The aim of the present study was to synthesize silver (Ag) and titanium dioxide (TiO 2 ) nanoparticles (NPs) using green synthesis from aqueous leaf extract of Euphorbia prostrata as antileishmanial agents and to explore the underlying molecular mechanism of induced cell death. In vitro antileishmanial activity of synthesized NPs was tested against promastigotes of Leishmania donovani by alamarBlue and propidium iodide uptake assays. Antileishmanial activity of synthesized NPs on intracellular amastigotes was assessed by Giemsa staining. The leishmanicidal effect of synthesized Ag NPs was further confirmed by DNA fragmentation assay and by cell cycle progression and transmission electron microscopy (TEM) of the treated parasites. TEM analysis of the synthesized Ag NPs showed a spherical shape with an average size of 12.82 ؎ 2.50 nm, and in comparison to synthesized TiO 2 NPs, synthesized Ag NPs were found to be most active against Leishmania parasites after 24 h exposure, with 50% inhibitory concentrations (IC 50 ) of 14.94 g/ml and 3.89 g/ml in promastigotes and intracellular amastigotes, respectively. A significant increase in G 0 /G 1 phase of the cell cycle with a subsequent decrease in S (synthesis) and G 2 /M phases compared to controls was observed. The growth-inhibitory effect of synthesized Ag NPs was attributed to increased length of S phase. A decreased reactive oxygen species level was also observed, which could be responsible for the caspase-independent shift from apoptosis (G 0 /G 1 arrest) to massive necrosis. High-molecular-weight DNA fragmentation as a positive consequence of necrotic cell death was also visualized. We also report that the unique trypanothione/trypanothione reductase (TR) system of Leishmania cells was significantly inhibited by synthesized Ag NPs. The green-synthesized Ag NPs may provide promising leads for the development of costeffective and safer alternative treatment against visceral leishmaniasis.

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BackgroundLeptomonas is monogenetic kinetoplastid parasite of insects and is primitive in comparison to Leishmania. Comparative studies of these two kinetoplastid may share light on the evolutionary transition to dixenous parasitism in Leishmania. In order to adapt and survive within two hosts, Leishmania species must have acquired virulence factors in addition to mechanisms that mediate susceptibility/resistance to infection in the pathology associated with disease. Rab proteins are key mediators of vesicle transport and contribute greatly to the evolution of complexity of membrane transport system. In this study we used our whole genome sequence data of these two divergent kinetoplastids to analyze the orthologues/paralogues of Rab proteins.ResultsDuring change of lifestyle from monogenetic (Leptomonas) to digenetic (Leishmania), we found that the prenyl machinery remained unchanged. Geranylgeranyl transferase-I (GGTase-I) was absent in both Leishmania and its sister Leptomonas. Farnesyltransferase (FTase) and geranylgeranyl transferase-II (GGTase-II) were identified for protein prenylation. We predict that activity of the missing alpha-subunit (α-subunit) of GGTase-II in Leptomonas was probably contributed by the α-subunit of FTase, while beta-subunit (β-subunit) of GGTase-II was conserved and indicated functional conservation in the evolution of these two kinetoplastids. Therefore the β-subunit emerges as an excellent target for compounds inhibiting parasite activity in clinical cases of co-infections. We also confirmed that during the evolution to digenetic life style in Leishmania, the parasite acquired capabilities to evade drug action and maintain parasite virulence in the host with the incorporation of short-chain dehydrogenase/reductase (SDR/MDR) superfamily in Rab genes.ConclusionOur study based on whole genome sequences is the first to build comparative evolutionary analysis and identification of prenylation proteins in Leishmania and its sister Leptomonas. The information presented in our present work has importance for drug design targeted to kill L. donovani in humans but not affect the human form of the prenylation enzymes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0538-3) contains supplementary material, which is available to authorized users.

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The aim of the present investigation was to synthesize silver (Ag) and titanium dioxide (TiO 2) nanoparticles (NPs) using the aqueous leaves extract of Euphorbia prostrata as antileishmanial agents and to explore the mechanism of induced cell death. In vitro antileishmanial activity of synthesized NPs was tested against promastigotes of Leishmania donovani by alamar Blue ® cell viability reagent and propidium iodide uptake assay. The effective leishmanicidal activity of synthesized Ag NPs was further confirmed by cell cycle progression, externalized phosphatidylserine, DNA fragmentation assay, reactive oxygen species (ROS) level, intracellular non-protein thiols and transmission electron microscopy (TEM) of the treated parasites. TEM analysis of the synthesized Ag NPs and TiO 2 NPs showed spherical shape with an average size of 12.82 ± 2.50 and 83.22 ± 1.50 nm, respectively. Ag NPs was found to be the most active agent against Leishmania parasites after 24 h exposure with IC 50 value of 14.94 μg/mL. A significant increase in G0/ G1 phase of the cell cycle with subsequent decrease in S and G2/M phases was observed when compared to control and thus confirming the growth inhibitory effect of synthesized Ag NPs. Decreased ROS level was also observed which could be responsible for caspase independent shift from apoptosis (G0/G1 arrest) to massive necrosis. High molecular weight DNA fragmentation as a positive consequence of necrotic cell death was also visualized. In the present study, the unique trypanothione/trypanothione reductase (TR) system of Leishmania cells was significantly inhibited by synthesized Ag NPs was reported. The green synthesized Ag NPs may provide promising leads for the development of cost effective and safer alternative treatment against visceral leishmaniasis.

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