Transient Down-Regulation of Nucleoside Transporter 3 Gene Expression as a Drug Target in Leishmania major Using Antisense RNA Technology
AbstractBackground: This study was aimed to silencing the Nucleoside transporter 3 (NT3) permease nucleobases involved in the salvage pathway of Leishmania in order to disrupt purine nucleotide uptake in the parasite and consequently, destruction of the parasite. Methods: Overall, 502 bp fragment of the NT3 gene sequence was designed to produce an antisense transcript upon entry of the vector into the parasite. The NT3 construct was transfected into L. major promastigotes and NT3 gene expression was studied in vivo and in vitro conditions. Results: Relative expression NT3 gene in transgenic Leishmania was decreased in tenth day. The percentages and the number of amastigotes infected macrophages with transgenic L. major were less than infected macrophages with wild-type strain. Our results in two groups of BALB/c female mice (wild-type strain and mutant, n=4 each group) were showed that size and number of ulcers in BALB/c mice infected with transgenic Leishmania promastigotes were less than the BALB/c mice infected with wild-type parasites. Conclusion: The results indicate the use of antisense RNA reduces of NT3 gene expression in L. major. More studies are required to obtain a new approach for treating Leishmania infection.
- Alvar J, Ve´lezI D, Bern C, Herrero M, Desjeux PH, Cano J, Jannin J, Boer M. The who Leishmaniasis control team, Leishmaniasis worldwide and global estimates of its incidence. PLOS ONE. 2012; 7 (5), e35671.
-Herwaldt BL. Leishmaniasis. Lancet. 1999; 354: 1191–1199.
-Murray HW, Berman JD, Davies CR, Saravia NG. Advances in Leishmaniasis. Lancet. 2005; 366(9496):1561-1577.
-Carter NS, Rager N, Ullman B. Purine and pyrimidine transport and metabolism. In Molecular and Medical Parasitology. Marr J.J, Nielsen T, Komuniecki R. (eds). London, Academic Press. 2003. p. 197–223.
-Berens R L, Krug E C, Marr J J. In Purine and Pyrimidine Metabolism. Marr J J and Muller M (eds), Academic, New York, USA. 1995. p. 89–117.
-Landfear SM, Ullman B, Carter N, Sanchez MA. Nucleoside and Nucleobase Transporters in parasitic protozoa. Eukaryo Cell. 2004: 245–254.
-Martinez S, Marr JJ. Allopurinol in the treatment of American cutaneous leishmaniasis. N Engl J Med.1992 .326: 741–744.
-Hediger MA. Bioparadigms. 2004. [WWW document]. URL http://www.bioparadigms.org.
-Stein A, Vaseduvan G, Carter NS, Ullman B, Landfear SM, Kavanaugh MP. Equilibrative nucleoside transporter family members from Leishmania donovani are electrogenic proton symporters. J of Biochem Chem. 2003. 278 (37): 35127-35134.
-TCDB. Transporters classification database. 2007. [WWWdocument]. URL http://www.tcdb.org.
-Sanchez MA, Tryon R, Pierce S, Vasudevan G, Landfear SM. Functional expression and characterization of a purine nucleobase transporter gene from Leishmania major. Mol Membr Biol. 2004. 21: 11–18.
-Nelson DJ, Bugge CJ, Elion GB, Berens RL, Marr JJ. Metabolism of pyrazolo (3, 4-d) Pyrimidines in leishmania braziliensis and leishmania donovani. Allopurinol, Oxipurinol and 4-aminopyrazolo (3, 4-d) pyrimidine.J Biol Chem. 1979.254:3959-3964.
-Iovannisci DM, Kaur K, Young L, Ullman B. Genetic analysis of nucleoside transport in Leishmania donovani. Mol Cell Biol. 1984. 4: 1013–1019.
-Ortiz D, Sanchez MA, Pierce S, Herrmann T, Kimblin N, Archie Bouwer, HG, Landfear, S.M. Molecular genetic analysis of purine nucleobase transport in Leishmania major.Mol Microbio. 2007. 64(5):1228–1243.
-BeverleySM, Clayton C. Transfection of Leishmania and Trypanosoma brucei by electroporation. Methods Mol Biol. 1993. 21:333-48.
-Potter H, Heller R. Transfection by electroporation. Curr Protoc Mol Biol. 2003. U–9.3.
-Liang X, Potter J, Kumar S, Zou Y, Quintanilla R, Sridharan M, Carte J, Chen W, Roark N, Ranganathan S, Ravinder N, Chesnut J.D. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J of Biotechno. 2015. 208: 44-53.
-Ouakad M, Bahi-Jaber N, Chenik M, Dellagi K, Louzir H.Selection of endogenous reference genes for gene expression analysis in Leishmania major developmental stages. Parasitol Res. 2007. 101(2): 473-7.
-Sambrook J, Fritsch E, Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, Cold Spring Harbor Laboratory 1, New York, N.Y., USA. 1989.
-Liu W, Boitz JM, Galazka J, Arendt CS, Carter NS, Ullman B. Functional characterization of nucleoside transporter gene repiacements in Leishmania donovani. Mol Biochem parasitol. 2006. 150(2): 300-307.
-Zhang WW, Matlashewski G. Loss of virulence in Leishmania donovani deficient in an amastigote-specific protein, A2. Proc Natl Acad Sci USA. 1997. 94: 8807–8811.
-Nicolas L, Prina E, Lang T, Milon G. Real-Time PCR for detection and quantitation of Leishmania in mouse tissues. J of clinical Microbio. 2002. 40(5), 1666–1669.
-Dias N, Stein CA. Antisense oligonucleotides: Basic concepts and mechanisms. Mol Cancer Therapeu.2002. 1: 347-355.
-Mallinson DJ, Coombs GH. Interaction of Leishmania metacyclics with macrophages. Int J Parasitol. 1989. 19: 647–656.
-Van Assche T, Deschacht M, da Luz RA, Maes L, Cos P. Leishmania-macrophage interactions: insights into the redox biology. Free Radic Biol Med. 2011.51:337–351.
-Mottram JC, Souza AE, Hutchison JE, Carter R, Frame MJ, Coombs GH. Evidence from disruption of the lmcpb gene array of Leishmania mexicana that cysteine proteinases are virulence factors. Proc Natl Acad Sci USA. 1996. 93(12): 6008-13.