Cloning and Expression of TRYP6 Gene from Leishmania major (MRHO/IR/75/ER)
-
G Eslami
,
R Salehi
,
H Hejazi
,
A Khamesipour
,
B Kazemi
Abstract
Background: Leishmania, needs to detoxify the macrophage derived potent peroxides (H2O2). Tryparedoxin pathway contains tryparedoxin peroxidase (TSA or TRYP). The aim of the study was to detect the full-length gene sequence and its encoded protein of the LmTRYP6 gene (EU251502), and comparison the gene sequence with LmTRYP6 (LmjF15.1140), another previously reported member of this gene family.
Methods: L.major (MRHO/IR/75/ER) promastigotes were cultured, DNA and RNA were extracted and the interested gene was amplified using PCR and RT-PCR methods. PCR/ RT-PCR fragments were purified and cloned first in pTZ57R/T and then in pET15b expression vector. The expressed protein was verified using western blot method. Characterization of the expressed protein was performed bioinformatically.
Results: Molecular evaluation revealed that the cloned LmTRYP6 gene (EU251502) encoded a predicted 184 amino acid long protein with a theoretical isoelectric point of 6.1101. Alignment showed a number of changes in amino acid composition including the replacement of highly conserved Trp177 by Cys in LmTRYP6 (ABX26130).
Conclusion: So far no study has been done on this group, i.e. TRYP6 gene, from tryparedoxin peroxidase family. The low homology with LmTRYP6 (LmjF15.1140) and vast array of differences observed in the gene under study (LmTRYP6; EU251502) could open new windows in the field of anti-Leishmania combat. Based on its important role in the viability and successful establishment of the parasite in the host organism it looks to be very good candidate for vaccine development and any other sort of novel drug development.
Bahar K, Dowlati Y, Shidani B, Alimoham- madian MH, Khamesipour A, Ehsasi S, Hashemi-Fesharki R, Ale-Agha S, Modabber F (1996) Comparative safety and immunogenicity trial of two killed Leishmania major with or without BCG in human volunteers. Clin Dermatol. 14 (5): 489-495.
Bryk R, Griffin P, Nathan C (2000) Peroxyni- trite reductase activity of bacterial per- oxiredoxins. Nature. 407(6801): 211–215.
Castro H, Sousa C, Novais M, Santos M, Budde H, Cordeiro-da-Silva A, Flohe L, Tomas AM (2004) Two linked genes of Leishmania infantum encode try- paredoxins localised to cytosol and Mito- chondrion. Mol Biochem Parasitol. 136:137-147.
Chauhan R, Mande SC (2002) Site-directed mutagenesis reveals a novel catalytic mechanism of Mycobacterium tuber- culosis alkylhydroperoxidase. C Biochem J. 367 (Pt 1): 255-61.
Chen L, Xie QW, Nathan C (1998) Alkyl hy- droperoxide reductase subunit C (AhpC) protects bacterial and human cells against reactive nitrogen intermediates. Mol Cell.1 (6): 795-805.
Choi HJ, Kang SW, Yang CH, Rhee SG, Ryu SE (1998) Crystal structure of a novel human peroxidase enzyme at 2.0 A resolution. Nat Struct Biol. 5(5): 400-6.
Eisenberger CL, Jaffe CL (1999) Leishma- nia: Identification of old world species using a permissively primed intergenic polymorphic-polymerase chain reaction. Exp Parasitol. 91: 70-77.
Guimarães BG, Souchon H, Honoré N,Saint-Joanis B, Brosch R, Shepard W, Cole ST, Alzari PM (2005) Structure and mechanism of the alkyl hydrop- eroxidase AhpC, a key element of the Mycobacterium tuberculosis defense sys- tem against oxidative stress. J Biol Chem.280 (27): 25735-42.
Harder S, Bente M, Isermann K, Bruchhaus I (2006) Expression of a mitochondrial peroxiredoxin prevents programmed cell death in Leishmania donovani. Eu- karyot Cell. 5 (5): 861–870.
Hendricks LD, Wood DE, Hajduk ME (1978) Haemoflagellates commercially available liquid media for rapid cultiva- tion. Parasitology. 76: 309–316.
Khalil EAG, El Hassan Zijlstra EE, Mukhtat MM, Ghalib HW, Musa B, Ibrahim ME, Kamil A, Elsheikh M, Modabber F (2000) Autoclaved L. major vaccine for prevention of visceral leishmaniasis: a randomised, double-blind, BCG-controlled trial in Sudan. Lancet. 356: 1565-69.
