KO T cells revealed an intrinsic defect in CD4(+) T cell proliferation associated with reduced IL-2 receptor expression, but operating independently of APC function and IL-2 production. Critically, the frequency of proliferating KO CD4(+) T cells secreting IFN-gamma matched that of wild-type cells, suggesting that NF-kappaB1 was not required for efficient transcription of the IFN-gamma gene. Taken together, these results identify a novel role for NF-kappaB1 in CD4(+) T cell proliferation and the development of Th1 cell responses required for protective immunity against intracellular pathogens. PMID:12574369

  9. 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.

  10. First detection of Leishmania major DNA in Sergentomyia (Sintonius) clydei (Sinton, 1928, Psychodidae: Phlebotominae), from an outbreak area of cutaneous leishmaniasis in Tunisia.

    PubMed

    Ayari, Chiraz; Ben Othman, Souad; Chemkhi, Jomaa; Tabbabi, Ahmed; Fisa, Roser; Ben Salah, Afif; BenAbderrazak, Souha

    2016-04-01

    In recent years there has been growing interest in Sergentomyia species. Their role in the spread of mammalian leishmaniasis appears repeatedly in the literature and the possibility of its implication in Leishmania transmission to humans remains controversial. Sergentomyia (Sintonius) clydei is one of several cryptic species sharing therefore common morphologic criteria with others species of the subgenera Sintonius. Little is known about this specie in Tunisia. We sampled and identified different specimens including four specimens of S. clydei collected from Sidi Bouzid and Kairouan areas (center of Tunisia) using morphological tools. Male Sergentomyia clydei and Sergentomyia christophersi are known to share several morphological characters and can be mistaken for. Consequently we took advantage of 5 male S. christophersi available in our collection (Tataouin, South of Tunisia). In our study morphological tools were completed by molecular study of cytochrome b gene to identify S. clydei. For the detection of Leishmania spp. that might infect our specimens, Leishmania DNA was analyzed by amplification of kinetoplast minicircle DNA using real-time PCR and nested-PCR. Obtained result was confirmed by restriction analysis of the amplified ribosomal internal transcribed spacer 1 (ITS1). We provide in our study, the first molecular identification of S. clydei, in Tunisia. Our Neighbor Joining tree based on mitochondrial cytochrome b gene shows two different clusters. The first includes the Tunisians S. clydei and other specimens from Africa, Middle East and the Arabic peninsula, and the second cluster containing the specimens from Seychelle. Unexpectedly, we also demonstrate the infection of one anthropophilic female S. clydei by Leishmania major DNA. This finding shows that more attention should be paid when identifying parasites by molecular tools within sandfly vector. PMID:26538476

  11. Infection with arginase deficient Leishmania major reveals a parasite number-dependent and cytokine-independent regulation of host cellular arginase activity and disease pathogenesis1

    PubMed Central

    Muleme, Helen M; Reguera, Rosa M; Berard, Alicia; Azinwi, Richard; Jia, Ping; Okwor, Ifeoma B; Beverley, Stephen; Uzonna, Jude E

    2009-01-01

    The balance between the products of L-arginine metabolism in macrophages regulates the outcome of Leishmania major infection. L-arginine can be oxidized by host inducible nitric oxide synthase (iNOS) to produce nitric oxide (NO), which contributes to parasite killing. In contrast, L-arginine hydrolysis by host arginase blocks NO generation and provides polyamines, which can support parasite proliferation. Additionally, Leishmania encode their own arginase which has considereable potential to modulate infectivity and disease pathogenesis. Here, we compare the infectivity and impact on host cellular immune response in vitro and in vivo of wild-type (WT) L. major with that of a parasite arginase null mutant (arg-). We found that arg- L. major are impaired in their macrophage infectivity in vitro independent of host iNOS activities. As with in vitro results, the proliferation of arg- L. major in animal infections was also significantly impaired in vivo resulting in delayed onset of lesion development, attenuated pathology and low parasite burden. Despite this attenuated pathology, the production of cytokines by cells from the draining lymph node of mice infected with WT and arg- L. major was similar at all times tested. Interestingly, in vitro and in vivo arginase levels were significantly lower in arg- than in WT infected cases and were directly correlated with parasite numbers inside infected cells. These results suggest that Leishmania-encoded arginase enhances disease pathogenesis by augmenting host cellular arginase activities leading and that contrary to previous in vitro studies, the host cytokine response does not influence host arginase activity. PMID:19923451

  12. Human Dendritic Cells exhibit a pronounced type I IFN signature following Leishmania major infection that is required for IL-12 induction1.

    PubMed Central

    Favila, Michelle A.; Geraci, Nicholas S.; Zeng, Erliang; Harker, Brent; Condon, David; Cotton, Rachel N.; Jayakumar, Asha; Tripathi, Vinita; McDowell, Mary Ann

    2014-01-01

    Leishmania major infected human dendritic cells (DCs) exhibit a marked induction of IL-12, ultimately promoting a robust Th1-mediated response associated with parasite killing and protective immunity. The host cell transcription machinery associated with the specific IL-12 induction observed during L. major infection remains to be thoroughly elucidated. In this study, we utilized Affymetrix Genechips to globally assess the host cell genes and pathways associated with early L. major infection in human myeloid-derived DCs. Our data revealed 728 genes were significantly differentially expressed and molecular signaling pathway revealed that the type I IFN pathway was significantly enriched. Addition of a neutralizing type I IFN decoy receptor blocked the expression of IRF7 and IL-12p40 during DC infection, indicating the L. major induced expression of IL-12p40 is dependent upon the type I IFN signaling pathway. In stark contrast, IL-12p40 expression is not elicited by Leishmania donovani, the etiological agent of deadly visceral leishmaniasis. Therefore, we examined the gene expression profile for several IFN response genes in L. major versus L. donovani DC infections. Our data revealed that L. major, but not L. donovani, induces expression of IRF2, IRF7, and IFIT5, implicating the regulation of type I IFN associated signaling pathways as mediating factors toward the production of IL-12. PMID:24808365

  13. 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

  14. Leishmania major Glycosylation Mutants Require Phosphoglycans (lpg2−) but Not Lipophosphoglycan (lpg1−) for Survival in Permissive Sand Fly Vectors

    PubMed Central

    Svárovská, Anna; Ant, Thomas H.; Seblová, Veronika; Jecná, Lucie; Beverley, Stephen M.; Volf, Petr

    2010-01-01

    Background Sand fly species able to support the survival of the protozoan parasite Leishmania have been classified as permissive or specific, based upon their ability to support a wide or limited range of strains and/or species. Studies of a limited number of fly/parasite species combinations have implicated parasite surface molecules in this process and here we provide further evidence in support of this proposal. We investigated the role of lipophosphoglycan (LPG) and other phosphoglycans (PGs) in sand fly survival, using Leishmania major mutants deficient in LPG (lpg1−), and the phosphoglycan (PG)-deficient mutant lpg2−. The sand fly species used were the permissive species Phlebotomus perniciosus and P. argentipes, and the specific vector P. duboscqi, a species resistant to L. infantum development. Principal Findings The lpg2− mutants did not survive well in any of the three sand fly species, suggesting that phosphoglycans and/or other LPG2-dependent molecules are required for parasite development. In vitro, all three L. major lines were equally resistant to proteolytic activity of bovine trypsin, suggesting that sand fly-specific hydrolytic proteases or other factors are the reason for the early lpg2− parasite killing. The lpg1− mutants developed late-stage infections in two permissive species, P. perniciosus and P. argentipes, where their infection rates and intensities of infections were comparable to the wild type (WT) parasites. In contrast, in P. duboscqi the lpg1− mutants developed significantly worse than the WT parasites. Conclusions In combination with previous studies, the data establish clearly that LPG is not required for Leishmania survival in permissive species P. perniciosus and P. argentipes but plays an important role in the specific vector P. duboscqi. With regard to PGs other than LPG, the data prove the importance of LPG2-related molecules for survival of L. major in the three sand fly species tested. PMID:20084096

  15. Comparison of Parasite Burden Using Real-Time Polymerase Chain Reaction Assay and Limiting Dilution Assay in Leishmania major Infected Mouse

    PubMed Central

    GHOTLOO, Somayeh; HAJI MOLLAHOSEINI, Mostafa; NAJAFI, Ali; YEGANEH, Farshid

    2015-01-01

    Background: Limiting dilution assay is considered as the gold standard method for quantifying the number of parasites in the animal model of Leishmania infection. Nowadays, real-time PCR is being increasingly applied to quantify infectious agents. In the present study, a real-time PCR assay was developed to estimate parasite burdens in lymph nodes of Leishmania major infected BALB/C mice. Enumeration of parasites was also performed by limiting dilution assay and compared with the results of real-time PCR based quantification. Methods: The SYBR Green based real-time PCR assay was performed to amplify a 75 bp fragment of superoxide dismutase B1 gene in the lymph nodes of L. major infected BALB/C mice 8 weeks post infection. Mice were infected subcutaneously at the base of their tail with 2 × 105 L. major promastigotes in the stationary phase of growth. To compare parasite burdens obtained by real-time PCR assay with those of limiting dilution assay, twelve 8-fold serial dilutions of the lymph node homogenates were prepared in the Schneider medium and incubated at 26°C. After 7 days, wells containing motile parasites were identified by direct observation under an inverted light microscope and the total number of parasites was estimated using the ELIDA software. Results: Spearman’s correlation coefficient of the parasite burdens between real-time PCR and limiting dilution assay was 0.72 (P value = 0.008). Conclusion: Real-time PCR assay is an appropriate replacement to existing limiting dilution assay in quantifying parasite burden in the experimental model of Leishmania infection. PMID:26811723

  16. Lymphocytic Choriomeningitis Virus Expands a Population of NKG2D+CD8+ T Cells That Exacerbates Disease in Mice Coinfected with Leishmania major.

