Detection and characterization of an albumin-like protein in Leishmania donovani.

The protozoan parasite, Leishmania donovani, undergoes several molecular adaptations and secretes many effector molecules for host cell manipulation and successful parasitism. The current study identifies an albumin-like secretory protein, expressed in its extracellular promastigote forms. A leishmanial complementary DNA sequence of a partial gene has been cloned, and the encoded peptide (14 kD) is used for the production of polyclonal antibody. This targeted antibody identifies a large native protein (66.421 kD), expressed stage-specifically in promastigotes. Through electron microscopic studies, the native protein is found to be localized in the flagellar pocket and flagella and at the surface of the promastigotes. This native protein is purified with the same customized antibody for future characterization and sequencing. The sequence analysis reveals its homology with the mammalian serum albumin. It is evidenced from in silico studies that this albumin-like protein remains associated with long-chain fatty acids while in vitro studies indicate its close association with membrane cholesterol. Since antibody-mediated blocking compromises the parasite infectivity, these leishmanial albumin-like molecules are hereby proposed to play an instrumental role in the infectivity of L. donovani to peripheral blood monocyte cells. Thus, identification and characterization of an albumin-like protein in L. donovani promastigotes may be interpreted as a molecular adaptation candidate. It may be hypothesized that the parasite mimics the mammalian system for importing fatty acids into the intracellular amastigotes, facilitating its host cell infectivity.

Cholesterol-lowering drug, in combination with chromium chloride, induces early apoptotic signals in intracellular L. donovani amastigotes, leading to death.

Leishmania establishes a successful parasitism by evading both oxidative and non-oxidative killing pathways, and its drug resistance against the currently available therapeutics demands for a safe and cheap drug. Since the parasite synthesizes ergosterol instead of cholesterol, using the same biochemical pathway and enzymes, an inhibitor of HMG-CoA-Reductase, Lovastatin, has been tried for its anti-Leishmanial effect. Lovastatin, being an inhibitor of HMG-CoA-Reductase, inhibits infection by cholesterol depletion, while chromium chloride complexes, at their higher concentrations, are reported to exhibit cytotoxicity. In intracellular amastigotes, cytotoxicity has been checked by assessing various manifestation of cell death, viz. DNA fragmentation, AnnexinV-FITC binding and JC-1 fluorescence ratio. Release of hydrogen peroxide (HPO) and nitric oxide (NO) has been assessed in live cell. Lovastatin and CrCl3.6H2O in combination has appeared to be ineffective on promastigotes but has induced cytotoxic effect on the intracellular amastigotes through up-regulation of cellular signalling mechanisms. CrCl 3.6H2O stimulates generation of NO, leading to reduction of the number of intracellular amastigote, while Lovastatin shows HPO-mediated killing of the same, keeping the host cell unaffected. This novel therapeutic approach, involving two known safe compounds in suboptimal doses, may resolve human visceral Leishmaniasis.