Journal Article•
Molecular cloning, expression and enzymatic assay of pteridine reductase 1 from Iranian lizard Leishmania.
TL;DR: Iranian lizard Leishmania ptr1 gene was successfully amplified and cloned into expression vector, and enzymatic assay was performed successfully, indicating PTR1 is a good drug-target candidate for anti-Leishmania chemotherapy.
Abstract: Background: Currently, there are no effective vaccines against leishmaniasis, and treatment using pentavalent antimonial drugs is occasionally effective and often toxic for patients. The PTR1 enzyme, which causes antifolate drug resistance in Leishmania parasites encoded by gene pteridine reductase 1 (ptr1). Since Leishmania lacks pteridine and folate metabolism, it cannot synthesize the pteridine moiety from guanine triphosphate. Therefore, it must produce pteridine using PTR1, an essential part of the salvage pathway that reduces oxidized pteridines. Thus, PTR1 is a good drug-target candidate for anti-Leishmania chemotherapy. The aim of this study was the cloning, expression, and enzymatic assay of the ptr1 gene from Iranian lizard Leishmania as a model for further studies on Leishmania. Methods: Promastigote DNA was extracted from the Iranian lizard Leishmania, and the ptr1 gene was amplified using specific primers. The PCR product was cloned, transformed into Escherichia coli strain JM109, and expressed. The recombinant protein (PTR1 enzyme) was then purified and assayed. Results: ptr1 gene was successfully amplified and cloned into expression vector. Recombinant protein (PTR1 enzyme) was purified using affinity chromatography and confirmed by Western-blot and dot blot using anti-Leishmania major PTR1 antibody and anti-T7 tag monoclonal antibody, respectively. The enzymatic assay was confirmed as PTR1 witch performed using 6- biopterin as a substrate and nicotinamide adenine dinucleotide phosphate as a coenzyme. Conclusion: Iranian lizard Leishmania ptr1 was expressed and enzymatic assay was performed successfully.
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TL;DR: The objective was to find Leishmania parasites circulating in reservoir hosts, sand flies and human simultaneously and to describe the mechanisms leading to ZCL in Iran.
Abstract: Objectives
Only Leishmania major is well known as a causative agent of zoonotic cutaneous leishmaniasis (ZCL) in Iran. Our objective was to find Leishmania parasites circulating in reservoir hosts, sand flies and human simultaneously.
Methods
Sand flies, rodents and prepared smears of humans were sampled. DNA of Leishmania parasites was extracted, and two fragments of ITS-rDNA gene amplified by PCR. RFLP and sequencing were employed to identify Leishmania parasites.
Results
Leishmania major and L. turanica were identified unequivocally by targeting and sequencing ITS-rDNA from humans, rodents and sand flies. The new Leishmania species close to gerbilli (GenBank Accession Nos. EF413076; EF413087) was discovered only in sand flies.
Conclusion
Based on parasite detection of ITS-rDNA in main and potential reservoir hosts and vectors and humans, we conclude that at least two Leishmania species are common in the Turkmen Sahra ZCL focus. Phylogenetic analysis proved that the new Leishmania is closely related to Leishmania mammal parasites (Leishmania major, Leishmania turanica, Leishmania gerbilli). Its role as a principal agent of ZCL is unknown because it was found only in sand flies. Our findings shed new light on the transmission cycles of several Leishmania parasites in sand flies, reservoir hosts and humans.
Objectifs
Seul Leishmania major est bien connu comme agent causal de la leishmaniose cutanee zoonotique (LCZ) en Iran. Notre objectif etait de trouver des parasites de Leishmania circulant simultanement chez les hotes reservoirs, les phlebotomes et les humains.
Methodes
Les phlebotomes, les rongeurs et des frottis prepares a partir d'humains ont ete echantillonnes. L’ADN de Leishmania a ete extrait et deux fragments du gene ITS-ADNr ont ete amplifies par PCR. Le RFLP et le sequencage ont ete utilises pour identifier les parasites Leishmania.