    PubMed

    Crosby, Erika J; Clark, Megan; Novais, Fernanda O; Wherry, E John; Scott, Phillip

    2015-10-01

    Leishmaniasis is a significant neglected tropical disease that is associated with a wide range of clinical presentations and a lifelong persistent infection. Because of the chronic nature of the disease, there is a high risk for coinfection occurring in patients, and how coinfections influence the outcome of leishmaniasis is poorly understood. To address this issue, we infected mice with Leishmania major and 2 wk later with lymphocytic choriomeningitis virus (LCMV) and then monitored the course of infection. Leishmania parasites are controlled by production of IFN-γ, which leads to macrophage-mediated parasite killing. Thus, one might predict that coinfection with LCMV, which induces a strong systemic type 1 response, would accelerate disease resolution. However, we found that infection with LCMV led to significantly enhanced disease in L. major-infected animals. This increased disease correlated with an infiltration into the leishmanial lesions of NKG2D(+) CD8(+) T cells producing granzyme B, but surprisingly little IFN-γ. We found that depletion of CD8 T cells after viral clearance, as well as blockade of NKG2D, reversed the increased pathology seen in coinfected mice. Thus, this work highlights the impact a secondary infection can have on leishmaniasis and demonstrates that even pathogens known to promote a type 1 response may exacerbate leishmanial infections. PMID:26290604

  17. 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. PMID:26279524

  18. 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

  19. 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

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

    PubMed

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

    2006-10-01

    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(1)21/P3(2)21). The protein crystallized in two distinct trigonal crystal forms, with unit-cell parameters a = b = 111.0, c = 86.1 A and a = b = 111.0, c = 175.6 A, respectively. At a synchrotron beamline, the diffraction pattern extended to a resolution limit of 1.99 A. PMID:17012788

  1. 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. PMID:27260450

  2. In Vitro Infectivity Assessment by Drug Susceptibility Comparison of Recombinant Leishmania major Expressing Enhanced Green Fluorescent Protein or EGFP-Luciferase Fused Genes with Wild-Type Parasite

    PubMed Central

    Sadeghi, Somayeh; Seyed, Negar; Etemadzadeh, Mohammad-Hossein; Abediankenari, Saeid; Rafati, Sima; Taheri, Tahereh

    2015-01-01

    Leishmaniasis is a worldwide uncontrolled parasitic disease due to the lack of effective drug and vaccine. To speed up effective drug development, we need powerful methods to rapidly assess drug effectiveness against the intracellular form of Leishmania in high throughput assays. Reporter gene technology has proven to be an excellent tool for drug screening in vitro. The effects of reporter proteins on parasite infectivity should be identified both in vitro and in vivo. In this research, we initially compared the infectivity rate of recombinant Leishmania major expressing stably enhanced green fluorescent protein (EGFP) alone or EGFP-luciferase (EGFP-LUC) with the wild-type strain. Next, we evaluated the sensitivity of these parasites to amphotericin B (AmB) as a standard drug in 2 parasitic phases, promastigote and amastigote. This comparison was made by MTT and nitric oxide (NO) assay and by quantifying the specific signals derived from reporter genes like EGFP intensity and luciferase activity. To study the amastigote form, both B10R and THP-1 macrophage cell lines were infected in the stationary phase and were exposed to AmB at different time points. Our results clearly revealed that the 3 parasite lines had similar in vitro infectivity rates with comparable parasite-induced levels of NO following interferon-γ/lipopolysaccharide induction. Based on our results we proposed the more reporter gene, the faster and more sensitive evaluation of the drug efficiency. PMID:26323836

  3. The role of Montanide ISA 70 as an adjuvant in immune responses against Leishmania major induced by thiol-specific antioxidant-based protein vaccine.

    PubMed

    Khabazzadeh Tehrani, Narges; Mahdavi, Mehdi; Maleki, Fatemeh; Zarrati, Somayeh; Tabatabaie, Fatemeh

    2016-09-01

    Leishmaniasis is a parasitic disease caused by several species of the genus Leishmania. Montanide ISA 70 is an adjuvant composed of a natural metabolizable oil and a very refined emulsifier from the manide monooleate family. The TSA (thiol-specific antioxidant) is a important antigen of Leishmania major. The purpose of this work was protein-vaccine efficacy as an protection and excellent candidate in the presence Montanide. The expression of recombinant protein was confirmed with SDS (sodium dodecyl sulfate) page and Western bloting. 48 BALB/c mice were divided into four groups (TSA/Freund,TSA/Alum + BCG, TSA/Montanide and PBS groups) and immunized with 20 μg of vaccine subcutaneously three times intervals on days 0, 14 and 28. The mice were challenged with parasite 21 days after final immunization. The lymphocyte proliferation was evaluated with Brdu method. Cytokines and also total antibody and subclasses were evaluated with ELISA method. The vaccine formulated with the recombinant TSA protein with Montanide induced lymphocytes proliferation cytokines and total antibody and subclasses as compared with the control group. PMID:27605780

  4. Computational and Investigative Study of Flavonoids Active Against Typanosoma cruzi and Leishmania spp.

    PubMed

    Ribeiro, Frederico F; Junior, Francisco J B M; da Silva, Marcelo S; Scotti, Marcus Tullius; Scotti, Luciana

    2015-06-01

    Flavonoid compounds active against Trypanosoma cruzi and Leishmania species were submitted to several methodologies in silico: docking with the enzymes cruzain and trypanothione reductase (from T. cruzi), and N-myristoyltransferase, dihydroorotate dehydrogenase, and trypanothiona reductase (from Leishmania spp). Molecular maps of the complexes and the ligands were calculated. In order to compare and evaluate the antioxidant activity of the flavonoids with their antiprotozoal activity, quantum parameters were calculated. Considering the energies, interactions, and hydrophobic surfaces calculated, the flavonoids chrysin dimethyl ether against T. cruzi, and ladanein against Leishmania sp. presented the best results. The antioxidant activity did not show any correlation with anti-parasitic activity; only chrysin and its dimethyl ether showed favorable anti-parasitic results. This study hopes to contribute to existing research on these natural products against these tropical parasites. PMID:26197515

  5. Apoptotic cell clearance of Leishmania major-infected neutrophils by dendritic cells inhibits CD8+ T-cell priming in vitro by Mer tyrosine kinase-dependent signaling

    PubMed Central

    Ribeiro-Gomes, F L; Romano, A; Lee, S; Roffê, E; Peters, N C; Debrabant, A; Sacks, D

    2015-01-01

    Neutrophils are the predominant recruited and infected cells during the early stages of Leishmania major infection in the skin, and depletion of neutrophils promotes immunity to infection transmitted by sand fly bite. In order to better understand how the acute neutrophilic response suppresses immunity, we assessed the consequences of the interaction between neutrophils recovered from the skin-inoculation site and bone marrow-derived dendritic cells (DCs) in vitro. The capture of infected, apoptotic neutrophils by the DCs completely inhibited their cross-presentation function that was dependent on engagement of the receptor tyrosine kinase Mer on the DCs. The capture of uninfected neutrophils, or neutrophils infected with Toxoplasma gondii, had only slight immunomodulatory effects. These studies define the clearance of infected, apoptotic neutrophils by DCs and Mer receptor signaling as central to the early immune evasion strategies of L. major, with relevance to other vector-borne pathogens delivered by bite to the skin. PMID:26658192

  6. 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.

  7. 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. PMID:26896749

  8. 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.

  9. 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. PMID:27528683

  10. Characterization of Phlebotomus papatasi Peritrophins, and the Role of PpPer1 in Leishmania major Survival in its Natural Vector

    PubMed Central

    Coutinho-Abreu, Iliano V.; Sharma, Narinder K.; Robles-Murguia, Maricela; Ramalho-Ortigao, Marcelo

    2013-01-01

    The peritrophic matrix (PM) plays a key role in compartmentalization of the blood meal and as barrier to pathogens in many disease vectors. To establish an infection in sand flies, Leishmania must escape from the endoperitrophic space to prevent excretion with remnants of the blood meal digestion. In spite of the role played regarding Leishmania survival, little is known about sand fly PM molecular components and structural organization. We characterized three peritrophins (PpPer1, PpPer2, and PpPer3) from Phlebotomus papatasi. PpPer1 and PpPer2 display, respectively, four and one chitin-binding domains (CBDs). PpPer3 on the other hand has two CBDs, one mucin-like domain, and a putative domain with hallmarks of a CBD, but with changes in key amino acids. Temporal and spatial expression analyses show that PpPer1 is expressed specifically in the female midgut after blood feeding. PpPer2 and PpPer3 mRNAs were constitutively expressed in midgut and hindgut, with PpPer3 also being expressed in Malpighian tubules. PpPer2 was the only gene expressed in developmental stages. Interestingly, PpPer1 and PpPer3 expression are regulated by Le. major infection. Recombinant PpPer1, PpPer2 and PpPer3 were obtained and shown to display similar biochemical profiles as the native; we also show that PpPer1 and PpPer2 are able to bind chitin. Knockdown of PpPer1 led to a 44% reduction in protein, which in spite of producing an effect on the percentage of infected sand flies, resulted in a 39% increase of parasite load at 48 h. Our data suggest that PpPer1 is a component for the P. papatasi PM and likely involved in the PM role as barrier against Le. major infection. PMID:23516661

  11. Mechanistic studies on the bovine liver mitochondrial dihydroorotate dehydrogenase using kinetic deuterium isotope effects

    SciTech Connect

    Hines, V.; Johnston, M. )

    1989-02-07

    Dihydroorotates deuteriated at both C{sub 5} and C{sub 6} have been prepared and used to probe the mechanism of the bovine liver mitochondrial dihydroorotate dehydrogenase. Primary deuterium isotope effects on k{sub cat} are observed with both (6RS)-(5(S)-{sup 2}H)- and (6RS)-(6-{sup 2}H)dihydroorotates ({bold 3} and {bold 6}, respectively); these effects are maximal at low pH. At pH 6.6, {sup D}V = 3.4 for the C{sub 5}-deuteriated dihydroorotate ({bold 3}), and {sup D}V = 2.3 for the C{sub 6}-deuteriated compound ({bold 6}). The isotope effects approach unity at pH 8.8. Analysis of the pH dependence of the isotope effects on k{sub cat} reveals a shift in the rate-determining step of the enzyme mechanism as a function of pH. Dihydroorotate oxidation appears to require general base catalysis; this step is completely rate-determining at low pH and isotopically sensitive. Reduction of the cosubstrate, coenzyme Q{sub 6}, is rate-limiting at high pH and is isotopically insensitive; this step appears to require general acid catalysis. The results of double isotope substitution studies and analysis for substrate isotope exchange with solvent point toward a concerted mechanism for oxidation of dihydroorotate. This finding serves to distinguish further the mammalian dehydrogenase from its parasitic cognate, which catalyzes a stepwise oxidation reaction.

