In visceral leishmaniasis, Th1 types of immune responses correlate with recovery from and resistance to disease, and resolution of infection results in lifelong immunity against the disease. Leishmanial Ags that elicit proliferative and cytokine responses in PBMCs from cured/exposed/Leishmania patients have been characterized through proteomic approaches, and elongation factor-2 is identified as one of the potent immunostimulatory proteins. In this study, we report the cloning and expression of Leishmania donovani elongation factor-2 protein (LelF-2) and its immunogenicity in PBMCs of cured/exposed Leishmania-infected patients and hamsters (Mesocricetus auratus). Leishmania-infected cured/exposed patients and hamsters exhibited significantly higher proliferative responses to recombinant Lelf-2 (rLelF-2) than those with L. donovani-infected hosts. The soluble L. donovani Ag stimulated PBMCs of cured/exposed and Leishmania patients to produce a mixed Thl/Th2-type cytokine profile, whereas rLelF-2 stimulated the production of IFN-γ, IL-12, and TNF-α but not IL-4 or IL-10. Further, rLelF-2 downregulated LPS-induced IL-10 as well as soluble L. donovani Ag-induced IL-4 production by Leishmania patient PBMCs. The immunogenicity of rLelF-2 was also checked in hamsters in which rLelF-2 generates strong IL-12– and IFN-γ–mediated Th1 immune response. This was further supported by a remarkable increase in IgG2 Ab level. We further demonstrated that rLelF-2 was able to provide considerable protection (∼65%) to hamsters against L. donovani challenge. The efficacy was supported by the increased inducible NO synthase mRNA transcript and Th1-type cytokines IFN-γ, IL-12, and TNF-α and downregulation of IL-4, IL-10, and TGF-β. Hence, it is inferred that rLelF-2 elicits a Th1 type of immune response exclusively and confers considerable protection against experimental visceral leishmaniasis.

In the absence of effective and safe treatment for visceral leishmaniasis or Kala-azar – a devastating parasitic disease caused by Leishmania donovani – the search for anti-leishmanial agents from natural resources in common use is imperative. Recently, the comparative in vitro anti-leishmanial activity of methanolic extracts from two landraces of Piper betle – P. betle landrace Bangla Mahoba (PB-BM) and P. betle landrace Kapoori Vellaikodi (PB-KV) – has been reported. Here, the putative pathway responsible for death induced by the effective extract of PB-BM methanolic extract in promastigotes, as well as the intracellular amastigote form of L. donovani, was assessed using various biochemical approaches. It was found that PB-BM was capable of selectively inhibiting both stages of Leishmania parasites by accelerating apoptotic events by generation of reactive oxygen species targeting the mitochondria without any cytotoxicity towards macrophages. The study was extended to determine the presence or absence of activity of the methanolic extract of PB-BM and PB-KV on the basis of differences in essential oil composition present in the extract assessed by GC and MS. The essential oil from PB-BM was found to be rich in eugenol compared with that from PB-KV. The anti-leishmanial efficacy of PB-BM methanolic extract mediated through apoptosis is probably due to the higher content of eugenol in the active landrace. This observation emphasizes the need to extend studies related to traditional medicines from bioactive plants below the species level to the gender/landrace level for better efficacy and reproducibility.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT Over 60% of patients with visceral leishmaniasis (VL) in India and Sudan have become unresponsive to treatment with pentavalent antimonials, the first line of drugs for over 60 years. The drug resistance mechanism, studied so far in in vitro selected laboratory strains, has been attributed to various biochemical parameters. The resistance to Sb (V) in Leishmania field isolates is still unexplored. WHAT THIS STUDY ADDS In order to elucidate for the first time the mechanism of drug resistance in field isolates, this study was done in those clinically relevant field isolates which were either responsive or non responsive to SAG. A comparison of proteome profiles of membrane-enriched as well as cytosolic protein fractions of these isolates has pinpointed the multiple overexpressed proteins in resistant isolates. This study has indicated their possible essential role in antimony resistance of the parasite and provides a vast field to be exploited to find much needed novel treatment strategies against VL. AIMS This study aimed to identify differentially overexpressed membrane-enriched as well as cytosolic proteins in SAG sensitive and resistant clinical strains of L. donovani isolated from VL patients which are involved in the drug resistance mechanism. METHODS The proteins in the membrane-enriched as well as cytosolic fractions of drug-sensitive as well as drug-resistant clinical isolates were separated using two-dimensional gel electrophoresis and overexpressed identified protein spots of interest were excised and analysed using MALDI-TOF/TOF. RESULTS Six out of 12 overexpressed proteins were identified in the membrane-enriched fraction of the SAG resistant strain of L. donovani whereas 14 out of 18 spots were identified in the cytosolic fraction as compared with the SAG sensitive strain. The major proteins in the membrane-enriched fraction were ABC transporter, HSP-83, GPI protein transamidase, cysteine–leucine rich protein and 60S ribosomal protein L23a whereas in the cytosolic fraction proliferative cell nuclear antigen (PCNA), proteasome alpha 5 subunit, carboxypeptidase, HSP-70, enolase, fructose-1,6-bisphosphate aldolase, tubulin-beta chain have been identified. Most of these proteins have been reported as potential drug targets, except 60S ribosomal protein L23a and PCNA which have not been reported to date for their possible involvement in drug resistance against VL. CONCLUSION This study for the first time provided a cumulative proteomic analysis of proteins overexpressed in drug resistant clinical isolates of L. donovani indicating their possible role in antimony resistance of the parasite. Identified proteins provide a vast field to be exploited for novel treatment strategies against VL such as cloning and overexpression of these targets to produce recombinant therapeutic/prophylactic proteins.