Resultats
L. major et L. turanica ont ete identifies sans equivoque par le ciblage et le sequencage de l’ITS-ADNr chez les etres humains, les rongeurs et les phlebotomes. Nous avons decouvert une nouvelle espece de Leishmania chez les phlebotomes que nous avons nomme L. iranica (n° d'acces GenBank: EF413076. EF413087).
Conclusion
Sur base de la detection des parasites par l’ITS-ADNr chez les hotes reservoirs principaux et potentiels et les vecteurs et les humains, nous concluons qu'au moins deux especes de Leishmania sont courantes dans le foyer Turkemen Sahara ZCL. L'analyse phylogenetique a montre que la nouvelle espece Leishmania est etroitement liee a des parasites Leishmania de mammiferes (L. major, L. turanica, L. gerbilli). Son role en tant que principal agent de LCZ est inconnu car elle n'a ete retrouvee que dans les phlebotomes. Nos resultats apportent une nouvelle lumiere sur les cycles de transmission de plusieurs parasites Leishmania dans les phlebotomes, les hotes reservoirs et les humains.
Objetivos
Solo Leishmania major es un agente causal bien conocido de la Leishmaniasis cutanea zoonotica (LCZ) en Iran. Nuestro objetivo era encontrar parasitos de Leishmania circulando simultaneamente entre hospederos reservorios, moscas de arena y humanos.
Metodos
Se tomaron muestras de moscas de arena, roedores y frotis preparados de humanos. Se extrajo el ADN de Leishmania y mediante PCR se amplificaron dos fragmentos del gen ITS-ADNr. Se utilizaron el RFLP y la secuenciacion para identificar los parasitos de Leishmania.
Resultados
Se identifico a L. major y L. turanica de forma inequivoca mediante amplificacion y secuenciacion del ITS-ADNr de muestras de humanos, roedores y moscas de arena. En moscas, hemos descubierto una nueva especie de Leishmania, la cual hemos llamado L. iranica (numeros de acceso de GenBank EF413076; EF413087).
Conclusion
Basandonos en la deteccion del ITS-ADNr de parasitos en el hospedero principal y otros potenciales, en vectores y en humanos, concluimos que al menos dos especies de Leishmania son comunes en el foco de LCZ de Turkemen Sahara. El analisis filogenetico muestra que la nueva Leishmania esta estrechamente relacionada con especies de Leishmania que parasitan mamiferos (L. major, L. turanica, L. gerbilli). Su papel como el agente principal de LCZ es desconocido puesto que se encontro solamente en moscas de arena. Nuestros hallazgos aportan nuevos datos sobre los ciclos de transmision de varios parasitos de Leishmania en moscas de arena, hospederos reservorios y humanos.
20 citations
Cites background or result from "Molecular cloning, expression and e..."
...Finding variations in L. major and assessing the density of these parasites can be of practical value and experimental evidence for the epidemiological surveillance and control of leishmaniasis in humans, hosts and vectors in Iran (Ready & Rogers 2012; Ready 2013). mania (Motazedian et al. 1996; kazemi et al. 2010) in Turkmen Sahra region and other places in Iran, but in this study, Sauroleishmania was not found....
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...It is conceivable that had we put more purposeful efforts into investigating of Sergentomyia sintoni species, and the parasites might have appeared as Sauroleishmania; however, in our previous study, we searched for Leishmania parasite in Sergentomyia sand fly species but found none (Parvizi & Amirkhani 2008; Parvizi & Ready 2008)....
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...Some previous studies were carried out on Sauroleishmania (Motazedian et al. 1996; kazemi et al. 2010) in Turkmen Sahra region and other places in Iran, but in this study, Sauroleishmania was not found....