  12. Depletion of UDP-Glucose and UDP-Galactose Using a Degron System Leads to Growth Cessation of Leishmania major

    PubMed Central

    Bandini, Giulia; Zarnovican, Patricia; Buettner, Falk R.; Lüder, Carsten G. K.; Ferguson, Michael A. J.; Routier, Françoise H.

    2015-01-01

    Interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal) by the UDP-Glc 4´-epimerase intimately connects the biosynthesis of these two nucleotide sugars. Their de novo biosynthesis involves transformation of glucose-6-phosphate into glucose-1-phosphate by the phosphoglucomutase and subsequent activation into UDP-Glc by the specific UDP-Glc pyrophosphorylase (UGP). Besides UGP, Leishmania parasites express an uncommon UDP-sugar pyrophosphorylase (USP) able to activate both galactose-1-phosphate and glucose-1-phosphate in vitro. Targeted gene deletion of UGP alone was previously shown to principally affect expression of lipophosphoglycan, resulting in a reduced virulence. Since our attempts to delete both UGP and USP failed, deletion of UGP was combined with conditional destabilisation of USP to control the biosynthesis of UDP-Glc and UDP-Gal. Stabilisation of the enzyme produced by a single USP allele was sufficient to maintain the steady-state pools of these two nucleotide sugars and preserve almost normal glycoinositolphospholipids galactosylation, but at the apparent expense of lipophosphoglycan biosynthesis. However, under destabilising conditions, the absence of both UGP and USP resulted in depletion of UDP-Glc and UDP-Gal and led to growth cessation and cell death, suggesting that either or both of these metabolites is/are essential. PMID:26529232

  13. The PGE2/IL-10 Axis Determines Susceptibility of B-1 Cell-Derived Phagocytes (B-1CDP) to Leishmania major Infection.

    PubMed

    Arcanjo, Angélica F; LaRocque-de-Freitas, Isabel F; Rocha, Juliana Dutra B; Zamith, Daniel; Costa-da-Silva, Ana Caroline; Nunes, Marise Pinheiro; Mesquita-Santos, Fabio P; Morrot, Alexandre; Filardy, Alessandra A; Mariano, Mario; Bandeira-Melo, Christianne; DosReis, George A; Decote-Ricardo, Debora; Freire-de-Lima, Célio Geraldo

    2015-01-01

    B-1 cells can be differentiated from B-2 cells because they are predominantly located in the peritoneal and pleural cavities and have distinct phenotypic patterns and activation properties. A mononuclear phagocyte derived from B-1 cells (B-1CDP) has been described. As the B-1CDP cells migrate to inflammatory/infectious sites and exhibit phagocytic capacity, the microbicidal ability of these cells was investigated using the Leishmania major infection model in vitro. The data obtained in this study demonstrate that B-1CDP cells are more susceptible to infection than peritoneal macrophages, since B-1CDP cells have a higher number of intracellular amastigotes forms and consequently release a larger number of promastigotes. Exacerbated infection by L. major required lipid bodies/PGE2 and IL-10 by B-1CDP cells. Both infection and the production of IL-10 were decreased when PGE2 production was blocked by NSAIDs. The involvement of IL-10 in this mechanism was confirmed, since B-1CDP cells from IL-10 KO mice are more competent to control L. major infection than cells from wild type mice. These findings further characterize the B-1CDP cells as an important mononuclear phagocyte that plays a previously unrecognized role in host responses to L. major infection, most likely via PGE2-driven production of IL-10. PMID:25933287

  14. The PGE2/IL-10 Axis Determines Susceptibility of B-1 Cell-Derived Phagocytes (B-1CDP) to Leishmania major Infection

    PubMed Central

    Zamith, Daniel; Costa-da-Silva, Ana Caroline; Nunes, Marise Pinheiro; Mesquita-Santos, Fabio P.; Morrot, Alexandre; Filardy, Alessandra A.; Mariano, Mario; Bandeira-Melo, Christianne; DosReis, George A.; Decote-Ricardo, Debora; Freire-de-Lima, Célio Geraldo

    2015-01-01

    B-1 cells can be differentiated from B-2 cells because they are predominantly located in the peritoneal and pleural cavities and have distinct phenotypic patterns and activation properties. A mononuclear phagocyte derived from B-1 cells (B-1CDP) has been described. As the B-1CDP cells migrate to inflammatory/infectious sites and exhibit phagocytic capacity, the microbicidal ability of these cells was investigated using the Leishmania major infection model in vitro. The data obtained in this study demonstrate that B-1CDP cells are more susceptible to infection than peritoneal macrophages, since B-1CDP cells have a higher number of intracellular amastigotes forms and consequently release a larger number of promastigotes. Exacerbated infection by L. major required lipid bodies/PGE2 and IL-10 by B-1CDP cells. Both infection and the production of IL-10 were decreased when PGE2 production was blocked by NSAIDs. The involvement of IL-10 in this mechanism was confirmed, since B-1CDP cells from IL-10 KO mice are more competent to control L. major infection than cells from wild type mice. These findings further characterize the B-1CDP cells as an important mononuclear phagocyte that plays a previously unrecognized role in host responses to L. major infection, most likely via PGE2-driven production of IL-10. PMID:25933287

  15. Validity and Reliability of Enzyme Immunoassays Using Leishmania major or L. infantum Antigens for the Diagnosis of Canine Visceral Leishmaniasis in Brazil

    PubMed Central

    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

    Background 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. Methods 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. Results 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. Interpretation 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. PMID:23922884

  16. Functional identification of galactosyltransferases (SCGs) required for species-specific modifications of the lipophosphoglycan adhesin controlling Leishmania major-sand fly interactions.

    PubMed

    Dobson, Deborah E; Scholtes, Luella D; Valdez, Kelli E; Sullivan, Deborah R; Mengeling, Brenda J; Cilmi, Salvatore; Turco, Salvatore J; Beverley, Stephen M

    2003-05-01

    Lipophosphoglycan (LPG) is an abundant surface molecule that plays key roles in the infectious cycle of Leishmania major. The dominant feature of LPG is a polymer of phosphoglycan (PG) (6Galbeta1,4Manalpha1-PO(4)) repeating units. In L. major these are extensively substituted with Gal(beta1,3) side chains, which are required for binding to midgut lectins and survival. We utilized evolutionary polymorphisms in LPG structure and cross-species transfections to recover genes encoding the LPG side chain beta1,3-galactosyltransferases (betaGalTs). A dispersed family of six SCG genes was recovered, whose predicted proteins exhibited characteristics of eukaryotic GalTs. At least four of these proteins showed significant LPG side chain betaGalT activity; SCG3 exhibited initiating GalT activity whereas SCG2 showed both initiating and elongating GalT activity. However, the activity of SCG2 was context-dependent, being largely silent in its normal genomic milieu, and different strains show considerable variation in the extent of LPG galactosylation. Thus the L. major genome encodes a family of SCGs with varying specificity and activity, and we propose that strain-specific LPG galactosylation patterns reflect differences in their expression. PMID:12604613

  17. Lysophosphatidylcholine exacerbates Leishmania major-dendritic cell infection through interleukin-10 and a burst in arginase1 and indoleamine 2,3-dioxygenase activities.

    PubMed

    Tounsi, Nabila; Meghari, Soraya; Moser, Muriel; Djerdjouri, Bahia

    2015-03-01

    Leishmania major is an obligate intracellular parasite hosted by phagocytes, including dendritic cells (DCs). Lysophosphatidylcholine (LPC) a pro-oxidant by-product of phospholipase A2 activity can modulate the maturation and function of DCs. However, little is known about its role in L. major infection. This study examined the effects of LPC and lipopolysaccharide (LPS) in BALB/c mouse-derived DC infection by L. major promastigotes, in vitro. Our results showed early divergent effects of LPS and LPC, which lasted up to 24h. In contrast to LPS, LPC worsened DC infection by reversing the immune balance IL-10 vs. TNF-α and IL-6, and inducing a sharp down regulation of CD40 and iNOsynthase activity. In addition, LPC potentiated xanthine oxidase stress, the production of kynurenine by indoleamine 2,3 dioxygenase (IDO), and arginase1 activity in the expense of iNOsynthase. Taken together, our results highlight some biochemical events bypassing the protective Th1 response. They suggest that LPC could facilitate the proliferation of this obligate intracellular parasite by neutralizing oxidative and nitrosative stresses and sustaining both IDO and arginase1 activities. PMID:25601495

  18. Structural modeling and docking studies of ribose 5-phosphate isomerase from Leishmania major and Homo sapiens: a comparative analysis for Leishmaniasis treatment.

    PubMed

    Capriles, Priscila V S Z; Baptista, Luiz Phillippe R; Guedes, Isabella A; Guimarães, Ana Carolina R; Custódio, Fabio L; Alves-Ferreira, Marcelo; Dardenne, Laurent E

    2015-02-01

    Leishmaniases are caused by protozoa of the genus Leishmania and are considered the second-highest cause of death worldwide by parasitic infection. The drugs available for treatment in humans are becoming ineffective mainly due to parasite resistance; therefore, it is extremely important to develop a new chemotherapy against these parasites. A crucial aspect of drug design development is the identification and characterization of novel molecular targets. In this work, through an in silico comparative analysis between the genomes of Leishmania major and Homo sapiens, the enzyme ribose 5-phosphate isomerase (R5PI) was indicated as a promising molecular target. R5PI is an important enzyme that acts in the pentose phosphate pathway and catalyzes the interconversion of d-ribose-5-phosphate (R5P) and d-ribulose-5-phosphate (5RP). R5PI activity is found in two analogous groups of enzymes called RpiA (found in H. sapiens) and RpiB (found in L. major). Here, we present the first report of the three-dimensional (3D) structures and active sites of RpiB from L. major (LmRpiB) and RpiA from H. sapiens (HsRpiA). Three-dimensional models were constructed by applying a hybrid methodology that combines comparative and ab initio modeling techniques, and the active site was characterized based on docking studies of the substrates R5P (furanose and ring-opened forms) and 5RP. Our comparative analyses show that these proteins are structural analogs and that distinct residues participate in the interconversion of R5P and 5RP. We propose two distinct reaction mechanisms for the reversible isomerization of R5P to 5RP, which is catalyzed by LmRpiB and HsRpiA. We expect that the present results will be important in guiding future molecular modeling studies to develop new drugs that are specially designed to inhibit the parasitic form of the enzyme without significant effects on the human analog. PMID:25528729

  19. Zoonotic Disease in a Peripheral Population: Persistence and Transmission of Leishmania major in a Putative Sink-Source System in the Negev Highlands, Israel

    PubMed Central

    Berger, Ruti; Warburg, Alon; Orshan, Laor; Kotler, Burt P.

    2014-01-01

    Abstract 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. PMID:25072990

  20. 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. PMID:25072990

  1. Stability and toxicity of tris-tolyl bismuth(V) dicarboxylates and their biological activity towards Leishmania major.

    PubMed

    Ong, Yih Ching; Blair, Victoria L; Kedzierski, Lukasz; Tuck, Kellie L; Andrews, Philip C

    2015-11-01

    A series of 29 tris-tolyl bismuth(v) di-carboxylato complexes of composition [Bi(Tol)3(O2CR)2] involving either ortho, meta or para substituted tolyl ligands have been synthesized and characterised. Of these 15 were assessed for their toxicity towards Leishmania promastigotes and human fibroblast cells, with ten then being subsequently assessed against parasite amastigotes. The carboxylate ligands are drawn from a series of substituted and biologically relevant benzoic acids which allow a comparison with earlier studies on [BiPh3(O2CR)2] and analogous Sb(v) [SbAr3(O2CR)2] (Ar = Ph and Tol) complexes. Twelve complexes have been structurally characterized by single crystal X-ray diffraction and shown to adopt a typical trigonal bipyramidal geometry in which the three tolyl ligands occupy the equatorial plane. NMR studies on two illustrative examples indicate that the complexes are stable in D2O and DMSO but only have a half-life of 1.2 hours in culture medium, with glucose being a contributing factor in decomposition and reduction to Bi(Tol)3. Despite their short lifetime many complexes show significant toxicity towards promastigotes at low concentration (<6 μM) and at that concentration provide for good selectivity indices (parasite vs. mammalian cells), for example 114 for [Bi(o-Tol)3(O2CC6H3(2-OH,5-C6H3(2,4-F2)))2] and 838 for [Bi(m-Tol)3(O2CC6H4(2-OAc))2]. Best activity and selectivity is observed with complexes containing o- and m-tolyl ligands, and it appears the primary influence on fibroblast toxicity is the Ar ligand while the carboxylate influences promastigote toxicity. The complexes are less effective in vitro against the parasite amastigotes, where longer incubation times and harsher chemical and biological environments are encountered in the assay. Nevertheless, there were some statistically relevant differences at 1 μM against the positive controls with the best performing complexes being [Bi(o-Tol)3(O2CC6H4(2-EtO))2] and [Bi(m-Tol)3(O2CC6H4(2-OAc))2

  2. 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.

  3. Molecular cloning and characterization of a novel repeat-containing Leishmania major gene, ppg1, that encodes a membrane-associated form of proteophosphoglycan with a putative glycosylphosphatidylinositol anchor.

    PubMed

    Ilg, T; Montgomery, J; Stierhof, Y D; Handman, E

    1999-10-29

    Leishmania parasites secrete a variety of proteins that are modified by phosphoglycan chains structurally similar to those of the cell surface glycolipid lipophosphoglycan. These proteins are collectively called proteophosphoglycans. We report here the cloning and sequencing of a novel Leishmania major proteophosphoglycan gene, ppg1. It encodes a large polypeptide of approximately 2300 amino acids. The N-terminal domain of approximately 70 kDa exhibits 11 imperfect amino acid repeats that show some homology to promastigote surface glycoproteins of the psa2/gp46 complex. The large central domain apparently consists exclusively of approximately 100 repetitive peptides of the sequence APSASSSSA(P/S)SSSSS(+/-S). Gene fusion experiments demonstrate that these peptide repeats are the targets of phosphoglycosylation in Leishmania and that they form extended filamentous structures reminiscent of mammalian mucins. The C-terminal domain contains a functional glycosylphosphatidylinositol anchor addition signal sequence, which confers cell surface localization to a normally secreted Leishmania acid phosphatase, when fused to its C terminus. Antibody binding studies show that the ppg1 gene product is phosphoglycosylated by phosphoglycan repeats and cap oligosaccharides. In contrast to previously characterized proteophosphoglycans, the ppg1 gene product is predominantly membrane-associated and it is expressed on the promastigote cell surface. Therefore this membrane-bound proteophosphoglycan may be important for direct host-parasite interactions. PMID:10531342

  4. 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

  5. Polymerase chain reaction detection and inducible nitric-oxide synthase expression of Leishmania major in mice inoculated by two different routes

    PubMed Central

    Mahmoud, Abeer E; Attia, Rasha AH; Eldeek, Hanan EM; Farrag, Haiam Mohammed Mahmoud; Makboul, Rania

    2016-01-01

    Introduction: Leishmania major needs a sensitive and specific method for proper diagnosis. This study aims to study the course and histopathology of L. major in certain tissues of experimentally infected BALB/c mice after subcutaneous (sc) and intraperitoneal (ip) inoculation. Materials and Methods: After infecting BALB/c mice using sc and ip inoculation, the histopathology was studied. The kinetoplastic DNA polymerase chain reaction (PCR) for its molecular detection and detect the inducible nitric-oxide synthase (iNOS) pattern during the first 3 months of infection. Result: PCR could detect the presence of L. major in all spleens, lymph nodes, and skin ulcers by both inoculation routes while (33%) and (42%) of livers were positive after sc and ip routes, respectively. Chronic inflammatory cell infiltrates with capsulitis was found in the spleen, lymph nodes, and liver. Granulomas were found in the spleen and liver. There was a statistically significant difference in iNOS expression along the experiment in the spleen and lymph nodes by both routes and in the liver by ip only. Apart from the liver, iNOS could not be detected on the 2nd week postinfection and was high after 1 month for both routes in all samples; a moderate decrease at 2 months and the highest decrease were detected after 3 months. Conclusions: L. major inoculation by both routes produce visceral disease in mice, and kinetoplastic DNA PCR can detect its presence from the 2nd week up to the 3rd month postinfection. The iNOS expression was high at 1 and 2 months and remained throughout the 3 months of the experiment; which plays an important role in the disease course and control. PMID:26998433

  6. The stage-regulated HASPB and SHERP proteins are essential for differentiation of the protozoan parasite Leishmania major in its sand fly vector, Phlebotomus papatasi

    PubMed Central

    Sádlová, Jovana; Price, Helen P; Smith, Barbara A; Votýpka, Jan; Volf, Petr; Smith, Deborah F

    2010-01-01

    The stage-regulated HASPB and SHERP proteins of Leishmania major are predominantly expressed in cultured metacyclic parasites that are competent for macrophage uptake and survival. The role of these proteins in parasite development in the sand fly vector has not been explored, however. Here, we confirm that expression of HASPB is detected only in vector metacyclic stages, correlating with the expression of metacyclic-specific lipophosphoglycan and providing the first definitive protein marker for this infective sand fly stage. Similarly, SHERP is expressed in vector metacyclics but is also detected at low levels in the preceding short promastigote stage. Using genetically modified parasites lacking or complemented for the LmcDNA16 locus on chromosome 23 that contains the HASP and SHERP genes, we further show that the presence of this locus is essential for parasite differentiation to the metacyclic stage in Phlebotomus papatasi. While wild-type and complemented parasites transform normally in late-stage infections, generating metacyclic promastigotes and colonizing the sand fly stomodeal valve, null parasites accumulate at the earlier elongated nectomonad stage of development within the abdominal and thoracic midgut of the sand fly. Complementation with HASPB or SHERP alone suggests that HASPB is the dominant effector molecule in this process. PMID:20636473

  7. The IL-6-deficient mouse exhibits impaired lymphocytic responses to a vaccine combining live Leishmania major and CpG oligodeoxynucleotides

    PubMed Central

    Wu, Wenhui; Weigand, Luise; Mendez, Susana

    2009-01-01

    We have previously reported that vaccination with CpG oligodeoxynucleotides delivered concomitantly with live Leishmania major (Lm/CpG) eliminates lesions associated with live vaccination in C57BL/6 mice. The absence of lesions is at least in part a result of the CpG DNA-mediated activation of dermal dendritic cells to produce cytokines such as interleukin (IL)-6. Wild-type C57BL/6 mice and IL-6−/− mice were immunized with the Lm/CpG vaccine and monitored for the development of lesions. IL-6−/− mice developed extensive, nonhealing lesions following live vaccination. The analysis of the inoculation site and draining lymph nodes of the IL-6−/− mice revealed a constitutive reduction in lymphocyte numbers, particularly CD4+ T cells. Live vaccination resulted in the specific expansion of CD4+Foxp3+ regulatory T cells in the knockout mice, and in a decrease of CD4+ IFN-γ -producing cells. These results indicate that IL-6−/− mice may have collateral immune defects that could influence the development of the natural immune response to pathogens, vaccines, or other inflammatory stimuli. PMID:19767842

  8. Genome-wide analysis of small nucleolar RNAs of Leishmania major reveals a rich repertoire of RNAs involved in modification and processing of rRNA.

    PubMed

    Eliaz, Dror; Doniger, Tirza; Tkacz, Itai Dov; Biswas, Viplov Kumar; Gupta, Sachin Kumar; Kolev, Nikolay G; Unger, Ron; Ullu, Elisabetta; Tschudi, Christian; Michaeli, Shulamit

    2015-01-01

    Trypanosomatids are protozoan parasites and the causative agent of infamous infectious diseases. These organisms regulate their gene expression mainly at the post-transcriptional level and possess characteristic RNA processing mechanisms. In this study, we analyzed the complete repertoire of Leishmania major small nucleolar (snoRNA) RNAs by performing RNA-seq analysis on RNAs that were affinity-purified using the C/D snoRNA core protein, SNU13, and the H/ACA core protein, NHP2. This study revealed a large collection of C/D and H/ACA snoRNAs, organized in gene clusters generally containing both snoRNA types. Abundant snoRNAs were identified and predicted to guide trypanosome-specific rRNA cleavages. The repertoire of snoRNAs was compared to that of the closely related Trypanosoma brucei, and 80% of both C/D and H/ACA molecules were found to have functional homologues. The comparative analyses elucidated how snoRNAs evolved to generate molecules with analogous functions in both species. Interestingly, H/ACA RNAs have great flexibility in their ability to guide modifications, and several of the RNA species can guide more than one modification, compensating for the presence of single hairpin H/ACA snoRNA in these organisms. Placing the predicted modifications on the rRNA secondary structure revealed hypermodification regions mostly in domains which are modified in other eukaryotes, in addition to trypanosome-specific modifications. PMID:25970223

  9. Leishmania major: anti-leishmanial activity of Nuphar lutea extract mediated by the activation of transcription factor NF-κB.

    PubMed

    Ozer, Lital; El-On, Joseph; Golan-Goldhirsh, Avi; Gopas, Jacob

    2010-12-01

    Here we report the effect of a partially purified alkaloid fraction (NUP) of Nuphar lutea on nuclear factor kappa B (NF-κB) expression and studied its mechanism of toxicity against Leishmania major in C3H mice peritoneal macrophages. NUP was found to be a mixture of thermo-stable dimeric sesquiterpene thioalkaloids containing mainly thionupharidines. The anti-leishmanial activity was shown to be mediated through the activation of NF-κB and increased iNOS production. Additionally, the nitric oxide inhibitor, N(G)-monomethyl-L-arginine (0.5mM) totally reverted the anti-leishmanial effect of NUP (0.25 and 0.5μg/ml). NUP was also shown to act as an anti-oxidant, almost completely inhibiting the macrophage respiratory burst activity. However, no elevated lysozyme (EC3.2.1.17) or β-galactosidase (EC3.2.1.23) activities were demonstrated in macrophages treated with NUP. This study suggests, that the activity of NUP is mediated by NF-κB activation and the production of nitric oxide which is dependent on the L-arginine:NO pathway. PMID:20515684

  10. Characterization of a compensatory mutant of Leishmania major that lacks ether lipids but exhibits normal growth, and G418 and hygromycin resistance.

    PubMed

    Zufferey, Rachel; Bibis, Stergios S; Zhu, Tongtong; Dhalladoo, Subbhalakshmi

    2012-03-01

    Ether glycerolipid biosynthesis in Leishmania major initiates with the acylation of dihydroxyacetonephosphate by the glycosomal dihydroxyacetonephosphate acyltransferase LmDAT. We previously reported that a null mutant of LmDAT is severely affected in logarithmic growth, survival during stationary phase, and in virulence in mice. In addition, it lacks all ether glycerolipids, produces altered forms of the ether-lipid based virulence factors lipophosphoglycan and increased levels of GPI-anchored protein gp63. Here, we describe the characterization of a compensatory mutant of a null strain of LmDAT, Δlmdat/Δlmdat(rev). Similarly to the null mutant, the Δlmdat/Δlmdat(rev) strain formed altered forms of lipophosphoglycan and increased levels of gp63, and was avirulent in mice infection. Further, dihydroxyacetonephosphate acyltransferase activity was absent in the revertant clone, indicating that a mutation in another acyltransferase gene did not confer dihydroxyacetonephosphate specificity. In contrast, the revertant grew normally but still exhibited poor survival during stationary phase. In addition, agarose gel analysis of its genomic DNA failed to detect any amplified DNA. Surprisingly, its sensitivity to aminoglycoside based antibiotics G418 and hygromycin was lower than that of the null mutant, wild type and complemented line. PMID:22306069

  11. 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

  12. In vitro efficacy of ethanolic extract of Artemisia absinthium (Asteraceae) against Leishmania major L. using cell sensitivity and flow cytometry assays.

    PubMed

    Azizi, Kourosh; Shahidi-Hakak, Fatemeh; Asgari, Qasem; Hatam, Gholam Reza; Fakoorziba, Mohammad Reza; Miri, Ramin; Moemenbellah-Fard, Mohammad Djaefar

    2016-09-01

    Leishmaniasis is one of the most neglected human diseases with an estimated global burden ranking second in mortality and fourth in morbidity among the tropical infections. Chemotherapy involving the use of drugs like glucantime is the mainstay treatment in endemic areas of Iran. Drug resistance is increasingly prevalent, so search for alternative therapy is gathering pace. Medicinal herbs, like wormwood Artemisia, have chemical compounds effective against a number of pathogens. In this study, the efficacy of ethanol extract from Artemisia absinthium (Asteraceae) against Leishmania major L. was investigated in vitro. The outcome of different effective doses (1-40 mg/ml) of ethanol extracts from this medicinal herb, A. absinthium, on a standard Iranian parasite strain of L. major was examined. The L. major promastigote cell sensitivity and mortality or viability effects due to the addition of herbal extract were measured using the MTT assay and the flow cytometry technique, respectively. There was complete agreement between the two assays. The lethal concentration (LC50) was measured as 101 mg/ml. Some contrasting relationships between the medicinal herb concentrations and the viability of parasites were observed; so that there was an increased multiplication of the parasite at low concentrations of the drug, but an anti-parasitic apoptotic effect was seen at high concentrations of A. absinthium. It was concluded that there might be one or more chemical constituents within the herbal extract of wormwood which at high concentration controlled cell division and affected the relevant activity within the only one giant mitochondrion in this flagellate parasite. At low doses, however, it showed the opposite effect of leading to mitotic cell divisions. PMID:27605775

  13. Atenolol Reduces Leishmania major-Induced Hyperalgesia and TNF-α Without Affecting IL-1β or Keratinocyte Derived Chemokines (KC).

    PubMed

    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

  14. 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

  15. 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.

  16. The identity of Leishmania isolated from sand flies and vertebrate hosts in a major focus of cutaneous leishmaniasis in Baturite, northeastern Brazil.

    PubMed

    Vasconcelos, I A; Vasconcelos, A W; Fe Filho, N M; Queiroz, R G; Santana, E W; Bozza, M; Sallenave, S M; Valim, C; David, J R; Lopes, U G

    1994-02-01

    During a field investigation carried out in Baturite, Brazil from 1989 to 1991, sand flies, sympatric rodents, domestic dogs and humans were surveyed for leishmaniasis. Twenty strains of Leishmania were isolated by in vitro culture from Lutzomyia whitmani, three strains were obtained from Rattus rattus, two strains from dogs, and five strains from humans. The isolates were characterized by isoenzyme electrophoresis by hybridization with kinetoplast DNA-specific probes. All the samples were identified as L. (Viannia) braziliensis. The importance of these results in the dynamics of the Leishmania infection in this focus is discussed. PMID:8116807

  17. 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

  18. 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%.

  19. Deficiency of prolactin-inducible protein leads to impaired Th1 immune response and susceptibility to Leishmania major in mice.

