Designing and Cloning Molecular Constructs to Knock Out N-Acetylglucosamine Phosphatidylinositol De-N-Acetylase (GPI12) Gene in Leishmania major (MRHO/IR/75/ER)
-
Pooya GHASEMI NEJAD ALMANI
,
Iraj SHARIFI
,
Bahram KAZEMI
,
Zahra BABAEI
,
Mojgan BANDEHPOUR
,
Samira SALARI
,
Ebrahim SAEDI DEZAKI
Abstract
Background: Leishmaniasis represents a major public health concern in tropical and sub-tropical countries. At present, there is no efficacious vaccine against the disease and new control methods are needed. One way to access this important goal is to knock out genes of specific macromolecules to evaluate the effect of deletion on the growth, multiplication, pathogenesis and immunity of the parasite. The aim of this study was to design and clone molecular constructs to knock out N-acetylglucosamine phosphatidylinositol de-N-acetylase (GPI12) gene in Leishmania major.
Methods: For designing and making molecular constructs, we used pLEXSY-neo2 and pLEXSY-hyg2 vectors. The molecular constructs were cloned in E. coli strain Top10. The molecular constructs were transfected by electroporation into L. major in two stages.
Results: The molecular constructs were confirmed by Colony PCR and sequencing. The recombinant strains were isolated by selective antibiotics, after which they were confirmed by PCR, Southern and Western blots.
Conclusion: Recombinant parasites were created and examined for subsequent study. With the use of molecular constructs, it was possible to remove and study gene GPI12 and to achieve a live recombinant Leishmania parasite that maintained the original form of the antigenic parasites. This achievement can be used as an experimental model for vaccine development studies. Further investigations are essential to check this model in a suitable host.Alvar J VI, Vélez ID, Bern C, Herrero M, Desjeux P, Cano J, Jannin J, den Boer M. WHO Leishmaniasis Control Team. Leishmaniasis Worldwide and Global Estimates of Its Incidence. PLoS ONE. 2012;7(5):e35671.
Postigo JAR. Leishmaniasis in the World Health Organization Eastern Mediterranean Region. Int J Antimicrob Agents. 2010;36(1):62-5.
Croft SL SS, Sundar S, Fairlamb AH. Drug resistance in leishmaniasis. Clin Microbiol Rev. 2006;19(1):111-26.
Khatami A FA, Firooz A, Gorouhi F, Dowlati Y. Treatment of acute Old World cutaneous leishmaniasis: a systematic review of the randomized controlled trials. J Am Acad Dermatol. 2007;57(2):335.
Späth GF EL, Epstein L Leader B, Singer SM, Avila HA, Turco SJ, Beverley SM. Lipophosphoglycan is a virulence factor distinct from related glycoconjugates in the protozoan parasite Leishmania major. Proc Natl Acad Sci U S A. 2000;97(16):9258-63.
Chawla B, Madhubala R. Drug targets in Leishmania. J Parasit Dis. 2010;34(1):1-13.
Silvestre R C-d-SA , Cordeiro-da-Silva A, Ouaissi A. Live attenuated Leishmania vaccines: a potential strategic alternative. Arch Immunol Ther Exp. 2008;56(2):123-6.
Hidekazu K. Enhancement of Homologous Recombination Efficiency by Homologous Oligonucleotides. Cell Interaction. 2012. p. 233.
Capecchi MR. Altering the genome by homologous recombination. Science. 1989;244(4910):1288-92.
Cruz A CC, C M Coburn , Beverley SM. Double targeted gene replacement for creating null mutants. Proc Natl Acad Sci USA. 1991;88(16):7170-4.
Crabb BS CB, Cooke BM ,Reeder JC, Waller RF, Caruana SR, Davern KM, Wickham ME, Brown GV, Coppel RL, Cowman AF. Targeted gene disruption shows that knobs enable malaria-infected red cells to cytoadhere under physiological shear stress. Cell. 1997;89(2):287-96.
Fulwiler AL SD, Soysa DR , Ullman B, Yates PA. A rapid, efficient and economical method for generating leishmanial gene targeting constructs. Mol Biochem Parasitol. 2011;175(2):209-12.
Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual: Cold Spring Harbor Laboratory Press; 2001.
Almani PG, Sharifi I, Kazemi B, Babaei Z, Bandehpour M, Salari S, et al. The role of GlcNAc-PI-de-N-acetylase gene by gene knockout through homologous recombination and its consequences on survival, growth and infectivity of Leishmania major in in vitro and in vivo conditions. Acta Trop. 2016;154:63-72.
Soltani M SA, Sadrebazzaz A, Nassiri M, Tahmoorespoor M. Cloning, Nucleotide Sequencing and Bioinformatics Study of NcSRS2 Gene, an Immunogen from Iranian Isolate of Neospora caninum. Iran J Parasitol. 2013;8(1):114-27.
Taheri T SA, Salmanian AH, Gholami E, Doustdari F, Zahedifard F, Rafati S. Leishmania major: disruption of signal peptidase type I and its consequences on survival, growth and infectivity. Exp Parasitol. 2010;126:135-45.
Huynh C SD, Sacks DL , Andrews NW. A Leishmania amazonensis ZIP family iron transporter is essential for parasite replication within macrophage phagolysosomes. J Exp Med. 2006;203(10):2363-75.
Bolhassani A TT, Taheri T, Taslimi Y, Zamanilui S, Zahedifard F, Seyed N, Torkashvand F, Vaziri B, Rafati S. Fluorescent Leishmania species: development of stable GFP expression and its application for in vitro and in vivo studies. Exp Parasitol. 2011;127(3):637-45.
Karcher S. Non-radioactive nucleic acid detection systems. In: Gelvin S, Schilperoort R, editors. Plant Molecular Biology Manual: Springer Netherlands; 1994. p. 309.
Li Q HY, Hu Y , Xu Y, Chen J, Fang L, Liu Z, Jiao X. A gene knock-in method used to purify plasmid pSPI12 from Salmonella enterica serovar Pullorum and characterization of IpaJ. J Microbiol Methods. 2014;98:128-33.
Kedzierski L ZY, Zhu Y, Handman E. Leishmania vaccines: progress and problems. Parasitology. 2006;133:87-112.
Mitchell GF HE, Handman E , Spithill TW. Vaccination against cutaneous leishmaniasis in mice using nonpathogenic cloned promastigotes of Leishmania major and importance of route of injection. Aust J Exp Biol Med Sci. 1984;62:145-53.
Gorczynski RM. Immunization of susceptible BALB/c mice against Leishmania braziliensis. II. Use of temperature-sensitive avirulent clones of parasite for vaccination purposes. Cell Immunol. 1985;94(1):11-20.
Kimsey PB TC, Theodos CM , Mitchen TK, Turco SJ, Titus RG. An avirulent lipophosphoglycan-deficient Leishmania major clone induces CD4+ T cells which protect susceptible BALB/c mice against infection with virulent L. major. Infect Immun. 1993;61(12):5205-13.
Rivier D SR, Shah R , Bovay P, Mauel J. Vaccine development against cutaneous leishmaniasis. Subcutaneous administration of radioattenuated parasites protects CBA mice against virulent Leishmania major challenge. Parasite Immunol. 1993;15(2):75-84.
Streit JA RT, Recker TJ ,Filho FG, Beverley SM, Wilson ME. Protective immunity against the protozoan Leishmania chagasi is induced by subclinical cutaneous infection with virulent but not avirulent organisms. J Immunol. 2001;166(3):1921-9.
Titus RG G-FF, F J Gueiros-Filho ,de Freitas LA, Beverley SM. Development of a safe live Leishmania vaccine line by gene replacement. Proc Natl Acad Sci U S A. 1995;92(22):10267-71.
Kwang-Poo Chang, Bradford S McGwire. Molecular determinants and regulation of Leishmania virulence. Kinetoplastid Biol Dis. 2002;1(1):1.
Chang KP RS, Reed SG , McGwire BS, Soong L. Leishmania model for microbial virulence: the relevance of parasite multiplication and pathoantigenicity. Acta Trop. 2003;85(3):375-90.
Seyed N ZF, Safaiyan S, Gholami E, Doustdari F, Azadmanesh K, et al. In silico analysis of six known Leishmania major antigens and in vitro evaluation of specific epitopes eliciting HLA-A2 restricted CD8 T cell response. PLoS Negl Trop Dis. 2013;5(9):e1295.
Seyed N, Taheri T, Vauchy C, Dosset M, Godet Y, Eslamifar A, et al. Immunogenicity evaluation of a rationally designed polytope construct encoding HLA-A*0201 restricted epitopes derived from Leishmania major related proteins in HLA-A2/DR1 transgenic mice: steps toward polytope vaccine. PLoS One. 2014;9(10):e108848.
| Files | ||
| Issue | Vol 11 No 4 (2016) | |
| Section | Original Article(s) | |
| Keywords | ||
| Leishmaniasis GPI12 Molecular constructs Cloning | ||
| Rights and permissions | |
|
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
