Molecular and Cellular Biochemistry

, Volume 359, Issue 1–2, pp 359–368 | Cite as

Identification of TLR inducing Th1-responsive Leishmania donovani amastigote-specific antigens

  • Ankita Srivastava
  • Nisha Singh
  • Manish Mishra
  • Vinod Kumar
  • Jalaj K. Gour
  • Surabhi Bajpai
  • Sangram Singh
  • Haushila P. Pandey
  • Rakesh K. Singh


Leishmania is known to elicit Th2 response that causes leishmaniasis progression; on the other hand, Th1 cytokines restricts amastigote growth and disease progression. In this study, we report the potential of two leishmanial antigens (65 and 98 kDa, in combination) which enhance strong macrophage effector functions, viz., production of respiratory burst enzymes, nitric oxide, and Th1 cytokines. The identification of antigens were done by resolving the crude soluble antigens on SDS-PAGE and eluted by reverse staining method. Further, RAW264.7 macrophages were challenged with eluted antigens, and the innate immune response was observed by detecting respiratory burst enzymes, nitric oxide (NOx), TNF-α, IFN-γ, IL-12, toll-like receptors (TLRs) gene expression, and TLR-signaling proteins. These antigens increased the production of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, superoxide dismutase, NOx, TNF-α, IFN-γ, IL-12, TLR2, and p38 mitogen-activated protein kinase. These antigens also induced human peripheral blood mononuclear cells proliferation and Th1 cytokine production. This study concludes that these antigens induce innate immune response as well as have prophylactic efficacy.


Leishmaniasis Antigens Toll-like receptors Th1 cytokines 



Financial assistance received from the Department of Science and Technology (SR/FT/LS/066/2007), and the Department of Biotechnology—DBT (BT/PR11177/MED/29/99/2008), New Delhi, India is gratefully acknowledged. AS and NS are highly thankful to the DBT, New Delhi for senior research fellowship.

Conflict of interest



  1. 1.
    Mishra BB, Singh RK, Srivastava A, Tripathi VJ, Tiwari VK (2009) Fighting against leishmaniasis: search of alkaloids as future true potential anti-leishmanial agents. Mini Rev Med Chem 9:107–123PubMedCrossRefGoogle Scholar
  2. 2.
    Desjeux P (2004) Leishmaniasis: current situation and new perspectives. Comp Immunol Microbiol Infect Dis 27:305–318PubMedCrossRefGoogle Scholar
  3. 3.
    Singh SP, Reddy DC, Rai M, Sundar S (2006) Serious underreporting of visceral leishmaniasis through passive case reporting in Bihar, India. Trop Med Int Health 11:899–905PubMedCrossRefGoogle Scholar
  4. 4.
    Singh RK, Pandey HP, Sundar S (2006) Visceral leishmaniasis (kala-azar): challenges ahead. Indian J Med Res 123:331–344PubMedGoogle Scholar
  5. 5.
    Kumar P, Pai K, Pandey HP, Sundar S (2002) NADH-oxidase, NADPH-oxidase and myeloperoxidase activity of visceral leishmaniasis patients. J Med Microbiol 51:832–836PubMedGoogle Scholar
  6. 6.
    Singh VK, Balaraman S, Tewary P, Madhubala R (2004) Leishmania donovani activates nuclear transcription factor-kappaB in macrophages through reactive oxygen intermediates. Biochem Biophys Res Commun 322:1086–1095PubMedCrossRefGoogle Scholar
  7. 7.
    Olivier M, Gregory DJ, Forget G (2005) Subversion mechanisms by which Leishmania parasites can escape the host immune response: a signaling point of view. Clin Microbiol Rev 18:293–305PubMedCrossRefGoogle Scholar
  8. 8.
    Bacellar O, D’Oliveira A Jr, Jeronimo S, Carvalho EM (2000) IL-10 and IL-12 are the main regulatory cytokines in visceral leishmaniasis. Cytokine 12:1228–1231PubMedCrossRefGoogle Scholar
  9. 9.
    Ghalib HW, Whittle JA, Kubin M, Hashim FA, el-Hassan AM, Grabstein KH, Trinchieri G, Reed SG (1995) IL-12 enhances Th1-type responses in human Leishmania donovani infections. J Immunol 154:4623–4629PubMedGoogle Scholar
  10. 10.
    Akira S (2003) Toll like receptor signalling. J Biol Chem 278:38105–38108PubMedCrossRefGoogle Scholar
  11. 11.
    Ghosh S, Bhattacharyya S, Sirkar M, Sa GS, Das T, Majumdar D, Roy S, Majumdar S (2002) Leishmania donovani suppresses activated protein 1 and NF-kappaB activation in host macrophages via ceramide generation: involvement of extracellular signal-regulated kinase. Infect Immun 70:6828–6838PubMedCrossRefGoogle Scholar
  12. 12.
    Kawai T, Akira S (2007) TLR signaling. Semin Immunol 19:24–32PubMedCrossRefGoogle Scholar
  13. 13.
    de Veer MJ, Curtis JM, Baldwin TM, Di Donato JA, Sexton A, McConville MJ, Handman E, Schofield L (2003) MyD88 is essential for clearance of Leishmania major: possible role for lipophosphoglycan and Toll-like receptor 2 signaling. Eur J Immunol 33:2822–2831PubMedCrossRefGoogle Scholar
  14. 14.
    Flandin JF, Chano F, Descoteaux A (2006) RNA interference reveals a role for TLR2 and TLR3 in the recognition of Leishmania donovani promastigotes by interferon-gamma-primed macrophages. Eur J Immunol 36:411–420PubMedCrossRefGoogle Scholar
  15. 15.
    Balanco JMF, Pral EMF, Da Silva S, Bijovsky AT, Mortara RA, Alfieri SC (1998) Axenic cultivation and partial characterization of Leishmania brazilensis amastigote like stages. Parasitology 116:103–113PubMedCrossRefGoogle Scholar
  16. 16.
    Lowry OH, Rosebrough NJ, Farr AL, Randal RJ (1951) Protein measurement with folin-phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  17. 17.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  18. 18.
    Castellanos-Serra LR, Fernandez-Patron C, Hardy E, Santana H, Huerta V (1997) High yield elution of proteins from sodium dodecyl sulfate-polyacrylamide gels at the low-picomole level application to N-terminal sequencing of a scarce protein and to in-solution biological activity analysis of on-gel renatured proteins. J Protein Chem 16:415–419PubMedCrossRefGoogle Scholar
  19. 19.
    Mahapatra SK, Chakraborty SP, Roy S (2010) In Vitro time dependent nicotine-induced free radical generation and status of glutathione cycle in murine peritoneal macrophage. Al Ameen J Med Sci 3:182–194Google Scholar
  20. 20.
    Misra HP, Fridovich I (1972) The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 247:3170–3175PubMedGoogle Scholar
  21. 21.
    Ding AH, Nathan CF, Stuehr DJ (1988) Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production. J Immunol 141:2407–2412PubMedGoogle Scholar
  22. 22.
    Mosmann TR, Coffman RL (1989) Th1 and Th2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol 7:145–173PubMedCrossRefGoogle Scholar
  23. 23.
    Abou Fakher FH, Rachinel N, Klimczak M, Louis J, Doyen N (2009) TLR9-dependent activation of dendritic cells by DNA from Leishmania major favors Th1 cell development and the resolution of lesions. J Immunol 182:1386–1396PubMedGoogle Scholar
  24. 24.
    Kropf P, Freudenberg MA, Modolell M, Price HP, Herath S, Antoniazi S, Galanos C, Smith DF, Muller I (2004) Toll-like receptor 4 contributes to efficient control of infection with the protozoan parasite Leishmania major. Infect Immun 72:1920–1928PubMedCrossRefGoogle Scholar
  25. 25.
    Ben-Othman R, Dellagi K, Guizani-Tabbane L (2009) Leishmania major parasites induced macrophage tolerance: implication of MAPK and NF-kappaB pathways. Mol Immunol 46:3438–3444PubMedCrossRefGoogle Scholar
  26. 26.
    Ben-Othman R, Guizani-Tabbane L, Dellagi K (2008) Leishmania initially activates but subsequently down-regulates intracellular mitogen-activated protein kinases and nuclear factor-kappaB signaling in macrophages. Mol Immunol 45:3222–3229PubMedCrossRefGoogle Scholar
  27. 27.
    Mathur RK, Awasthi A, Wadhone P, Ramanamurthy B, Saha B (2004) Reciprocal CD40 signals through p38MAPK and ERK-1/2 induce counteracting immune responses. Nat Med 10:540–544PubMedCrossRefGoogle Scholar
  28. 28.
    Murphy ML, Wille U, Villegas EN, Hunter CA, Farrell JP (2001) IL-10 mediates susceptibility to Leishmania donovani infection. Eur J Immunol 31:2848–2856PubMedCrossRefGoogle Scholar
  29. 29.
    Kumar R, Pai K, Sundar S (2001) Reactive oxygen intermediates, nitrite and IFN-gamma in Indian visceral leishmaniasis. Clin Exp Immunol 124:262–265PubMedCrossRefGoogle Scholar
  30. 30.
    Ghalib HW, Piuvezam MR, Skeiky YA W, Siddig M, Hasim FA, El-Hassan AM, Russo DM, Reed SG (1993) Interleukin 10 production correlates with pathology in human Leishmania donovani infections. J Clin Invest 92:324–329PubMedCrossRefGoogle Scholar
  31. 31.
    Sacks DL, Lal SL, Shrivastava SN, Blackwell J, Neva FA (1987) An analysis of T cell responsiveness in Indian kala-azar. J Immunol 138:908–913PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2011

Authors and Affiliations

  • Ankita Srivastava
    • 1
  • Nisha Singh
    • 1
  • Manish Mishra
    • 1
  • Vinod Kumar
    • 1
  • Jalaj K. Gour
    • 1
  • Surabhi Bajpai
    • 1
  • Sangram Singh
    • 2
  • Haushila P. Pandey
    • 1
  • Rakesh K. Singh
    • 1
  1. 1.Department of Biochemistry, Faculty of ScienceBanaras Hindu UniversityVaranasiIndia
  2. 2.Department of BiochemistryDr. R.M.L. Avadh UniversityFaizabadIndia

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