    PubMed

    Li, Jintao; Liu, Dong; Mou, Zhirong; Ihedioha, Olivia C; Blanchard, Anne; Jia, Ping; Myal, Yvonne; Uzonna, Jude E

    2015-04-01

    Although the strategic production of prolactin-inducible protein (PIP) at several ports of pathogen entry into the body suggests it might play a role in host defense, no study has directly implicated it in immunity against any infectious agent. Here, we show for the first time that PIP deficiency is associated with reduced numbers of CD4(+) T cells in peripheral lymphoid tissues and impaired CD4(+) Th1-cell differentiation in vitro. In vivo, CD4(+) T cells from OVA-immunized, PIP-deficient mice showed significantly impaired proliferation and IFN-γ production following in vitro restimulation. Furthermore, PIP-deficient mice were highly susceptible to Leishmani major infection and failed to control lesion progression and parasite proliferation. This susceptibility was associated with impaired NO production and leishmanicidal activity of PIP KO macrophages following IFN-γ and LPS stimulation. Collectively, our findings implicate PIP as an important regulator of CD4(+) Th1-cell-mediated immunity. PMID:25594453

  20. Genome-Wide Analysis of C/D and H/ACA-Like Small Nucleolar RNAs in Leishmania major Indicates Conservation among Trypanosomatids in the Repertoire and in Their rRNA Targets▿ †

    PubMed Central

    Liang, Xue-hai; Hury, Avraham; Hoze, Ehud; Uliel, Shai; Myslyuk, Inna; Apatoff, Avihay; Unger, Ron; Michaeli, Shulamit

    2007-01-01

    Small nucleolar RNAs (snoRNAs) are a large group of noncoding RNAs that exist in eukaryotes and archaea and guide modifications such as 2′-O-ribose methylations and pseudouridylation on rRNAs and snRNAs. Recently, we described a genome-wide screening approach with Trypanosoma brucei that revealed over 90 guide RNAs. In this study, we extended this approach to analyze the repertoire of the closely related human pathogen Leishmania major. We describe 23 clusters that encode 62 C/Ds that can potentially guide 79 methylations and 37 H/ACA-like RNAs that can potentially guide 30 pseudouridylation reactions. Like T. brucei, Leishmania also contains many modifications and guide RNAs relative to its genome size. This study describes 10 H/ACAs and 14 C/Ds that were not found in T. brucei. Mapping of 2′-O-methylations in rRNA regions rich in modifications suggests the existence of trypanosomatid-specific modifications conserved in T. brucei and Leishmania. Structural features of C/D snoRNAs, such as copy number, conservation of boxes, K turns, and intragenic and extragenic base pairing, were examined to elucidate the great variation in snoRNA abundance. This study highlights the power of comparative genomics for determining conserved features of noncoding RNAs. PMID:17189491

  1. Leishmania major-human macrophage interactions: cooperation between Mac-1 (CD11b/CD18) and complement receptor type 1 (CD35) in promastigote adhesion.

    PubMed Central

    Rosenthal, L A; Sutterwala, F S; Kehrli, M E; Mosser, D M

    1996-01-01

    It has been suggested that the developmental maturation of Leishmania major promastigotes can affect their interaction with human complement receptors. To study this, we measured the adhesion of metacyclic and logarithmic-phase L. major promastigotes to complement receptors expressed on primary macrophages, to recombinant receptors expressed on transfected cells, or to purified complement receptors in a cell-free system. We demonstrate that complement-opsonized promastigotes can bind to both Mac-1 and complement receptor type 1 (CR1) and that the transition of promastigotes from the noninfectious logarithmic phase of growth to the infectious metacyclic stage does not affect this interaction. Furthermore, we show that Mac-1 and CR1 can cooperate to mediate the efficient adhesion of complement-opsonized metacyclic promastigotes to cells expressing both receptors. On human monocyte-derived macrophages, Mac-1 appears to make a quantitatively greater contribution to this adhesion than does CR1, since blocking macrophage Mac-1 diminishes metacyclic promastigote adhesion to a greater extent than does blocking CR1. In addition, bovine monocytes lacking Mac-1 exhibit a dramatic decrease in complement-dependent promastigote adhesion, relative to normal monocytes. The predominance of Mac-1 in these interactions is due, at least in part, to the factor I cofactor activity of CR1, which facilitates the conversion of C3b to iC3b. The stable adhesion of complement-opsonized metacyclic promastigotes to Mac-1 is a prerequisite for phagocytosis by human monocyte-derived macrophages. Blocking Mac-1 on macrophages abrogates the majority of the complement-dependent phagocytosis of promastigotes, whereas blocking CR1 has no detectable effect on phagocytosis. In addition, bovine monocytes lacking Mac-1 exhibit a dramatic reduction in promastigote phagocytosis relative to normal bovine monocytes. We conclude, therefore, that the two complement receptors, Mac-1 and CR1, can cooperate to

  2. Identification of a compensatory mutant (lpg2-REV) of Leishmania major able to survive as amastigotes within macrophages without LPG2-dependent glycoconjugates and its significance to virulence and immunization strategies.

    PubMed

    Späth, Gerald F; Lye, Lon-Fye; Segawa, Hiroaki; Turco, Salvatore J; Beverley, Stephen M

    2004-06-01

    Different Leishmania species rely to different extents on abundant glycoconjugates, such as lipophosphoglycan (LPG) and related molecules, in mammalian infections. Previously, we showed that Leishmania major deletion mutants lacking the Golgi GDP-mannose transporter LPG2, which is required for assembly of the dominant phosphoglycan (PG) repeats of LPG, were unable to survive in macrophages. These lpg2- mutants, however, retained the ability to generate asymptomatic, persistent infections in mice. In contrast, Ilg and colleagues showed that Leishmania mexicana LPG2 mutants retained virulence for mice. Here we identified a partial revertant population of the L. major lpg2- mutants (designated lpg2(-)REV) that had regained the ability to replicate in macrophages and induce disease pathology through a compensatory change. Like the lpg2 parent, the lpg2(-)REV revertant was unable to synthesize LPG2-dependent PGs in the promastigote stage and thus remained highly attenuated in the ability to induce infection. However, after considerable delay lpg2(-)REV revertant-infected mice exhibited lesions, and amastigotes isolated from these lesions were able to replicate within macrophages despite the fact that they were unable to synthesize PGs. Thus, in some respects, the lpg2(-)REV amastigotes resemble L. mexicana amastigotes. Future studies of the gene(s) responsible may shed light on the mechanisms employed by L. major to survive in the absence of LPG2-dependent glycoconjugates and may also improve the potential of the lpg2- L. major line to serve as a live parasite vaccine by overcoming its tendency to revert toward virulence. PMID:15155672

  3. 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

  4. Insights into the mechanism of oxidation of dihydroorotate to orotate catalysed by human class 2 dihydroorotate dehydrogenase: a QM/MM free energy study.

    PubMed

    Alves, Cláudio Nahum; Silva, José Rogério A; Roitberg, Adrian E

    2015-07-21

    The dihydroorotate dehydrogenase (DHOD) enzyme catalyzes the unique redox reaction in the de novo pyrimidine biosynthesis pathway. In this reaction, the oxidation of dihydroorotate (DHO) to orotate (OA) and reduction of the flavin mononucleotide (FMN) cofactor is catalysed by DHOD. The class 2 DHOD, to which the human enzyme belongs, was experimentally shown to follow a stepwise mechanism but the data did not allow the determination of the order of bond-breaking in a stepwise oxidation of DHO. The goal of this study is to understand the reaction mechanism at the molecular level of class 2 DHOD, which may aid in the design of inhibitors that selectively impact the activity of only certain members of the enzyme family. In this paper, the catalytic mechanism of oxidation of DHO to OA in human DHOD was studied using a hybrid Quantum Mechanical/Molecular Mechanical (QM/MM) approach and Molecular Dynamics (MD) simulations. The free energy barriers calculated reveal that the mechanism in human DHOD occurs via a stepwise reaction pathway. In the first step, a proton is abstracted from the C5 of DHO to the deprotonated Ser215 side chain. Whereas, in the second step, the transfer of the hydride or hydride equivalent from the C6 of DHO to the N5 of FMN, where free energy barrier calculated by the DFT/MM level is 10.84 kcal mol(-1). Finally, a residual decomposition analysis was carried out in order to elucidate the influence of the catalytic region residues during DHO oxidation. PMID:26087682

  5. 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. PMID:26232657

  6. Characterization of Leishmania major phosphatidylethanolamine methyltransferases LmjPEM1 and LmjPEM2 and their inhibition by choline analogs.

    PubMed

    Bibis, Stergios S; Dahlstrom, Kelly; Zhu, Tongtong; Zufferey, Rachel

    2014-09-01

    Phosphatidylcholine (PC) is the most abundant phospholipid in the membranes of the human parasite Leishmania. It is synthesized via two metabolic routes, the de novo pathway that starts with the uptake of choline, and the threefold methylation of phosphatidylethanolamine. Choline was shown to be dispensable for Leishmania; thus, the methylation pathway likely represents the primary route for PC production. Here, we have identified and characterized two phosphatidylethanolamine methyltransferases, LmjPEM1 and LmjPEM2. Both enzymes are expressed in promastigotes as well as in the vertebrate form amastigotes, suggesting that these methyltransferases are important for the development of the parasite throughout its life cycle. These enzymes are maximally expressed during the log phase of growth which correlates with the demand of PC synthesis during cell multiplication. Immunofluorescence studies combined with cell fractionation have shown that both methyltransferases are localized at the endoplasmic reticulum membrane. Heterologous expression in yeast has demonstrated that LmjPEM1 and LmjPEM2 complement the choline auxotrophy phenotype of a yeast double null mutant lacking phosphatidylethanolamine methyltransferase activity. LmjPEM1 catalyzes the first, and to a lesser extent, the second methylation reaction. In contrast, LmjPEM2 has the capacity to add the second and third methyl group onto phosphatidylethanolamine to yield (lyso)PC; it can also add the first methyl group, albeit with very low efficiency. Finally, we have demonstrated using inhibition studies with choline analogs that miltefosine and octadecyltrimethylammonium bromide are potent inhibitors of this metabolic pathway. PMID:25176160

  7. Dihydroorotate dehydrogenase depletion hampers mitochondrial function and osteogenic differentiation in osteoblasts.

    PubMed

    Fang, JingXian; Yamaza, Haruyoshi; Uchiumi, Takeshi; Hoshino, Yoshihiro; Masuda, Keiji; Hirofuji, Yuta; Wagener, Frank A D T G; Kang, Dongchon; Nonaka, Kazuaki

    2016-06-01

    Mutation of the dihydroorotate dehydrogenase (DHODH) gene is responsible for Miller syndrome, which is characterized by craniofacial malformations with limb abnormalities. We previously demonstrated that DHODH was involved in forming a mitochondrial supercomplex and that mutated DHODH led to protein instability, loss of enzyme activity, and increased levels of reactive oxygen species in HeLa cells. To explore the etiology of Miller syndrome in more detail, we investigated the effects of DHODH inhibition in the cells involved in skeletal structure. Dihydroorotate dehydrogenase in MC3T3-E1 cells derived from mouse calvaria osteoblast precursor cells was knocked down by specific small interfering RNAs (siRNAs), and cell proliferation, ATP production, and expression of bone-related genes were investigated in these cells. After depletion of DHODH using specific siRNAs, inhibition of cell proliferation and cell cycle arrest occurred in MC3T3-E1 cells. In addition, ATP production was reduced in whole cells, especially in mitochondria. Furthermore, the levels of runt-related transcription factor 2 (Runx2) and osteocalcin (Ocn) mRNAs were lower in DHODH siRNA-treated cells compared with controls. These data suggest that depletion of DHODH affects the differentiation and maturation of osteoblasts. This study shows that mitochondrial dysfunction by DHODH depletion in osteoblasts can be directly linked to the abnormal bone formation in Miller syndrome. PMID:27086500

  8. Expression, purification and crystallization of Trypanosoma cruzi dihydroorotate dehydrogenase complexed with orotate.