Khamesipour A, Dowlati Y, Asilian A, Hashemi-Fesharki R, Javadi A, Noazin S, Modabber F (2005) Leishmaniza- tion: use of an old method for evalua- tion of candidate vaccines against leishma- niasis. Vaccine. 23 (28): 3642-8.
Koshkin A, Knudsen GM, Ortiz De Montellano PR (2004) Intermolecular interactions in the AhpC/AhpD antioxidant defense system of Mycobacterium tuberculosis. Arch Biochem Biophys. 427(1): 41-7.
Levick MP, Tetaud E, Fairlamb AH, Blackwell JM (1998) Identification and charac- terisation of a functional peroxidoxin from Leishmania major. Mol Biochem Parasitol. 96 (1-2): 125-37.
Lin YC, Hsu JY, Chiang SC, Lee ST (2005)Distinct overexpression of cytosolic and mitochondrial tryparedoxin peroxidases results in preferential detoxification of different oxidants in arsenite-resistant Leishmania amazonensis with and without DNA amplification. Mol Bio- chem Parasitol. 142 (1): 66-75.
McGonigle S, Dalton JP, James ER (1998) Peroxidoxins: a new antioxidant family. Parasitol Today. 14: 139–45.
Momeni AZ, Jalayer T, Emamjomeh M, Khamesipour A, Zicker F, Ghassemi RL, Dowlati Y, Sharifi I, Aminjavaheri M, Shafiei A, Alimohammadian MH, Hashemi-Fesharki R, Nasseri K, Godal T, Smith PG, Modabber F (1997) A randomised, double-blind, controlled trial of a killed L. major vaccine plus BCG against zoonotic cutaneous leishmaniasis in Iran. Vaccine. 17 (5): 466-72.
Montemartini M, Kalisz HM, Hecht HJ, Steinert P, Flohe L (1999) Activation of active-site cysteine residues in the peroxiredoxin-type tryparedoxin peroxi- dase of Crithidia fasciculata. Eur J Biochem. 264 (2): 516-24.
Muller S, Liebau E, Walter RD, Krauth-Siegel RL (2003) Thiol based redox metabolism of protozoan parasites. Trends Parasitol.19 (7): 320–328.
Nadim A, Javadian E (1998) Leishmaniza- tion in Iran. In: Research on Strategies for the Control of Leishmaniasis. Walton B, Wijeyaretne PM, Modabber F (Eds.), International Development Research Center, Ottawa. pp. 336-369.
Nogoceke E, Gommel DU, Kiess M, Kalisz HM, Flohe L (1997) A unique cascade of oxidoreductases catalyses trypan- othionemediated peroxide metabolism in Crithidia fasciculate. Biol Chem.378: 827–36.
Ozbilgin A, Tas S¸ Atambay M, Alkan Z, O¨ zbel Y (1995) Comparison of liquid and biphasic media in in vitro cultiva- tion of Leishmania infantum promas- tigotes. Acta Parasitol Turc. 19: 1-5.
Sharifi I, FeKri AR, Aflatonian MR, Khamesi-pour A, Nadim A, Mousavi MR, Momeni AZ, Dowlati Y, Godal T, Zicker F, Smith PG, Modabber F (1998) Randomised vaccine trial of single dose of killed Leishmania major plus BCG against anthroponotic cutaneous leishmaniasis in Bam, Iran. Lancet. 351 (9115): 1540-3.
Walker J, Acestor N, Gongora R, Quadroni M, Segura I, Fasel N, Saravia NG (2006) Comparative protein profiling identifies elongation factor-1beta and try- paredoxin peroxidase as factors associ- ated with metastasis in Leishmania guy- anensis. Mol Biochem Parasitol. 145(2):254-64.
Wilson ME, Andersen KA, Britigan BE (1994) Response of Leishmania cha- gasi promastigotes to oxidant stress. Infect Immun. 62: 5133–41.
Zarley JH, Britigan BE, Wilson ME (1991) Hydrogen peroxide-mediated toxicity for Leishmania donovani chagasi pro- mastigotes. Role of hydroxyl radical and protection by heat shock. J Clin Invest.88: 1511–21.
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| Issue | Vol 2 No 1 (2008) | |
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| Tryparedoxin peroxidase L. major Peroxiredoxin TRYP6 Cloning | ||
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