Characterization of Glycolytic Enzymes - rAldolase and rEnolase of Leishmania donovani, Identified as Th1 Stimulatory Proteins, for Their Immunogenicity and Immunoprophylactic Efficacies against Experimental Visceral Leishmaniasis

Reema Gupta,Mohammad Imran Siddiqi,Chandradev Pati Tripathi,Vikash Kumar,Pramod Kumar Kushawaha,Kalyan Mitra,Sumit Joshi,Shyam Sundar,Anuradha Dube,Amogh Anant Sahasrabuddhe

doi:10.1371/journal.pone.0086073

Reema Gupta, Mohammad Imran Siddiqi + Show 8 more

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https://doi.org/10.1371/journal.pone.0086073

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Abstract

Th1 immune responses play an important role in controlling Visceral Leishmaniasis (VL) hence, Leishmania proteins stimulating T-cell responses in host, are thought to be good vaccine targets. Search of such antigens eliciting cellular responses in Peripheral blood mononuclear cells (PBMCs) from cured/exposed/Leishmania patients and hamsters led to the identification of two enzymes of glycolytic pathway in the soluble lysate of a clinical isolate of Leishmania donovani - Enolase (LdEno) and aldolase (LdAld) as potential Th1 stimulatory proteins. The present study deals with the molecular and immunological characterizations of LdEno and LdAld. The successfully cloned and purified recombinant proteins displayed strong ability to proliferate lymphocytes of cured hamsters’ along with significant nitric-oxide production and generation of Th1-type cytokines (IFN-γ and IL-12) from stimulated PBMCs of cured/endemic VL patients. Assessment of their prophylactic potentials revealed ∼90% decrease in parasitic burden in rLdEno vaccinated hamsters against Leishmania challenge, strongly supported by an increase in mRNA expression levels of iNOS, IFN-γ, TNF-α and IL-12 transcripts along with extreme down-regulation of TGF-β, IL-4 and IL-10. However, animals vaccinated with rLdAld showed comparatively lesser prophylactic efficacy (∼65%) with inferior immunological response. Further, with a possible implication in vaccine design against VL, identification of potential T-cell epitopes of both the proteins was done using computational approach. Additionally, in-silico 3-D modelling of the proteins was done in order to explore the possibility of exploiting them as potential drug targets. The comparative molecular and immunological characterizations strongly suggest rLdEno as potential vaccine candidate against VL and supports the notion of its being effective T-cell stimulatory protein.

Highlights

The search for an effective vaccine against Visceral Leishmaniasis (VL), caused by Leishmania donovani producing a severe and potentially fatal systemic disease in which parasites invade the macrophages of liver, spleen and bone marrow causing serious illness, remains a challenging and elusive goal
In earlier studies from our laboratory, while carrying out classical activity based fractionation and sub-fractionation of soluble L. donovani, we have observed that the sub-fraction ranging from 89.9 to 97.1 kDa elicited significant Th1 stimulatory response in Peripheral blood mononuclear cells (PBMCs)/lymphocytes of cured Leishmania patients/hamsters
The presence of Enolase and aldolase in higher molecular weight range in proteomic studies, in contrast to its observed molecular mass could be attributed to the posttranslational modifications which are widely prevalent in Leishmania

Summary

Introduction

The search for an effective vaccine against Visceral Leishmaniasis (VL), caused by Leishmania donovani producing a severe and potentially fatal systemic disease in which parasites invade the macrophages of liver, spleen and bone marrow causing serious illness, remains a challenging and elusive goal. Human VL is characterized by a marked humoral response and impaired cell-mediated immunity (CMI), associated with an inability to control infection. Leishmanial antigens that predominantly stimulate Th1 responses in patient cells or rodents infected with the parasite have been accepted as ‘potential protective antigens’ and promising vaccine candidates. Further proteomic characterization of this subfraction led to the identification of 18 Th1 stimulatory proteins and among them Enolase and Fructose bisphosphate Aldolase (FBA), the vital proteins belonging to the glycolytic pathway, were present. Aldolase is a central glycolytic enzyme (E.C. 4.1.2.13) in carbohydrate metabolism, catalyzing the cleavage of fructose 1,6-bisphosphate into two triose sugars, glyceraldehyde 3-phosphate and dihydroacetone phosphate [7] whereas Enolase (2phospho-D-glycerate hydrolase, EC 4.2.1.11) is known to catalyse the reversible dehydration of D-2-phosphoglycerate (2PGA) to phosphoenolpyruvate (PEP) in both glycolysis and gluconeogenesis [8]

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