    PubMed

    Inaoka, Daniel Ken; Takashima, Eizo; Osanai, Arihiro; Shimizu, Hironari; Nara, Takeshi; Aoki, Takashi; Harada, Shigeharu; Kita, Kiyoshi

    2005-10-01

    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 A resolution using synchrotron radiation (lambda = 0.900 A). X-ray diffraction data were collected at 100 K and processed to 1.9 A resolution with 98.2% completeness and an overall Rmerge of 7.8%. The TcDHOD crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 67.87, b = 71.89, c = 123.27 A. The presence of two molecules in the asymmetric unit (2 x 34 kDa) gives a crystal volume per protein weight (VM) of 2.2 A3 Da(-1) and a solvent content of 44%. PMID:16511183

  9. Apoptotic cell clearance of Leishmania major-infected neutrophils by dendritic cells inhibits CD8⁺ T-cell priming in vitro by Mer tyrosine kinase-dependent signaling.

    PubMed

    Ribeiro-Gomes, F L; Romano, A; Lee, S; Roffê, E; Peters, N C; Debrabant, A; Sacks, D

    2015-01-01

    Neutrophils are the predominant recruited and infected cells during the early stages of Leishmania major infection in the skin, and depletion of neutrophils promotes immunity to infection transmitted by sand fly bite. In order to better understand how the acute neutrophilic response suppresses immunity, we assessed the consequences of the interaction between neutrophils recovered from the skin-inoculation site and bone marrow-derived dendritic cells (DCs) in vitro. The capture of infected, apoptotic neutrophils by the DCs completely inhibited their cross-presentation function that was dependent on engagement of the receptor tyrosine kinase Mer on the DCs. The capture of uninfected neutrophils, or neutrophils infected with Toxoplasma gondii, had only slight immunomodulatory effects. These studies define the clearance of infected, apoptotic neutrophils by DCs and Mer receptor signaling as central to the early immune evasion strategies of L. major, with relevance to other vector-borne pathogens delivered by bite to the skin. PMID:26658192

  10. A comparative study of the effects of venoms from five rear-fanged snake species on the growth of Leishmania major: identification of a protein with inhibitory activity against the parasite.

    PubMed

    Peichoto, María E; Tavares, Flávio L; Dekrey, Gregory; Mackessy, Stephen P

    2011-07-01

    Leishmania parasites of several species cause cutaneous and visceral disease to millions of people worldwide, and treatment for this vector-borne protozoan parasite typically involves administration of highly toxic antimonial drugs. Snake venoms are one of the most concentrated enzyme sources in nature, displaying a broad range of biological effects, and several drugs now used in humans were derived from venoms. In this study, we compared the effects of the venoms of the South American rear-fanged snakes Philodryas baroni (PbV), Philodryas olfersii olfersii (PooV) and Philodryas patagoniensis (PpV), and the North American rear-fanged snakes Hypsiglena torquata texana (HttV) and Trimorphodon biscutatus lambda (TblV), on the growth of Leishmania major, a causative agent of cutaneous leishmaniasis. Different concentrations of each venom were incubated with the log-phase promastigote stage of L. major. TblV showed significant anti-leishmanial activity (IC₅₀ of 108.6 μg/mL) at its highest concentrations; however, it induced parasite proliferation at intermediate concentrations. PpV was not very active in decreasing the parasitic growth, and a high final concentration (1.7 mg/mL) was necessary to inhibit proliferation by only 51.5% ± 3.6%. PbV, PooV and HttV, at final concentrations of 562, 524 and 438 μg/mL respectively, had no significant effect on L. major growth. The phospholipase A₂ of TblV (trimorphin) was isolated and assayed as for crude venom, and it also exhibited dose-dependent biphasic effects on the parasite culture, with potent cytotoxicity at higher concentrations (IC₅₀ of 0.25 μM; 3.6 μg/mL) and stimulation of proliferation at very low concentrations. Anti-leishmanial activity of TblV appears to be solely due to the action of trimorphin. This is the first report of anti-leishmanial activity of rear-fanged snake venoms, and these results suggest novel possibilities for discovering new protein-based drugs that might be used as possible agents

  11. 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

  12. Genomic organization and expression of the expanded SCG/L/R gene family of Leishmania major: internal clusters and telomeric localization of SCGs mediating species-specific LPG modifications.

    PubMed

    Dobson, Deborah E; Scholtes, Luella D; Myler, Peter J; Turco, Salvatore J; Beverley, Stephen M

    2006-04-01

    Stage-specific modifications to the abundant surface lipophosphoglycan (LPG) adhesin of Leishmania play critical roles in binding and release of the parasite during its infectious cycle in the sand fly, and control the ability of different fly species to transmit different parasite strains and species. In Leishmania major Friedlin V1, binding to a sand fly midgut lectin is mediated by side chain galactosyl (scGal) modifications of the LPG phosphoglycan (PG) repeats, while release occurs following arabinose-capping of scGals. Previously we identified a family of six SCG genes encoding PG scbeta-galactosyltransferases, and here we show that the extended SCG gene family (now termed SCG/L/R) encompasses 14 members in three subfamilies (SCG, SCGL and SCGR). Northern blot and RT-PCR analyses suggest that most of the SCG/L/R genes are expressed, with distinct patterns during the infectious cycle. The six SCGR subfamily genes are clustered and interspersed with the two SCA genes responsible for developmentally regulated arabinosylation of PG scGals; relationships amongst the SCGR revealed clear evidence of extensive gene conversion. In contrast, the seven SCG 'core' family members are localized adjacent to telomeres. These telomeres share varying amounts of sequence upstream and/or downstream of the SCG ORFs, again providing evidence of past gene conversions. Multiple SCG1-7 RNAs were expressed simultaneously within parasite populations. Potentially, telomeric localization of SCG genes may function primarily to facilitate gene conversion and the elaboration of functional evolutionary diversity in the degree of PG sc-galactosylation observed in other strains of L. major. PMID:16464509

  13. Identification and characterization of new Leishmania promastigote surface antigens, LaPSA-38S and LiPSA-50S, as major immunodominant excreted/secreted components of L. amazonensis and L. infantum.

    PubMed

    Bras-Gonçalves, Rachel; Petitdidier, Elodie; Pagniez, Julie; Veyrier, Renaud; Cibrelus, Prisca; Cavaleyra, Mireille; Maquaire, Sarah; Moreaux, Jérôme; Lemesre, Jean-Loup

    2014-06-01

    We have previously demonstrated that sera from dogs vaccinated with excreted/secreted antigens (ESA) of Leishmania infantum promastigotes (LiESAp) mainly recognized an immunodominant antigen of 54 kDa. An anti-LiESAp-specific IgG2 humoral response was observed and associated to Th1-type response in vaccinated dogs. This response was highly correlated with a long-lasting and strong LiESAp-vaccine protection toward L. infantum experimental infection. In addition, it was also shown that dogs from the vaccinated group developed a selective IgG2 response against an immunodominant antigen of 45 kDa of Leishmania amazonensis ESA promastigotes (LaESAp). In order to identify and characterize these immunodominant antigens, a mouse monoclonal antibody (mAb F5) was produced by immunization against LaESAp. It was found to recognize the major antigenic targets of both LaESAp and LiESAp. Analysis with mAb F5 of L. amazonensis amastigote and promastigote cDNA expression libraries enabled the identification of clones encoding proteins with significant structural homology to the promastigote surface antigens named PSA-2/gp-46. Among them, one clone presented a full-length cDNA and encoded a novel L. amazonensis protein of 38.6 kDa calculated molecular mass (LaPSA-38S) sharing an amino acid sequence consistent with that of the PSA polymorphic family and a N-terminal signal peptide, characteristic of a secreted protein. We then screened a L. infantum promastigote DNA cosmid library using a cDNA probe derived from the LaPSA-38S gene and identified a full-length clone of a novel excreted/secreted protein of L. infantum with a calculated molecular mass of 49.2 kDa and named LiPSA-50S. The fact that a significant immunological reactivity was observed against PSA, suggests that these newly identified proteins could have an important immunoregulatory influence on the immune response. This hypothesis is supported by the fact that (i) these proteins were naturally excreted/secreted by viable

  14. Novel selective and potent inhibitors of malaria parasite dihydroorotate dehydrogenase: discovery and optimization of dihydrothiophenone derivatives.

    PubMed

    Xu, Minghao; Zhu, Junsheng; Diao, Yanyan; Zhou, Hongchang; Ren, Xiaoli; Sun, Deheng; Huang, Jin; Han, Dongmei; Zhao, Zhenjiang; Zhu, Lili; Xu, Yufang; Li, Honglin

    2013-10-24

    Taking the emergence of drug resistance and lack of effective antimalarial vaccines into consideration, it is of significant importance to develop novel antimalarial agents for the treatment of malaria. Herein, we elucidated the discovery and structure-activity relationships of a series of dihydrothiophenone derivatives as novel specific inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH). The most promising compound, 50, selectively inhibited PfDHODH (IC50 = 6 nM, with >14,000-fold species-selectivity over hDHODH) and parasite growth in vitro (IC50 = 15 and 18 nM against 3D7 and Dd2 cells, respectively). Moreover, an oral bioavailability of 40% for compound 50 was determined from in vivo pharmacokinetic studies. These results further indicate that PfDHODH is an effective target for antimalarial chemotherapy, and the novel scaffolds reported in this work might lead to the discovery of new antimalarial agents. PMID:24073986

  15. 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

  16. 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.

