Comparison of p27 Gene Expression of Promastigote and Amastigote Forms of Leishmania major (MRHO/IR/75/ER) by Real-time RT-PCR (original) (raw)
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Molecular Microbiology, 2010
Leishmaniasis is caused by the dimorphic protozoan parasite Leishmania. Differentiation of the insect form, promastigotes, to the vertebrate form, amastigotes, and survival inside the vertebrate host accompanies a drastic metabolic shift. We describe a gene first identified in amastigotes that is essential for survival inside the host. Gene expression analysis identified a 27kDa protein encoding gene (Ldp27) that was more abundantly expressed in amastigotes and metacyclic promastigotes than in procyclic promastigotes. Immunofluorescence and biochemical analysis revealed that Ldp27 is a mitochondrial membrane protein. Co-imunoprecipitation using antibodies to the cytochrome c oxidase (COX) complex, present in the inner mitochondrial membrane, placed the p27 protein in the COX complex. Ldp27 gene deleted parasites (Ldp27 −/−) showed significantly less COX activity and ATP synthesis than wild type in intracellular amastigotes. Moreover, the Ldp27 −/− parasites were less virulent both in human macrophages and in BALB/c mice. These results demonstrate that Ldp27 is an important component of an active COX complex enhancing oxidative phosphorylation specifically in infectious metacyclics and amastigotes and promoting parasite survival in the host. Thus, Ldp27 can be explored as a potential drug target and parasites devoid of the p27 gene could be considered as a live attenuated vaccine candidate against visceral leishmaniasis.
Expression analysis of viscerotropic leishmaniasis gene in Leishmania species by real-time RT-PCR
Acta Parasitologica, 2016
Viscerotropic leishmaniasis (VTL) is a parasitic disease with non-specific manifestations caused by Leishmania tropica. Specific antigens produced by Viscerotropic leishmaniasis gene have been used for diagnosis of VTL. The aim of this study was to compare the expression level of VTL gene among the viscerotropic L. tropica isolates (n: 3) and visceral L. infantum isolates (n: 4). Also, the expression level was compared in L. tropica (n: 21) and L. major (n: 8) isolates, the main causes of cutaneous leishmaniasis in Iran by real time-RT-PCR. Results showed viscerotropic leishmaniasis gene was expressed in all 3 species; L. tropica, L. major and L. infantum. The most expression rate was in L. tropica and L. major as the cutaneous species and the lowest in visceral isolates including L. infantum and viscerotropic L. tropica strains respectively.Conclusion: Results revealed that VTL gene can play an important role in visceralization process of L. tropica although there are other mechani...
Genetic analysis of clinical isolates of Leishmania major from Isfahan, Iran
Journal of vector borne diseases, 2012
BACKGROUND & OBJECTIVES Leishmaniasis is a geographically widespread severe disease which includes visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). There are 350 million people at risk in over 80 countries. In the Old World, CL is usually caused by Leishmania major, L. tropica, and L. aetiopica complex of which 90% of cases occur in Afghanistan, Algeria, Iran, Iraq, Saudi Arabia, Syria, Brazil and Peru. Recently, Eslami et al (2011) reported a novel TRYP6 gene encoding tryparedoxin peroxidase from an Iranian L. major strain exhibiting homology with the related gene in a divergent genus of Kinetoplastida, the Crithidia. This prompted us to analyze the mentioned gene in 100 isolates obtained from patients with suspected CL. Consequently, we analyzed internal transcribed spacer 1 (ITS1) region, RNA polymerase II largest subunit (RPOIILS) and the mitochondrial DNA polymerase beta (DPOLB). METHODS After obtaining samples from 100 patients, DNA extraction was performed and TR...
Molecular Cloning and Expression of Iranian Leishmania major Pteridine Reductase 1
Iranian Journal of Parasitology, 2008
Leishmaniasis is an endemic disease in 88 countries. Reports on Leishmania drug resistance are growing in number. The mechanism of unresponsiveness against glucantime in Iranian cutaneous leishmaniasis has not yet been char- acterized. To begin the first step in finding an anti-Leishmania chemotherapy, we prepared recombinant L. major PTR1 enzyme and characterized its activity by enzymatic assay. Methods: Leishmania promastigote DNA was extracted and the ptr1 gene amplified using specific primers. The PCR prod- uct was cloned in pQE30 expression vector, transformed into E.coli and expressed. The recombinant protein was purified, its enzymatic activity was assayed and anti-PTR1 antibody prepared in rabbit. Results: The PCR product of ptr1 gene was sequenced and deposited in GenBank. The amino acid sequence of Iranian L.major PTR1 was compared with other Leishmania PTR1 and showed some identities and diversities. Purified protein was reacted by anti PTR1 antibody in gel diffusion and ...
Subcloning and Expression of Leishmania major (MRHO/IR/75/ER) P4 Gene
Journal of Archives in Military Medicine, 2013
Background: Leishmania major p4 gene is localized in the endoplasmic reticulum at the intracellular amastigote stage. Objectives: We expressed this gene for possible future vaccine preparation, drug target studies, and leishmaniasis serodiagnosis test. Materials and Methods: The Leishmania major (MRHO/IR/75/ER) p4 gene, which had been subcloned into the pQE-30 expression vector, was incluced by IPTG. Recombinant protein was confirmed by SDS-PAGE followed by a double diffusion and western blot using an anti His-tag antibody or human antibody. Results: Production of protein with approximately 35 kDa molecular weight in E. coli M15 transformed cells was confirmed using SDS-PAGE, which reacted with antibodies of leishmaniasis serum using double diffusion and western blot tests and either for anti His-tag antibody using western blot. Conclusions: We have expressed the Iranian L. major p4 gene successfully and are ready to continue the research for vaccine production. Positive results from the western blot and double diffusion test suggest the hypothesis of using this Ag in diagnostic tests. We used the pQE-30 plasmid as an expression vector, which has high quantity of expressed p4 protein.
The Open Tropical Medicine Journal, 2011
We primarily identified Leishmania donovani parasites from eastern Sudan using species-specific primers that amplify a whole length minicircle. Based on the amplification of a cytochrome oxidase II fragment (COII), heteroduplex analysis (HDA) was performed. In HDA, the appearance of the extra bands with molecular weights higher than 540 bp indicates the presence of mismatched bases in the selected samples. Such bands were detected when hybridization was between reference strains and clinical isolates, as well as between the reference strains themselves, while no heteroduplexes were detected between the clinical isolates. Moreover, an RFLP assay using the restriction enzyme Ssp1 was performed on the original 540 bp products to discern an A-G transition, which differentiates between members of the Leishmania (L) infantum and those of Leishmania (L) donovani subspecies. The proposed minicircle genes-based analysis was rapid and easy to perform method for the characterization of Leishmania donovani complex isolates and with a potential to be extended to characterization of other species of Leishmania.
Veterinary World, 2018
Aim: Cutaneous leishmaniasis (CL) is one of the most important health problems that are capable of involving both tropical and subtropical areas, especially in Iran. This cross-sectional study aimed to differentiate the species that are able to cause CL in Zahedan city by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Materials and Methods: It was conducted on 145 suspected CL patients in Zahedan city between 2014 and 2016. The smears were initially prepared, air-dried, fixed with absolute methanol, and stained with 10% Giemsa. Then, we examined the stained samples by a light microscope under 1000× magnifications. PCR assay targeted cytochrome b (cyt b) gene using LCBF1 and LCBR2 primers and the products digested by Ssp1 enzymes. Results: From 145 suspected CL patients, 76 (52.4%) were positive in microscopic examination. In addition, we detected gene of interest (cyt b) in 98 (67.5%). The results of PCR-RFLP indicated that 53/98 (54%) cases were Leishmania major and 45/98 (46%) were Leishmania tropica, and the main species in these areas was L. major. Conclusion: We concluded that the microscopic examination is not sensitive enough and is not able to distinguish between different Leishmania species. Instead, molecular methods like PCR-RFLP can be appropriately used with promising results.
Parasitology, 2007
Leishmania donovani causes visceral disease (kala-azar), a major health problem throughout the tropics with 500 000 new cases every year. Leishmania differentiates from the promastigote to the amastigote form to establish infection in a mammalian host. To understand the process of differentiation, we assessed the global variation in gene expression in promastigotes, an intermediate stage of differentiation (PA24) and axenic amastigotes in culture using an L. donovani genomic microarray with 4224 clones printed in triplicate. During an intermediate stage of differentiation 24 h after shifting the promastigotes into amastigotes (PA24), there were 41 (y1%) clones with expression o2 . 0-fold higher than promastigotes, whereas in terminally differentiated amastigotes there were 130 (y3%) such clones. Of particular interest were certain genes that exhibited a transient increase or decrease in expression at the PA24 stage. Kinases showed a transient increase, and surface molecules, PSA and amino acid permease, were prominent clones among those showing a brief decrease at the PA24 stage. The microarray results have been validated using Northern blots or RT-PCR. In summary, our results provide important clues about the genes involved in the differentiation process of L. donovani that may contribute to virulence.
Veterinary world, 2018
Cutaneous leishmaniasis (CL) is one of the most important health problems that are capable of involving both tropical and subtropical areas, especially in Iran. This cross-sectional study aimed to differentiate the species that are able to cause CL in Zahedan city by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. It was conducted on 145 suspected CL patients in Zahedan city between 2014 and 2016. The smears were initially prepared, air-dried, fixed with absolute methanol, and stained with 10% Giemsa. Then, we examined the stained samples by a light microscope under 1000× magnifications. PCR assay targeted cytochrome (cyt ) gene using LCBF1 and LCBR2 primers and the products digested by Ssp1 enzymes. From 145 suspected CL patients, 76 (52.4%) were positive in microscopic examination. In addition, we detected gene of interest (cyt ) in 98 (67.5%). The results of PCR-RFLP indicated that 53/98 (54%) cases were and 45/98 (46%) were , and the main sp...