Gene Regulation of Pteridine Reductase 1 in Leishmania Promastigotes and Amastigotes Using a Full-Length Antisense Construct
-
F Kheirandish
,
M Bandehpour
,
N Davoudi
,
N Mosaffa
,
S Dawood
,
B Kazemi
,
A Haghighi
,
A Khamesipour
,
H Masjedi
,
M Mohebali
,
F Mahboudi
Abstract
Background: Pteridine metabolic pathway is unusual features of Leishmania, which is necessary for the growth of parasite. Leishmania has evolved a complex and versatile pteridine salvage network which has the ability of scavenging a wide area of the conjugated and unconjugated pteridines espe-cially folate and biopterin. In this study, we focus on the inhibition of ptr1 gene expression.
Methods: L. major ptr1 gene was cloned into pcDNA3 and digested using KpnI and BamHI. The gene was subcloned so that antisense will transcribe and called pcDNA-rPTR. Leishmania major was cultured and late logarithmic-phase promastigotes were harvested. The promastigotes were divided into two groups. One group was transfected with 50 μg of pcDNA-rPTR, whereas the other group was transfected with pcDNA3. Transfected cells were cultured and plated onto semi-solid media. Mouse pritonean macrophages were transfected using pcDNA-rPTR–tansfected promastigotes. Western blotting was performed on mouse transfected pritonean macrophages and extracts from transfected promastigotes of L. major using a L. major ptr1 antibody raised in rabbits.
Results: The PTR1 protein was not expressed in pcDNA-rPTR– tansfected promastigotes and mouse macrophage transfected with pcDNA-rPTR– tansfected promastigotes.
Conclusion: This approach might be used to study the pteridine salvage pathway in Leishmania or to assess the possibility of using gene expression inhibition in the treatment of leishmaniasis.
Ouellette M, Drummelsmith J, Elfadidi A, Kundig C, Richard D, Roy G. Pterin transport and metabolism in Leishmania and related trypanosomatid parasites. Int J Parasitol. 2002; 32 (4): 385- 398.
Scott DA, Coombs GH, Sanderson BE. Fo-late utilisation by Leishmania species and the identification of intracellular derivatives and folate- metabolizing enzymes. Mol Biochem Parasitol. 1987; 23 (2): 139-149.
Callahan HL, Beverley SM. A member of the aldoketo reductase family confers methotrex-ate resistance in Leishmania. J Boil Chem. 1992; 267 (34): 24165- 24168.
Cunningham ML, Beverley SM. Pteridine sal-vage throughout the Leishmania infectious cy-cle: implications for antifolate chemotherapy. Mol Biochem Parasitol. 2001;113 (2). 199-213.
Nare B, Uuba J, Hardy L, Beverley SM. New approach to Leishmania chemotherapy: pteri-dine reductase 1 (ptr1) as a target and modula-tor of antifolate sensitivity. Parasitol. 1997; 114 Suppl: S101 – 110.
Hardy LW, Matthews W, Nare B, Beverley SM. Biochemical and genetic tests for inhibi-tors of Leishmania pteridine pathways. Exp Parasitol. 1997; 87(3): 158- 170.
Beverley SM. Gene amplification in Leishma-nia. Annu Rev Microbiol. 1991; 45: 417-444.
Nichol CA, Smith GK, Duch DS. Biosynthe-sis and metabolism of tetrahydrobiopterin and molybdopterin. Ann Rev Biochem. 1985; 54: 729- 764.
Beck J, Ullman B. Biopterin conversion to re-duced folates by Leishmania donovani promasti-gotes. Mol Biochem Parasitol. 1991; 49(1): 21.
Bello AR, Nare B, Freedman D, Hardy L, Beverley SM. PTR1: A reductase mediating salvage of oxidized pteridines and methotrex-ate resistance in the protozoan parasite Leishmania major. Proc Natl Acad Sci USA. 1994; 91(24): 11442- 11446.
Papadopoulou B, Roy G, Ouellette M. A nov-el antifolate resistance gene on the amplified H circle of Leishmania. EMBO J. 1992; 11(10): 3601- 3608.
Whiteley JM, Xuong NH, Varughese KI. Is dihydropteridine reductase an anomalous dihydrofolate reductase, a flavin-like enzyme, or a short-chain dehydrogenase? Adv Exp Med Biol. 1993; 338: 115-121.
Gourley DG, Luba J, Hardy LW, Beverley SM, Hunter WN. Crystallization of recombi-nant Leishmania major pteridine reductase 1 (ptr1). Acta Crystallogr D Biol Crystallogr. 1999; 55 (Pt 9): 1608 – 1610.
Kapler GM, Coburn CM, Beverley SM. Stable transfection of the human parasite Leishmania major delineates a 30 – kilo base region suffi-cient for extra chromosomal replication and expression. Mol Cell Biol. 1990; 10 (3): 1084.
Joshi PB, Sacks DL, Modi G, McMaster WR. Targeted gene deletion of Leishmania major genes encoding developmental stage – specific leishmanolysin (Gp63). Mol Microbiol. 1998; 27(3): 519-530.
Beverley SM, Clayton CE. Transfection of Leishmania and Trypanosome brucei by electro-poration. Methods Mol Biol. 1993; 21: 333.
Kelly JM, Ward HM, Miles MA, Kendall G. A shuttle vector which facilitates the expression of transfected genes in Trypanosoma cruzi and Leishmania. Nucleic Acids Res. 1992; 20 (15): 3963–3969.
Rangarajan D, Gokool S, McCrossan MV, Smith DF. The gene B protein localizes to the surface of Leishmania major parasites in the ab-sence of metacyclic stage lipophosphoglycan. J Cell Sci. 1995; 108 (Pt 11): 3359- 3366.
Shewry PR, Fido RJ. Protein blotting, Princi-pels and Applications. In: Molecular Biomethods handbook. Editted by Ralph Rapley and John M. Walker. Published by Humana. USA. 1998; Press pp: 435 – 444.
Kheirandish F, Bandehpour M, Haghighi A, Mahboudi F, Mohebali M, Mosaffa N, Ka-zemi B. Molecular cloning and expression of Iranian Leishmania major pteridine reductase 1. Iranian J Parasitol. 2008; 3 (2): 1-9.
Robello C, Navarro P, Castanys S, Gamarro F. A pteridine reductase gene ptr1 contiguous to a P- glycoprotein confers resistance to antifo-lates in Trypanosoma cruzi. Mol Biochem Parasi-tol. 1997; 90 (2): 525- 535.
White JC. Reversal of methotrexate binding to dihydrofolate reductase by dihydrofolate. Stu-dies with pure enzyme and computer model-ing using network thermodynamics. J Bio Chem. 1979; 254(21): 10889- 10895.
El Fadili A, Kündig C, Roy G, Ouellette M. Inactivation of the Leishmania tarentolae pterin transporter (BT1) and reductase (ptr1) genes leads to viable parasites with changes in folate metabolism and hypersensitivity to the antifo-late methotrexate. J Biol Chem. 2004; 279 (18): 18575-18582.
Hadighi R, Mohebali M, Boucher P, Hajjaran H, Khamesipour A, Ouellette M. Unrespon-siveness to Glucantime Treatment in Iranian Cutaneous Leishmaniasis due to Drug-Resistant Leishmania tropica Parasites. PLoS Med. 2006; 3: e162.
Chen DQ, Kolli BK, Yadava N, Lu HG, Gil-man-Sachs A, Peterson DA, Chang KP. Epi-somal expression of specific sense and anti-sense mRNAs in Leishmania amazonensis: mod-ulation of gp63 level in promastigotes and their infection of macrophages in vitro. Infect Immun. 2000; 68 (1): 80-86.
Joshi PB, Kelly BL, Kamhawi S, Sacks DL, McMaster WR. Targeted gene deletion in Leishmania major identifies leishmanolysin (GP63) as a virulence factor. Mol Biochem Parasitol. 2002; 120 (1): 33-40.
Dumas C, Chow C, Muller M, Papadopoulou B. A novel class of developmentally regulated noncoding RNAs in Leishmania. Eukaryot Cell. 2006; 5 (12): 2033-2046.
Liang XH, Liu Q, Michaeli S. Small nucleolar RNA interference induced by antisense or double-stranded RNA in trypanosomatids. Proc Natl Acad Sci USA. 2003; 100 (13): 7521- 7526.
Dasgupta D, Adhya S, Basu MK. The effect of beta-tubulin-specific antisense oligonucleo-tide encapsulated in different cationic lipo-somes on the suppression (correction of sup-pression) of intracellular L. donovani parasites in vitro. J Biochem. 2002; 132 (1): 23-27.
Compagno D, Toulme JJ. Antisense effects of oligonucleotides complementary to the hairpin of the Leishmania mini-exon RNA. Nucleo-sides Nucleotides. 1999; 18 (6-7): 1701-1704.
Chakraborty R, Dasgupta D, Adhya S, Basu MK. Cationic liposome-encapsulated anti-sense oligonucleotide mediates efficient killing of intracellular Leishmania. Biochem J. 1999; 340 (Pt 2): 393-396.
Compango D, Lampe JN, Bourge, C, Kutya-vin IV, Yurchenko L, Lukhtanov EA, Gorn VV, Gamper JR, Toulme JJ. Antisense oligo-nucleotides containing modified bases inhibit in vitro translation of Leishmania amazonensis mRNAs by invading the mini-exon hairpin. J Biol Chem. 1999; 274 (12): 8191-8198.
Pascolo E, Blonskim C, Shire D, Toulme JJ. Antisense effect of oligodeoxynucleotides complementary to the mini-exon sequence of the protozoan parasite Leishmania amazonensis. Biochimie. 1993; 75 (1-2): 43-47.
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| Issue | Vol 8 No 2 (2013) | |
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| Antisense Gene regulation Inhibition Leishmania Pteridine reductase 1 | ||
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