Comparison of p27 Gene Expression of Promastigote and Amastigote Forms of Leishmania major (MRHO/IR/75/ER) by Real-time RT-PCR
Abstract
Background: Cutaneous leishmaniasis (CL) is one of the world health problems. Leishmania major is the etiological agent of zoonotic cutaneous leishmaniasis (ZCL). Promastigote and amastigote are two morphological forms of Leishmania parasites that express different proteins and p27 is an important gene encoding cytochrome c oxidase (COX) component. P27 gene expresses a 27 kDa protein that essential in ATP synthesis. This study aimed to compare p27 gene expression in promastigote and amastigote forms in Iranian strain of L. major (MRHO/IR/75/ER).
Methods: This study was conducted in 2015. Clinical isolates of CL patients from north, center, west and south parts of Iran were collected and identified by PCR-RFLP. After RNA extraction of promastigotes and amastigotes and cDNA synthesis, the expression level of p27 gene was compared by real-time RT-PCR.
Results: By comparison of expression level between amastigote and promastigote forms of Iranian strain of L. major, up-regulation of p27 gene (2.73 fold) was observed in amastigotes. Moreover, there was no significant difference in p27 gene expression between L. major isolates.
Conclusion: p27 gene and protein can be considered as a target in recombinant vaccine production and treatment process.
Alvar J, Vélez ID, Bern C, et al. Leishman-iasis worldwide and global estimates of its incidence. PLoS One. 2012; 7(5):e35671.
Martínez-García M, Campos-Salinas J, Cabello-Donayre M et al. LmABCB3, an atypical mitochondrial ABC transporter essential for Leishmania major virulence, acts in heme and cytosolic iron/sulfur clusters biogenesis. Parasit Vectors. 2016; 9:7.
Shirzadi MR, Esfahania SB, Mohebali M et al. Epidemiological status of leishmaniasis in the Islamic Republic of Iran, 1983–2012. East Mediterr Health J. 2015; 21(10):736-42.
Dey R, Meneses C, Salotra P, Kamhawi S, Nakhasi HL, Duncan R. Characterization of a Leishmania stage-specific mitochondrial membrane protein that enhances the activ-ity of cytochrome c oxidase and its role in virulence. Mol Microbiol. 2010; 77(2):399-414.
Alexander J, Satoskar AR, Russell DG. Leishmania species: models of intracellular parasitism. J Cell Sci. 1999; 112 Pt 18:2993-3002.
McConville MJ, Ralton JE. Developmen-tally regulated changes in the cell surface architecture of Leishmania parasites. Behring Inst Mitt. 1997 ;(99):34-43.
McConville MJ, de Souza D, Saunders E, Likic VA, Naderer T. Living in a phagoly-sosome; metabolism of Leishmania amastigotes. Trends Parasitol. 2007; 23(8):368-75.
Naderer T1, McConville MJ. The Leishma-nia–macrophage interaction: a metabolic perspective. Cell Microbiol. 2008; 10(2):301-8.
Hart DT, Vickerman K, Coombs GH. Respiration of Leishmania mexicana amastigotes and promastigotes. Mol Bio-chem Parasitol. 1981; 4(1-2):39-51.
Van Hellemond JJ, Tielens AG. Inhibition of the respiratory chain results in a re-versible metabolic arrest in Leishmania promastigotes. Mol Biochem Parasitol. 1997;85(1):135-8.
Santhamma KR, Bhaduri A. Characteriza-tion of the respiratory chain of Leishmania donovani promastigotes. Mol Biochem Para-sitol. 1995; 75(1):43-53.
Hellemond JJ, Bakker BM, Tielens AG. Energy metabolism and its compartmenta-tion in Trypanosoma brucei. Adv Microb Physiol. 2005; 50:199-226.
Zíková A, Panigrahi AK, Uboldi AD, Dal-ley RA, Handman E, Stuart K. Structural and functional association of Trypanosoma brucei MIX protein with cytochrome c oxi-dase complex. Eukaryot Cell. 2008; 7(11):1994-2003.
Hajjaran H, Mohebali M, Teimouri A, Oshaghi MA et al. Identification and phy-logenetic relationship of Iranian strains of various Leishmania species isolated from cutaneous and visceral cases of leishmani-asis based on N-acetylglucosamine-1-phosphate transferase gene. Infect Genet Evol. 2014; 26:203-12.
Esmaeili J, Mohebali M, Edrissian Gh, Rezayat S, Ghazi-Khansari M, Charehdar S. Evaluation of miltefosine against Leishma-nia major (MRHO/IR/75/ER): in vitro and in vivo studies. Acta Med Iran. 2008; 46(3):191-6.
Filardy AA, Costa-da-Silva AC, Koeller CM, Guimarães-Pinto K, Ribeiro-Gomes FL, Lopes MF, Heise N, Freire-de-Lima CG, Nunes MP, DosReis GA. Infection with Leishmania major induces a cellular stress response in macrophages. PLoS One. 2014; 9(1):e85715.
Majumder S, Dey R, Bhattacharjee S, Rub A, Gupta G, Bhattacharyya Majumdar S, Saha B, Majumdar S. Leishmania-induced biphasic ceramide generation in macro-phages is crucial for uptake and survival of the parasite. J Infect Dis. 2012; 205(10):1607-16.
Ribeiro-Gomes FL, Otero AC, Gomes NA, Moniz-De-Souza MC, Cysne-Finkelstein L, Arnholdt AC, Calich VL, Coutinho SG, Lopes MF, DosReis GA. Macrophage interactions with neutrophils regulate Leishmania major infection. J Im-munol. 2004; 172(7):4454-62.
Moreno ML, de Meirelles Mde N. In vitro method for isolation of amastigote forms of Leishmania amazonensis from J774G8 macrophage induced by temperature shift-ing. Mem Inst Oswaldo Cruz. 1998; 93(1):99-102.
Kazemi-Rad E, Mohebali M, Khadem-Erfan MB et al. Overexpression of ubiqui-tin and amino acid permease genes in as-sociation with antimony resistance in Leishmania tropica field isolates. Korean J Parasitol. 2013; 51(4):413-9.
Pfaffl MW, Horgan GW, Dempfle L. Rela-tive expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in re-al-time PCR. Nucleic Acids Res. 2002; 30(9):e36.
Kazemi-Rad E, Mohebali M, Khadem-Erfan MB et al. Identification of antimony resistance markers in Leishmania tropica field isolates through a cDNA-AFLP approach. Exp Parasitol. 2013; 135(2):344-9.
Livak KJ, Schmittgen TD. Analysis of rela-tive gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001; 25(4):402-8.
Alves-Ferreira EV, Toledo JS, De Oliveira AH, Ferreira TR et al. Differential gene ex-pression and infection profiles of cutane-ous and mucosal Leishmania braziliensis iso-lates from the same patient. PLoS Negl Trop Dis. 2015; 9(9):e0004018.
Rochette A, Raymond F, Corbeil J, Ouel-lette M, Papadopoulou B. Whole-genome comparative RNA expression profiling of axenic and intracellular amastigote forms of Leishmania infantum. Mol Biochem Parasi-tol. 2009; 165(1):32-47.
Saxena A, Lahav T, Holland N et al. Anal-ysis of the Leishmania donovani transcrip-tome reveals an ordered progression of transient and permanent changes in gene expression during differentiation. Mol Bi-ochem Parasitol. 2007; 152(1):53-65.
Srividya G, Duncan R, Sharma P, Raju BV, Nakhasi HL, Salotra P. Transcriptome analysis during the process of in vitro dif-ferentiation of Leishmania donovani using ge-nomic microarrays. Parasitology. 2007; 134(Pt 11):1527-39.
Rochette A, McNicoll F, Girard J, Breton M, Leblanc E, Bergeron MG, Papadopou-lou B. Characterization and developmental gene regulation of a large gene family en-coding amastin surface proteins in Leish-mania spp. Mol Biochem Parasitol. 2005; 140(2):205-20.
McKean PG, Denny PW, Knuepfer E, Keen JK, Smith DF. Phenotypic changes associated with deletion and overexpres-sion of a stage-regulated gene family in Leishmania. Cell Microbiol. 2001; 3(8):511-23.
Dobson DE, Scholtes LD, Myler PJ, Tur-co SJ, Beverley SM. 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. Mol Biochem Parasitol. 2006; 146(2):231-41.
Akopyants NS, Matlib RS, Bukanova EN, Smeds MR, Brownstein BH, Stormo GD, Beverley SM. Expression profiling using random genomic DNA microarrays iden-tifies differentially expressed genes associ-ated with three major developmental stag-es of the protozoan parasite Leishmania ma-jor. Mol Biochem Parasitol. 2004; 136(1):71-86.
Belli S, Formenton A, Noll T, Ivens A, Jacquet R, Desponds C, Hofer D, Fasel N. Leishmania major: Histone H1 Gene Ex-pression from thesw3 Locus. Exp Parasi-tol. 1999; 91(2):151-60.
Burchmore RJ, Landfear SM. Differential regulation of multiple glucose transporter genes in Leishmania mexicana. J Biol Chem. 1998; 273(44):29118-26.
Kelly BL, Nelson TN, McMaster WR. Stage-specific expression in Leishmania conferred by 3′ untranslated regions of L. major leishmanolysin genes (GP63). Mol Biochem Parasitol. 2001; 116(1):101-4.
Yao C, Donelson JE, Wilson ME. The major surface protease (MSP or GP63) of Leishmania sp. Biosynthesis, regulation of expression, and function. Mol Biochem Parasitol.2003; 132(1):1-16.
Files | ||
Issue | Vol 13 No 2 (2018) | |
Section | Original Article(s) | |
Keywords | ||
p27 gene Real-time RT-PCR Leishmania major Amastigote Promastigote |
Rights and permissions | |
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |