Skip to main content Accessibility help

Co-factor-independent phosphoglycerate mutase of Leishmania donovani modulates macrophage signalling and promotes T-cell repertoires bearing epitopes for both MHC-I and MHC-II



Immunoactivation depends upon the antigen potential to modulate T-cell repertoires. The present study has enumerated the effect of 61 kDa recombinant Leishmania donovani co-factor-independent phosphoglycerate mutase (rLd-iPGAM) on mononuclear cells of healthy and treated visceral leishmaniasis subjects as well as on THP-1 cell line. rLd-iPGAM stimulation induced higher expression of interleukin-1β (IL-1β) in the phagocytic cell, its receptor and CD69 on T-cell subsets. These cellular activations resulted in upregulation of host-protective cytokines IL-2, IL-12, IL-17, tumour necrosis factor-α and interferon-γ, and downregulation of IL-4, IL-10 and tumour growth factor-β. This immune polarization was also evidenced by upregulation of nuclear factor-κ light-chain enhancer of activated B cells p50 and regulated expression of suppressor of mother against decapentaplegic protein-4. rLd-iPGAM stimulation also promoted lymphocyte proliferation and boosted the leishmaniacidal activity of macrophages by upregulating reactive oxygen species. It also induced 1·8-fold higher release of nitric oxide (NO) by promoting the transcription of inducible nitric oxide synthase gene. Besides, in silico analysis suggested the presence of major histocompatibility complex class I and II restricted epitopes, which can proficiently trigger CD8+ and CD4+ cells, respectively. This study reports rLd-iPGAM as an effective immunoprophylactic agent, which can be used in future vaccine design.


Corresponding author

*Corresponding author: Microbiology Division, RMRIMS (ICMR), Agamkuan, Patna-800007, India. E-mail:,


Hide All
Agallou, M., Margaroni, M. and Karagouni, E. (2011). Cellular vaccination with bone marrow-derived dendritic cells pulsed with a peptide of Leishmania infantum KMP-11 and CpG oligonucleotides induces protection in a murine model of visceral leishmaniasis. Vaccine 29, 50535064.
Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z. and Miller, W. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25, 33893402.
Amit, A., Dikhit, M. R., Mahantesh, V., Chaudhary, R., Singh, A. K., Singh, A., Singh, S. K., Das, V. N. R., Pandey, K., Ali, V., Narayan, S., Sahoo, G. C., Das, P. and Bimal, S. (2016). Immunomodulation mediated through Leishmania donovani protein disulfide isomerase by eliciting CD8+ T-cell in cured visceral leishmaniasis subjects and identification of its possible HLA class-1 restricted T-cell epitopes. Journal of Biomolecular Structure and Dynamics 35, 135.
Anselmo, S. S., Giudice, A., Pereira, J. M. B., Guimaraes, L. H., De Jesus, A. R., Tatiana, R. M., Mary, E. W., Edgar, M. C. and Roque, P. A. (2009). Resistance of Leishmania (Viannia) braziliensis to nitric oxide: correlation with antimony therapy and TNF-α production. BMC Infectious Disease 10, 209.
Aseffa, A., Gumy, A., Launois, P., MacDonald, H. B., Louis, J. A. and Fabienne, T. C. (2002). The early IL-4 response to Leishmania major and the resulting Th2 cell maturation steering progressive disease in BALB/c mice are subject to the control of regulatory CD4+CD25+ T cells. The Journal of Immunology 169, 32323241.
Bisti, S., Konidou, G., Boelaert, J., Lebastard, M. and Soteriadou, K. (2006). The prevention of the growth of L. major progeny in BALB/c iron-loaded mice: a process coupled to increased oxidative burst, the amplitude and duration of which depend on initial parasite development stage and dose. Microbes and Infection 8, 14641472.
Carrillo, E., Crusat, M., Nieto, J., Chicharro, C., Thomas, Mdel. C. and Martinez, E. (2008). Immunogenicity of HSP-70, KMP-11 and PFR-2 leishmanial antigens in the experimental model of canine visceral leishmaniasis. Vaccine 26, 19021911.
Chakravarty, J., Kumar, S. and Trivedi, S. (2011). A clinical trial to evaluate the safety and immunogenicity of the LEISHF1+MPL-SE vaccine for use in the prevention of visceral leishmaniasis. Vaccine 29, 35313537.
Chevalier, N., Rigden, D. J., van Roy, J., Opperdoes, F. R. and Michels, P. A. M. (2000). Trypanosoma brucei contains a 2, 3-bisphosphoglycerate independent phosphoglycerate mutase. European Journal of Biochemistry 267, 14641472.
Christopher, L. K., El-Safi, S. H., Wynn, T. A., Maria, M. H., Satti, M. M. H., Kordofani, A. M., Hashim, F. A., Ali, M. H., Neva, F. A., Nutman, T. B. and Sacks, D. L. (1993). In vivo cytokine profiles in patients with Kala-azar marked elevation of both interleukin-10 and interferon-gamma. Journal of Clinical Investigation 91, 16441648.
Coler, R. N., Goto, Y., Vanitha, L. B. and Steven, G. R. (2007). Leish-111f, a recombinant polyprotein vaccine that protects against visceral leishmaniasis by elicitation of CD4+ T cells. Infection and Immunity 75, 46484654.
Coler, R. N., Duthie, M. S., Hofmeyer, K. A., Guderian, J., Jayashankar, L., Vergara, J., Rolf, T., Misquith, A., Laurance, J. D., Raman, V. S., Bailor, H. R., Cauwelaert, N. D., Reed, S. J., Vallur, A., Favila, M., Orr, M. T., Ashman, J., Ghosh, P., Mondal, D. and Reed, S. G. (2015). From mouse to man: safety, immunogenicity and efficacy of a candidate leishmaniasis vaccine LEISH-F3+GLA-SE. Clinical and Translational Immunology 4, e35.
Dikhit, M. R., Kumar, S., Sahoo, B. R., Mansuri, R., Amit, A. and Ansari, M. Y. (2015). Computational elucidation of potential antigenic CTL epitopes in Ebola virus . Infection, Genetics and Evolution 36, 369375.
Djalma, S. L., Diego, L. C., Vanessa, C., Larissa, D. C., Alexandre, L. N. S., Tiago, W. P. M., Fredy, R. S. G., Maria, B., Karina, R. B., Richard, A. F., Marcelo, T. B. and Dario, S. Z. (2013). Inflammasome-derived IL-1β production induces nitric oxide-mediated resistance to Leishmania . Nature Medicine 19, 909915.
Gannt, K. R., Goldman, T. L., McCormick, M. L., Miller, M. A. and Jeronimo, S. M. (2001). Oxidative responses of human and murine macrophage during phagocytosis of Leishmania chagasi . The Journal of Immunonology 167, 893901.
Garg, R., Gupta, S. K., Tripathi, P., Naik, S. and Sundar, S. (2005). Immunostimulatory cellular responses of cured leishmania infected patients and hamsters against the integral membrane proteins and non-membranous soluble protein of recent clinical isolate of Leishmania donovani . Clinical and Experimental Immunology 140, 149156.
Ghalib, H. W., Whittle, J. A., Kubin, M., Hashim, F. A., el-Hassan, A. M., Grabstein, K. H., Trinchieri, G. and Reed, S. G. (1995). IL-12 enhances Th1-type responses in human Leishmania donovani infections. The Journal of Immunology 154, 46234629.
Ghosh, A., Zhang, W. W. and Matlashewski, G. (2001). Immunization with A2 protein results in a mixed Th1/Th2 and a humoral response which protects mice against Leishmania donovani infections. Vaccine 20, 5966.
Goto, Y., Bogatzki, L. Y., Bertholet, S., Coler, R. N. and Reed, S. G. (2007). Protective immunization against visceral leishmaniasis using Leishmania sterol 24-c methyltransferase formulated in adjuvant. Vaccine 25, 74507458.
Howard, J. G. and Liew, F. Y. (1984). Mechanisms of acquired immunity in leishmaniasis. Philosophical Transactions of Royal Society of London, Series B: Biological Science 307, 8798.
Huber, M., Heink, S., Pagenstecher, A., Reinhard, K., Ritter, J., Visekruna, A., Guralnik, A., Bollig, N., Jeltsch, K., Heinemann, C., Wittmann, E., Buch, T., Prazeres da Costa., O., Brüstle, A., Brenner, D., Mak, T. W., Mittrücker, H. W., Tackenberg, B., Kamradt, T. and Lohoff, M. (2013). IL-17A secretion by CD8+ T cells supports Th17-mediated autoimmune encephalomyelitis. Journal of Clinical Investigation 123, 247260.
Kamhawi, S., Oliveira, F. and Valenzuela, J. G. (2014). Using humans to make a human leishmaniasis vaccine. Science Translational Medicine 6, fs218.
Kar, R. K., Ansari, M. Y., Suryadevara, P., Sahoo, B. R., Sahoo, G. C. and Dikhit, M. R. (2013). Computational elucidation of structural basis for ligand binding with Leishmania donovani adenosine kinase. BioMed Research International 2013, 114. doi: 10.1155/2013/609289.
Kira, R. G., Schultz-Cherry, S., Rodriguez, N., Jeronimo, S. M. B., Nascimento, E. T., Goldman, T. L., Recker, T. J., Miller, M. A. and Wilson, M. E. (2003). Activation of TGF-β by Leishmania chagasi: importance for parasite survival in macrophages. The Journal of Immunology 170, 26132620.
Kumar, R. and Engwerda, C. (2014). Vaccine to prevent leishmaniasis. Clinical and Translational Immunology 2014, e13.
Kumar, R., Pai, K. and Sundar, S. (2001). Reactive oxygen intermediates, nitric and IFN-gamma in Indian visceral leishmanisis. Clinical and Experimental Immunology 124, 262265.
Kumar, P., Pai, K., Pandey, H. P. and Sundar, S. (2002). NADH-oxidase, NADPH-oxidase and myeloperoxidase activity of visceral leishmaniasis patient. Journal of Medical Microbiology 51, 832836.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature 227, 680685.
Liew, F. Y. (1991a). Role of cytokines in killing of intracellular pathogens. Immunology Letter 30, 193197.
Liew, F. Y. (1991b). The effector mechanism and vaccination against cutaneous leishmaniasis. Behring Institute Mitteilungen 1991, 239243.
Murray, H. W. (1981). Susceptibility of Leishmania to oxygen intermediates and killing by normal macrophages. Journal of Experimental Medicine 153, 13021315.
Murray, H. W., Miralles, G. D., Mark, Y. S. and McDermott, D. F. (1993). Role and effect of IL-2 in experimental visceral leishmaniasis. The Journal of Immunonology 151, 929938.
Murray, H. W., Christine, W., Jianguo, L. and Xiaojing, M. (2006). Responses to Leishmania donovani in mice deficient in interleukin- 12 (IL-12), IL-12/IL-23, or IL-18. Infection and Immunity 74, 43704374.
Naederer, T., Ellis, M. A., Sernee, M. F., De Souza, D. P., Curtis, J., Handman, E. and McConville, M. J. (2006). Virulence of Leishmania major in macrophage and mice requires the glucogenic enzyme fructose-1,6-bisphosphotase. Proceeding of the National Academy of Sciences of the United States of America 103, 55025507.
Nascimento, M. S., Carregaro, V., Junior, D. S. L., Costa, D. L., Ryffel, B., Duthie, M. S., Jesus, A., Almedia, R. P. D. and Silva, J. S. D. (2011). Interleukin 17A acts synergistically with interferon γ to promote protection against Leishmania infantum infection. Journal of Infectious Disease 211, 10151026.
Nateghi, R. M., Keshavarz, H., Edalat, R., Sarrafnejad, A. and Shahrestani, T. (2010). CD8+ t cells as a source of IFN-γ production in human cutaneous leishmaniasis. PLoS Neglected Tropical Diseases 4, e845. doi: 10.1371/journal.pntd.0000845.
Nielsen, M. and Lund, O. (2009). NN-align. An artificial neural network-based alignment algorithm for MHC class II peptide binding prediction. BioMed Central Bioinformatics 10, 296.