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...…evidence for the epidemiological surveillance and control of leishmaniasis in humans, hosts and vectors in Iran (Ready & Rogers 2012; Ready 2013). mania (Motazedian et al. 1996; kazemi et al. 2010) in Turkmen Sahra region and other places in Iran, but in this study, Sauroleishmania was not found....
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Journal Article•
TL;DR: The chromosomal and mitochondrial genomes of Leishmania spp.
Abstract: Leishmania is a protozoan parasite belonging to the family Trypanosomatidae, which is found among 88 different countries. The parasite lives as an amastigote in vertebrate macro- phages and as a promastigote in the digestive tract of sand fly. It can be cultured in the laboratory using appropriate culture media. Although the sexual cycle of Leishmania has not been observed during the promastigote and amastigote stages, it has been reported by some researchers. Leishmania has eukaryotic cell organization. Cell culture is convenient and cost effective, and because posttranslational modifications are common processes in the cultured cells, the cells are used as hosts for preparing eukaryotic recombinant proteins for research. Several transcripts of rDNA in the Leishmania genome are suitable regions for conducting gene transfer. Old World Leishmania spp. has 36 chromosomes, while New World Leishmania spp. has 34 or 35 chromo- somes. The genomic organization and parasitic characteristics have been investigated. Leishmania spp. has a unique genomic organization among eukaryotes; the genes do not have introns, and the chromosomes are smaller with larger numbers of genes confined to a smaller space within the nucleus. Leishmania spp. genes are organized on one or both DNA strands and are transcribed as polycistronic (prokaryotic-like) transcripts from undefined promoters. Regulation of gene expres- sion in the members of Trypanosomatidae differs from that in other eukaryotes. The trans-splic- ing phenomenon is a necessary step for mRNA processing in lower eukaryotes and is observed in Leishmania spp. Another particular feature of RNA editing in Leishmania spp. is that mitochon- drial genes encoding respiratory enzymes are edited and transcribed. This review will discuss the chromosomal and mitochondrial (kinetoplast) genomes of Leishmania spp. as well as the phenomenon of RNA editing in the kinetoplast genome.
10 citations
Cites methods from "Molecular cloning, expression and e..."
...It should be noted that most Leishmania genes have no introns (40), and that chromosomal DNA is used as the template for cloning by PCR (41-45)....
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TL;DR: Mice immunized with ILL+CpG were protected against the development of the dermal lesion and showed a significant reduction in the parasite load, in comparison to the control groups, indicating that ILL with an appropriate adjuvant would be a suitable choice for vaccination against leishmaniasis.
Abstract: Background and Objectives: The live non-pathogenic Leishmania tarantolae has recently provided a promising approach as an effective vaccine candidate against experimental leishmaniasis (ILL). Here, we evaluated the immunoprotective potential of the live Iranian Lizard Leishmania mixed with CpG adjuvant against L. major infection in BALB/c mice. Methods: Four groups of female BALB/c mice were included in the study. The first and second groups received PBS and CpG, respectively. The immunized groups received 2 × 105 ILL promastigotes and the CpG-mixed ILL (ILL+CpG). Injections were performed subcutaneously in the right footpad. Three weeks later, all mice were challenged with 2 × 105 metacyclic promastigotes of Leishmania major EGFP ; inoculation was done in the left footpad. The measurement of footpad swelling and in vivo fluorescent imaging were used to evaluate disease progress during infection course. Eight weeks after challenge, all mice were sacrificed and the cytokines levels (IFN-γ, IL-4, and IL-10) and sera antibodies concentrations (IgG2a and IgG1) using ELISA assay, nitric oxide production using Griess assay, and arginase activity in cultured splenocytes, were measured. In addition, direct fluorescent microscopy analysis and qPCR assay were used to quantify the splenic parasite burden. Result: The results showed that mice immunized with ILL+CpG were protected against the development of the dermal lesion. Moreover, they showed a significant reduction in the parasite load, in comparison to the control groups. The observed protection was associated with higher production of IFN-γ, as well as a reduction in IL-4 level. Additionally, the results demonstrated that arginase activity was decreased in ILL+CpG group compared to other groups. Conclusion: Immunization using ILL+CpG induces a protective immunity; indicating that ILL with an appropriate adjuvant would be a suitable choice for vaccination against leishmaniasis.
9 citations
Cites background from "Molecular cloning, expression and e..."
...Leishmania promastigotes that previously were isolated in other countries (28, 29)....
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References
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TL;DR: Possible strategies to validate several purine salvage enzymes as targets for chemotherapeutic manipulation with special reference to adenosine kinase of Leishmania donovani are discussed.
Abstract: Distinguishable differences between infectine organisms and their respective hosts with respect to metabolism and macromolecular structure provide scopes for detailed characterization of target proteins and/or macromolecules as the focus for the development of selective inhibitors. In order to develop a rational approach to antiparasitic chemotherapy, finding differences in the biochemical pathways of the parasite with respect to the host it infects is therefore of primary importance. Like most parasitic protozoan, the genus Leishmania is an obligate auxotroph of purines and hence for requirement of purine bases depends on its own purine salvage pathways. Among various purine acquisition routes used by the parasite, the pathway involved in assimilation of adenosine nucleotide is unique and differs significantly in the extracellular form of the parasite (promastigotes) from its corresponding intracellular form (amastigotes). Adenosine kinase (AdK) is the gateway enzyme of this pathway and displays stage-specific activity pattern. Therefore, understanding the catalytic mechanism of the enzyme, its structural complexities and mode of its regulation have emerged as one of the major areas of investigation. This review, in general, discusses possible strategies to validate several purine salvage enzymes as targets for chemotherapeutic manipulation with special reference to adenosine kinase of Leishmania donovani. Systemic endotheliosis, commonly known as Kala-azar in India, is caused by the parasitic protozoon Leishmania donovani. The spread of leishmaniases follows the distribution of these vectors in the temperate, tropical and subtropical regions of the world leading to loss of thousands of human lives.' WHO has declared leishmaniasis among one of the six major diseases namely leishmaniasis, malaria, amoebiasis, filariasis, Chagas disease and schistosomiasis in its Special Programme for Research and Training in Tropical Diseases. Strategies for better prophylaxis and urgent therapies must be therefore devised to control this menace among poor and under privileged population. However, the possible availability of antiparasitic vaccines appears remote in near future. Therefore, chemotherapy remains the mainstay for the treatment of most parasitic diseases. Selectivity of an antiparasitic compound must depend upon its mode of specific inhibition of parasite replication leaving host processes unaffected. In principle, these agents are expected to exert their selective actions against growth of the invading organisms by having one or both of the following properties: (i) Selective activation of compounds in question by enzyme (s) from the invading organisms, which are not present in the uninfected cells. (ii) Selective inhibition of vital enzyme(s), which are essential for replication of the parasites. In order to design specific compounds with the above characteristics, it is essential to have a thorough knowledge of the properties of the enzyme(s) and/or macromolecules which are unique to the parasite. Phylogenetic studies suggested that trypanosomatid parasites are relatively early-branching eukaryotic cells and indeed their cellular organization differs considerably from their mammalian hosts counterpart. Various enzymes, metabolites or proteins identified in parasites and known to be absent from or strikingly different in the mammalian hosts were considered as ideal drug targets. Among the various metabolic pathways that are presently being studied for their prospects to be exploited as the target for chemotherapeutic manipulation, the most important are (i) purine salvage (ii) polyamine and thiol metabolism (iii) folate biosynthesis (iv) DNA replication (v) glycolytic and (vi) fatty acid biosynthetic pathways etc. A number of excellent reviews, describing the prospects and efficacies of these pathways, already exist in the literature. Our laboratory is engaged in studying the pathways responsible for synthesis and assimilation ofpurine nucleotides in the parasitic protozoon Leishmania donovani. Therefore, we shall, for the constraint of space, try to restrict the discussion mostly with the purine salvage pathways of various Leishmania parasites with particular reference to the unique features of one of the enzymes of the purine salvage pathway viz AdK and its prospects as the chemotherapeutic target. However, contributions of other workers will also be discussed whenever essential and analogy will be drawn in order to make the reading coherent. The Leishmania genus goes through a dimorphic life cycle. It exists as a promastigote (extracellular form) in the sand fly vector but is converted to an amastigote (intracellular form) upon entry into mammalian macrophages. During this transformation process, the activities of a large number of proteins and/or enzymes have been reported to be stage-specifically altered and hence they could be prospective targets for development of chemotherapeutic regimen based on the exploitable differences of the parasitic proteins from their respective host counterpart.
45 citations
"Molecular cloning, expression and e..." refers background in this paper
...No effective vaccine is available and treatment by pentavalent antimonial drugs is only occasionally effective and often toxic for patients [2]....
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TL;DR: Using reversed-phase high-performance liquid chromatography, the ability of intact L. donovani to transform [3H]biopterin into tetrahydrofolates was demonstrated and supported the hypothesis that folate transport deficiency in mutant organisms is associated with an inability to transform pterins to reduced folates.
36 citations
"Molecular cloning, expression and e..." refers background in this paper
..., human, are similar, it is thought that therapeutic manipulation of pyrimidine metabolism in Leishmania would be less effective as compared to manipulation of the purine salvage pathway [6-8]....
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TL;DR: The Leishmania short-chain dehydrogenase/reductase gene ptr1 has been isolated while characterizing antifolate-resistant leishmania mutants by site-directed mutagenesis as mentioned in this paper.
Abstract: The Leishmania short-chain dehydrogenase/reductase gene ptr1 has been isolated while characterizing antifolate-resistant Leishmania mutants. PTR1 is active as a tetramer and can reduce pterins and folates. PTR1 has several of the hallmarks of the short chain dehydrogenase/reductase family, including a glycine rich co-factor binding site at its N-terminus, and the consensus catalytic site TyrXaa3Lys. To start probing the structure/function of PTR1, we have generated by site-directed mutagenesis five mutants, either in the co-factor-binding site, Y38D or in the catalytic site Y195F, Y195W, K199R or in a PTR1-specific region, Y175F. The mutated versions of PTR1 were studied in vivo in Leishmania and at the biochemical level using purified proteins. The Y175F mutant showed properties similar to wild-type PTR1 in every aspect tested, but all the other mutants were inactive even if they were purified as tetramers. To test the ability of the mutated PTR1 versions to bind its co-factor and substrates, trypsin digestion experiments were carried out under conditions upon which binding will prevent wild-type PTR1 of being digested by trypsin. The wild-type PTR1 as well as Y175F and Y195F mutants were protected against trypsin digestion whereas Y38D and K199R mutants were not. Mutations in regions involved in co-factor binding (Y38D) or in catalytic site (K199R) altered the binding of the ligands, explaining why those protein versions are inactive.
17 citations
"Molecular cloning, expression and e..." refers background in this paper
...[28] isolated a Leishmania ptr1 gene and showed...
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TL;DR: It is suggested that PTR1 degradation during the stationary phase of growth is mediated by the proteasome, which leads to lower levels of H4-biopterin, which favors metacyclogenesis, and subsequently results in a highly infective stage of the parasite.
17 citations
Additional excerpts
...[26] suggested that PTR1 is degraded...
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17 citations
"Molecular cloning, expression and e..." refers background in this paper
...As an additional discovery, Iranian lizard Leishmania promastigote was found in the host bloodstream [14], Cavazzuti et al....
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...However, we have isolated a lizard Leishmania promastigote [14] which differs from lizard Leishmania isolated previously in other...
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