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Background Resistance emergence against antileishmanial drugs, particularly Sodium Antimony Gluconate (SAG) has severely hampered the therapeutic strategy against visceral leishmaniasis, the mechanism of resistance being indistinguishable. Cysteine leucine rich protein (CLrP), was recognized as one of the overexpressed proteins in resistant isolates, as observed in differential proteomics between sensitive and resistant isolates of L. donovani. The present study deals with the characterization of CLrP and for its possible connection with SAG resistance. Methodology and Principal Findings In pursuance of deciphering the role of CLrP in SAG resistance, gene was cloned, over-expressed in E. coli system and thereafter antibody was raised. The expression profile of CLrP and was found to be over-expressed in SAG resistant clinical isolates of L. donovani as compared to SAG sensitive ones when investigated by real-time PCR and western blotting. CLrP has been characterized through bioinformatics, immunoblotting and immunolocalization analysis, which reveals its post-translational modification along with its dual existence in the nucleus as well as in the membrane of the parasite. Further investigation using a ChIP assay confirmed its DNA binding potential. Over-expression of CLrP in sensitive isolate of L. donovani significantly decreased its responsiveness to SAG (SbV and SbIII) and a shift towards the resistant mode was observed. Further, a significant increase in its infectivity in murine macrophages has been observed. Conclusion/Significance The study reports the differential expression of CLrP in SAG sensitive and resistant isolates of L. donovani. Functional intricacy of CLrP increases with dual localization, glycosylation and DNA binding potential of the protein. Further over-expressing CLrP in sensitive isolate of L. donovani shows significantly decreased sensitivity towards SAG and increased infectivity as well, thus assisting the parasite in securing a safe niche. Results indicates the possible contribution of CLrP to antimonial resistance in L. donovani by assisting the parasite growth in the macrophages., Author Summary Leishmania causes complex of pathologies called Leishmaniasis and among the several forms visceral leishmaniasis is the precarious one as being fatal, if left untreated. Emergence of resistance against several antileishmanials particularly Sodium Antimony Gluconate (SAG) has severely battered the therapeutic strategy against VL and the resistance mechanism is still vague. Thus, to apprehend the underlying mechanism, previously, a differential proteomics of SAG unresponsive versus SAG sensitive isolates of L.donovani was done wherein overexpression of Cysteine Leucine Rich protein (CLrP), a member of Leucine rich repeat superfamily, was observed. To scrutinize its involvement in the SAG resistance mechanism, which is till date not investigated, the characterization of CLrP was carried out which revealed its post-translational modification along with its dual existence in the nucleus and in the membrane of the parasite. Further investigation using a ChIP assay confirmed its DNA binding potential. Over-expression of CLrP in sensitive isolate of L. donovani significantly decreased its SAG sensitivity. CLrP overexpressed parasites have increased infectivity. These results point out towards the CLrP’s contribution to antimonial resistance in L. donovani by facilitating parasites growth through macrophages. Further studies are required to depict CLrP as a potential drug target to strengthen the present arsenal against L donovani.

Background The development of a vaccine conferring long-lasting immunity remains a challenge against visceral leishmaniasis (VL). Immunoproteomic characterization of Leishmania donovani proteins led to the identification of a novel protein NAD+-dependent Silent Information regulatory-2 (SIR2 family or sirtuin) protein (LdSir2RP) as one of the potent immunostimulatory proteins. Proteins of the SIR2 family are characterized by a conserved catalytic domain that exerts unique NAD-dependent deacetylase activity. In the present study, an immunobiochemical characterization of LdSir2RP and further evaluation of its immunogenicity and prophylactic potential was done to assess for its possible involvement as a vaccine candidate against leishmaniasis. Methodology/Principal Findings LdSir2RP was successfully cloned, expressed and purified. The gene was present as a monomeric protein of ~45 kDa and further established by the crosslinking experiment. rLdSir2RP shown cytosolic localization in L. donovani and demonstrating NAD+-dependent deacetylase activity. Bioinformatic analysis also confirmed that LdSir2RP protein has NAD binding domain. The rLdSir2RP was further assessed for its cellular response by lymphoproliferative assay and cytokine ELISA in cured Leishmania patients and hamsters (Mesocricetus auratus) in comparison to soluble Leishmania antigen and it was observed to stimulate the production of IFN-γ, IL-12 and TNF-α significantly but not the IL-4 and IL-10. The naïve hamsters when vaccinated with rLdSir2RP alongwith BCG resisted the L. donovani challenge to the tune of ~75% and generated strong IL-12 and IFN-γ mediated Th1 type immune response thereof. The efficacy was further supported by remarkable increase in IgG2 antibody level which is indicative of Th1 type of protective response. Further, with a possible implication in vaccine design against VL, identification of potential T-cell epitopes of rLdSir2RP was done using computational approach. Conclusion/Significance The immunobiochemical characterization strongly suggest the potential of rLdSir2RP as vaccine candidate against VL and supports the concept of its being effective T-cell stimulatory antigen., Author Summary Visceral Leishmaniasis (VL) is the most fatal form of leishmaniasis disease in Indian subcontinent. Through proteomic approaches, NAD-dependent Silent information regulator-2 was identified as one of the potent immunostimulatory proteins. Herein, it was first reported the cloning, expression, purification and immunobiochemical characterization of a NAD+-dependent protein from Leishmania donovani. The gene encodes a monomeric protein (LdSir2RP) of approximately 45 kDa and showed NAD+-dependent deacetylase activity. LdSir2RP was immunodetected in whole cell lysate of L. donovani and further it immunolocalized in cytoplasm of the Leishmania parasite. Recombinant protein rLdSir2RP shown immunogenicity in PBMCs of cured Leishmania patients and hamsters (Mesocricetus auratus). rLdSir2RP stimulated the production of IFN-γ, IL-12 and TNF-α but not IL-4 and IL-10. This was further supported by remarkable increase in IgG2 antibody level. It was further demonstrated that rLdSir2RP was able to provide considerable protection to hamsters against L. donovani challenge. These results supported by the increased iNOS mRNA transcript and the specific Th1-type cytokines—IFN-γ, IL-12 and TNF-α and down-regulation of IL-4, IL-10 and TGF-β. Hence, it is inferred that rLdSir2RP confer significant protection against experimental VL and considered as potential vaccine targets against visceral leishmaniasis.