Nowicki, M. W., Kuaprasert, B., McNae, I. W., Morgan, H. P., Harding, M. M., Michels, P. A. M., Fothergill-Gilmore, L. A. and Walkinshaw, M. D. (2009). Crystal structures of Leishmania maxicana phosphoglycerate mutase suggest a one metal mechanism and a new enzyme subclass. Journal of Molecular Biology 394, 535543.
Pandya, S. K., Verma, R. K., Khare, P., Tiwari, B., Srinivasarao, D. A., Dube, A., Goyal, N. and Mishra, A. (2016). Supplementation of host response by targeting nitric oxide to the macrophage cytosol is efficacious in the hamster model of visceral leishmaniasis and adds to efficacy of amphotericin B. International Journal of Parasitology: Drugs and Drug Resistance 6, 125132.
Parker, K. C., Bednarek, M. A. and Coligan, J. E. (1994). BIMAS: scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains. Journal of Immunology 152, 163175.
Pitta, M. G. R., Romano, P. A., Cabantous, S., Henri, S., Hammad, A., Kouriba, B., Argiro, L., El Kheir, M., Bucheton, B., Mary, C., El-Safi, S. H. and Dessein, A. (2009). IL-17 and IL-22 are associated with protection against human kala azar caused by Leishmania donovani . Journal of Clinical Investigation 119, 23792387.
Qila, S., Woodward, J. and Suzuki, Y. (2013). IL-2 Produced by CD8+ immune T cells can augment their IFN-γ production independently from their proliferation in the secondary response to an intracellular pathogen. Journal of Immunology 190, 21992207.
Rafati, S., Zahedifard, F. and Nazgouee, F. (2006). Prime-boost vaccination using cysteine proteinasestype I and II of leishmania infantum confers protective immunity in murine visceral leishmaniasis. Vaccine 24, 21692175.
Rammensee, H., Bachmann, J., Emmerich, N. P., Bachor, O. A. and Stevanovic, S. (1999). SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 50, 213219.
Rebecca, J., Faleiro, R. K., Louise, M. H. and Christian, R. E. (2014). Immune regulation during chronic visceral leishmaniasis. PLoS Neglected Tropical Disease 8(7), e2914. doi: 10.1371/journal.pntd.0002914.
Reche, P. A., Glutting, J-P., Zhang, H. and Reinherz, E. L. (2004). Enhancement to the RANKPEP resource for the prediction of peptide binding to MHC molecules using profiles. Immunogenetics 56, 405419.
Roelen, B. A., Cohen, O. S., Raychowdhury, M. K., Chadee, D. N., Zhang, Y. and Kyriakis, J. M. (2003). Phosphorylation of threonine 276 in Smad4 is involved in transforming growth factor-beta-induced nuclear accumulation. American Journal of Physiology, Cell Physiology 285, C823C830.
Schuler, M. M., Nastke, M. D. and Stevanović, S. (2007). SYFPEITHI: database for searching and T-cell epitope prediction. Immunoinformatics 409, 7593.
Singh, S. K., Bimal, S., Narayan, S., Jee, C., Bimal, D., Das, P. and Bimal, R. (2011). Leishmania donovani: assessment of leishmanicidal effects of herbal extracts obtained from plants in the visceral leishmaniasis endemic area of Bihar, India. Experimental Parasitology 127, 552558.
Singh, P. K., Kushwaha, S., Rana, A. K. and Bhattacharya, S. M. (2014). Cofactor independent phosphoglycerate mutase of Brugia malayi induces a mixed Th1/Th2 type immune response and inhibits larval development in the host. BioMed Research International 2014, 119.
Skeiky, Y. A., Kennedy, M., Kaufman, D., Borges, M. M., Guderian, J. A. and Scholler, J. K. (1998). LeIF: a recombinant Leishmania protein that induces an IL-12-mediated Th1 cytokine profile. Journal of Immunology 161, 61716179.
Srivastav, S., Saha, A., Barua, J., Ukil, A. and Das, P. K. (2015). IRAK-M regulates the inhibition of TLR-mediated macrophage immune response during late in vitro Leishmania donovani infection. European Journal of Immunology 45, 27872797.
Stager, S., Smith, D. F. and Kaye, P. M. (2000). Immunization with a recombinant stage-regulated surface protein from Leishmania donovani induces protection against visceral leishmaniasis. Journal of Immunology 165, 70647071.
Swain, S. L., Weinberg, A. D., English, M. and Huston, G. (1990). IL-4 directs the development of Th2-like helper effectors. Journal of Immunology 145, 37963806.
Towbin, H., Staehelin, T. and Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gel to nitrocellulose sheet: procedure and some application. Proceeding of National Academy of Science 27, 43504354.


Type Description Title
Supplementary materials

Singh et al supplementary material
Tables S1-S3

 Word (23 KB)
23 KB


Altmetric attention score

Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed