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Journal Article

Molecular cloning, expression and enzymatic assay of pteridine reductase 1 from Iranian lizard Leishmania.

15 Nov 2010-Iranian biomedical journal (IRANIAN BIOMEDICAL JOURNAL)-Vol. 14, Iss: 3, pp 97-102

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.

AbstractBackground: 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.

Topics: Pteridine (58%), Leishmania (53%), Molecular cloning (52%), Expression vector (51%), Affinity chromatography (51%)

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Journal ArticleDOI
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.

18 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.

8 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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Journal ArticleDOI
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.

1 citations


Cites background from "Molecular cloning, expression and e..."

  • ...Leishmania promastigotes that previously were isolated in other countries (28, 29)....

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Journal ArticleDOI
TL;DR: A simple, one-step procedure for the preparation of competent Escherichia coli that uses a transformation and storage solution and can be frozen in TSS without addition of other components.
Abstract: We have developed a simple, one-step procedure for the preparation of competent Escherichia coli that uses a transformation and storage solution [TSS; 1 x TSS is LB broth containing 10% (wt/vol) polyethylene glycol, 5% (vol/vol) dimethyl sulfoxide, and 50 mM Mg2+ at pH 6.5]. Cells are mixed with an equal volume of ice-cold 2 x TSS and are immediately ready for use. Genetic transformation is equally simple: plasmid DNA is added and the cells are incubated for 5-60 min at 4 degrees C. A heat pulse is not necessary and the incubation time at 4 degrees C is not crucial, so there are no critical timing steps in the transformation procedure. Transformed bacteria are grown and selected by standard methods. Thus, this procedure eliminates the centrifugation, washing, and long-term incubation steps of current methods. Although cells taken early in the growth cycle (OD600 0.3-0.4) yield the highest transformation efficiencies (10(7)-10(8) transformants per micrograms of plasmid DNA), cells harvested at other stages in the growth cycle (including stationary phase) are capable of undergoing transformation (10(5)-10(7) transformants per micrograms of DNA). For long-term storage of competent cells, bacteria can be frozen in TSS without addition of other components. Our procedure represents a simple and convenient method for the preparation, transformation, and storage of competent bacterial cells.

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Journal ArticleDOI
TL;DR: This is the first report of proven resistant parasites contributing to treatment failure for cutaneous leishmaniasis and shows that primary Glucantime-resistant L. tropica field isolates are now frequent in Iran.
Abstract: Background Recent circumstantial evidence suggests that an increasing number of Iranian patients with cutaneous leishmaniasis are unresponsive to meglumine antimoniate (Glucantime), the first line of treatment in Iran. This study was designed to determine whether the clinical responses (healing, or non-healing) were correlated with the susceptibility of Leishmania parasites to Glucantime. Methods and Findings In vitro susceptibility testing was first performed on 185 isolated parasites in the intracellular mouse peritoneal macrophage model. A strong correlation between the clinical outcome and the in vitro effective concentration 50% (EC50) values was observed. Parasites derived from patients with non-healing lesions had EC50 values at least 4-fold higher than parasites derived from lesions of healing patients. A selection of these strains was typed at the molecular level by pulsed-field gels and by sequencing the pteridine reductase 1 (PTR1) gene. These techniques indicated that 28 out of 31 selected strains were Leishmania tropica and that three were Leishmania major. The L. major isolates were part of a distinct pulsed-field group, and the L. tropica isolates could be classified in three related additional pulsed-field groups. For each pulsed-field karyotype, we selected sensitive and resistant parasites in which we transfected the firefly luciferase marker to assess further the in vitro susceptibility of field isolates in the monocyte cell line THP1. These determinations confirmed unequivocally that patients with non-healing lesions were infected with L. tropica parasites resistant to Glucantime. Additional characterization of the resistant isolates showed that resistance is stable and can be reversed by buthionine sulfoximine, an inhibitor of glutathione biosynthesis. Conclusions To the authors' knowledge, this is the first report of proven resistant parasites contributing to treatment failure for cutaneous leishmaniasis and shows that primary Glucantime-resistant L. tropica field isolates are now frequent in Iran.

265 citations


"Molecular cloning, expression and e..." refers background or methods in this paper

  • ...tropica [3] and 864 bp for Leishmania donovani [27]....

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  • ...Because leishmaniasis presents a significant health problem in Iran and drug resistance and failure of glucantime treatment have been reported [3, 4], we cloned, expressed, and tested the enzymatic properties of Iranian lizard Leishmania PTR1 as a model for further...

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TL;DR: In the course of their cyclical development, flagellates of the genera Leishmania and trypanosoma pass through stages comparable with those of the monogenetic Trypanosomatidae and so it has been customary to refer to them by names derived from those genera in which the corresponding stages are the most characteristic forms.
Abstract: KNOWLEDGE of the structure and life cycles of the medically important Haemoflagellates of man and lower animals has increased during this century, and it became necessary to define the developmental stages of the leishmanias and trypanosomes in their mammalian and insect hosts. In the course of their cyclical development, flagellates of the genera Leishmania and Trypanosoma pass through stages comparable with those of the monogenetic Trypanosomatidae and so it has been customary to refer to them by names derived from those genera in which the corresponding stages are the most characteristic forms.

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"Molecular cloning, expression and e..." refers background in this paper

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Journal ArticleDOI
TL;DR: The results support the hypothesis that the H region of the Leishmania genome contains several drug resistance genes and that preferential amplification of this region has evolved as a defense mechanism against cytotoxic drugs.
Abstract: In several Leishmania spp., resistance to methotrexate and other drugs is often associated with amplification of the chromosomal H region in the form of extrachromosomal H circles. We report here that the H circle of Leishmania tarentolae contains an 867 bp open reading frame, ltdh, which mediates high levels of resistance to methotrexate and other antifolates, after transfection. The predicted amino acid sequence of the ltdh gene product has significant similarities to a family of short-chain dehydrogenases, enzymes that are involved in several oxido-reduction reactions in a wide range of organisms. To resist antifolates, Leishmania amplifies the ltdh gene as part of the H circle. We propose that LTDH might be involved in an alternative pathway for the synthesis of reduced folates and that ltdh overproduction represents a novel mechanism for resistance to antifolates. Our results support the hypothesis that the H region of the Leishmania genome contains several drug resistance genes and that preferential amplification of this region has evolved as a defense mechanism against cytotoxic drugs.

206 citations


"Molecular cloning, expression and e..." refers background in this paper

  • ...[10] recognized that the amplification of the H-circle in Leishmania species accompanies selection with methotrexate....

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  • ...ir enzyme PTR1 reduces pteridines, such as biopterin and folate [9-11], thereby reducing the effectiveness of methotrexate––a dihydrofolate reductase (DHFR) inhibitor––in Leishmania antifolate therapy...

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