  17. Immunogenicity of a Salmonella typhi CVD 908 candidate vaccine strain expressing the major surface protein gp63 of Leishmania mexicana mexicana.

    PubMed

    González, C R; Noriega, F R; Huerta, S; Santiago, A; Vega, M; Paniagua, J; Ortiz-Navarrete, V; Isibasi, A; Levine, M M

    1998-01-01

    Attenuated Salmonella typhi are attractive for use as live vector vaccines to express protozoal antigens and deliver them to the human immune system. The gene encoding the mature form of Leishmania mexicana mexicana gp63 under control of tac promoter was integrated into the delta aroC locus of the chromosome of attenuated delta aroC, delta aroD S. typhi strain CVD 908. After oral immunization of BALB/c mice with two 1 x 10(9) colony forming unit doses given 21 days apart, CVD 908 omega (delta aroC::Ptac-gp63) elicited a broad T cell-mediated immune response against L. m. mexicana gp63 as demonstrated by: (1) lymphoproliferative response to fixed whole L. m. mexicana promastigotes; (2) activation of IL-2 (but not IL-4)-producing lymphocytes; (3) appearance of cytotoxic T cells against mouse mastocytoma cells expressing gp63. This T-cell mediated immune response was associated with significant protection in F1 (BALB/cXC57Bl/6) mice challenged in their footpads with a wild type strain of L. m. mexicana. PMID:9682357

  18. Detection of Leishmania (Leishmania) amazonensis and Leishmania (Leishmania) infantum chagasi in Brazilian bats.

    PubMed

    Savani, Elisa San Martin Mouriz; de Almeida, Marilene Fernandes; de Oliveira Camargo, Maria Cecília Gibrail; D'Auria, Sandra Regina Nicoletti; Silva, Miriam Martos Sodré; de Oliveira, Maria Lúcia; Sacramento, Débora

    2010-02-26

    Although bats are one of the most abundant mammals in the new world and are present in virtually all ecosystems, including urban and peri-urban environments, few studies have investigated the role of these animals in the epidemiological chain of leishmaniosis. Here, we report a study of 683 bats captured in São Paulo county (southeastern from Brazil), which were screened by serology, parasitologic methods and polymerase chain reaction (PCR) for trypanosomatids. The indirect immunofluorescent antibody test demonstrated that 0.9% of bats react positively for leishmaniosis and PCR detected the presence of DNA of Leishmania (Leishmania) amazonensis in 18 bats and Leishmania (Leishmania) infantum chagasi in 3 specimens. These results indicate that further studies are necessary to evaluate the role of bats in maintenance of the Leishmania life cycle, especially in areas where these diseases are endemic. PMID:19939568

  19. 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

  20. Is lipophosphoglycan a virulence factor? A surprising diversity between Leishmania species.

    PubMed

    Turco, S J; Späth, G F; Beverley, S M

    2001-05-01

    Lipophosphoglycan is a prominent member of the phosphoglycan-containing surface glycoconjugates of Leishmania. Genetic tests enable confirmation of its role in parasite virulence and permit discrimination between the roles of lipophosphoglycan and related glycoconjugates. When two different lipophosphoglycan biosynthetic genes from Leishmania major were knocked out, there was a clear loss of virulence in several steps of the infectious cycle but, with Leishmania mexicana, no effect on virulence was found. This points to an unexpected diversity in the reliance of Leishmania species on virulence factors, a finding underscored by recent studies showing great diversity in the host response to Leishmania species. PMID:11323305

  1. Computational study of the mechanism of half-reactions in class 1A dihydroorotate dehydrogenase from Trypanosoma cruzi.

    PubMed

    Silva, Natália de Farias; Lameira, Jerônimo; Alves, Cláudio Nahum; Martí, Sergio

    2013-11-21

    Chagas' disease is considered to be a health problem affecting millions of people in Latin America. This disease is caused by the parasite Trypanosoma cruzi. Recently dihydroorotate dehydrogenase class 1A from Trypanosoma cruzi (TcDHODA) was shown to be essential for the survival and growth of T. cruzi and proposed as a drug target against Chagas' disease. This enzyme catalyzes the oxidation of (S)-dihydroorotate to orotate, with a proposed catalytic cycle consisting of two half-reactions. In the first half-reaction dihydroorotate is oxidized to orotate, with the consequent reduction of the flavin mononucleotide cofactor. In the second half-reaction fumarate is reduced to succinate. The first oxidation half-reaction may occur via a concerted or a stepwise mechanism. Herein, the catalytic mechanism of TcDHODA has been studied using hybrid Quantum Mechanical/Molecular Mechanical (QM/MM) Molecular Dynamics (MD) simulations. The free energy profiles derived from the bidimensional potential of mean force reveal more details for two half-reaction processes. PMID:24084894

  2. 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

  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. 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

  5. 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. PMID:25992245

  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. Biochemical and molecular characterization of the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase from Toxoplasma gondii.

    PubMed

    Hortua Triana, Miryam Andrea; Huynh, My-Hang; Garavito, Manuel F; Fox, Barbara A; Bzik, David J; Carruthers, Vern B; Löffler, Monika; Zimmermann, Barbara H

    2012-08-01

    The pyrimidine biosynthesis pathway in the protozoan pathogen Toxoplasma gondii is essential for parasite growth during infection. To investigate the properties of dihydroorotate dehydrogenase (TgDHOD), the fourth enzyme in the T. gondii pyrimidine pathway, we expressed and purified recombinant TgDHOD. TgDHOD exhibited a specific activity of 84U/mg, a k(cat) of 89s(-1), a K(m)=60μM for l-dihydroorotate, and a K(m)=29μM for decylubiquinone (Q(D)). Quinones lacking or having short isoprenoid side chains yielded lower k(cat)s than Q(D). As expected, fumarate was a poor electron acceptor for this family 2 DHOD. The IC(50)s determined for A77-1726, the active derivative of the human DHOD inhibitor leflunomide, and related compounds MD249 and MD209 were, 91μM, 96μM, and 60μM, respectively. The enzyme was not significantly affected by brequinar or TTFA, known inhibitors of human DHOD, or by atovaquone. DSM190, a known inhibitor of Plasmodium falciparum DHOD, was a poor inhibitor of TgDHOD. TgDHOD exhibits a lengthy 157-residue N-terminal extension, consistent with a potential organellar targeting signal. We constructed C-terminally c-myc tagged TgDHODs to examine subcellular localization of TgDHOD in transgenic parasites expressing the tagged protein. Using both exogenous and endogenous expression strategies, anti-myc fluorescence signal colocalized with antibodies against the mitochondrial marker ATPase. These findings demonstrate that TgDHOD is associated with the parasite's mitochondrion, revealing this organelle as the site of orotate production in T. gondii. The TgDHOD gene appears to be essential because while gene tagging was successful at the TgDHOD gene locus, attempts to delete the TgDHOD gene were not successful in the KU80 background. Collectively, our study suggests that TgDHOD is an excellent target for the development of anti-Toxoplasma drugs. PMID:22580100

  8. Biochemical and molecular characterization of the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase from Toxoplasma gondii

    PubMed Central

    Triana, Miryam Andrea Hortua; Huynh, My-Hang; Garavito, Manuel F.; Fox, Barbara A.; Bzik, David J.; Carruthers, Vern B.; Löffler, Monika; Zimmermann, Barbara H.

    2013-01-01

    Summary The pyrimidine biosynthesis pathway in the protozoan pathogen Toxoplasma gondii is essential for parasite growth during infection. To investigate the properties of dihydroorotate dehydrogenase (TgDHOD), the fourth enzyme in the T. gondii pyrimidine pathway, we expressed and purified recombinant TgDHOD. TgDHOD exhibited a specific activity of 84 U/mg, a kcat of 89 sec−1, a Km = 60 μM for L-dihydroorotate, and a Km = 29 μM for decylubiquinone (QD). Quinones lacking or having short isoprenoid side chains yielded lower kcats than QD. As expected, fumarate was a poor electron acceptor for this family 2 DHOD. The The IC50s determined for A77-1726, the active derivative of the human DHOD inhibitor leflunomide, and related compounds MD249 and MD209 were, 91 μM, 96 μM, and 60 μM, respectively. The enzyme was not significantly affected by brequinar or TTFA, known inhibitors of human DHOD, or by atovaquone. DSM190, a known inhibitor of Plasmodium falciparum DHOD, was a poor inhibitor of TgDHOD. TgDHOD exhibits a lengthy 157-residue N-terminal extension, consistent with a potential organellar targeting signal. We constructed C-terminally c-myc tagged TgDHODs to examine subcellular localization of TgDHOD in transgenic parasites expressing the tagged protein. Using both exogenous and endogenous expression strategies, anti-myc fluorescence signal colocalized with antibodies against the mitochondrial marker ATPase. These findings demonstrate that TgDHOD is associated with the parasite’s mitochondrion, revealing this organelle as the site of orotate production in T gondii. The TgDHOD gene appears to be essential because while gene tagging was successful at the TgDHOD gene locus, attempts to delete the TgDHOD gene were not successful in the KU80 background. Collectively, our study suggests that TgDHOD is an excellent target for the development of anti-Toxoplasma drugs. PMID:22580100

  9. 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

    Sánchez-Sampedro, Lucas; Gómez, Carmen Elena; Mejías-Pérez, Ernesto; Sorzano, Carlos Oscar S; 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

  10. 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