Neeloo Singh

Elevated levels of tryparedoxin peroxidase in antimony unresponsive Leishmania donovani field isolates

Enhancement of the anti-oxidant metabolism of Leishmania parasites, dependent upon the unique dithiol trypanothione, has been implicated in laboratory-generated antimony resistance. Here, the role of the trypanothione-dependent anti-oxidant pathway is studied in antimony-resistant clinical isolates. Elevated levels of tryparedoxin and tryparedoxin peroxidase, key enzymes in hydroperoxide detoxification, were observed in antimonial resistant parasites resulting in an increased metabolism of peroxides. These data suggest that enhanced anti-oxidant defences may play a significant role in clinical resistance to antimonials.

Novel Mechanism of Drug Resistance in Kala Azar Field Isolates

Clinical resistance to pentavalent antimonial drugs in the form of sodium antimony gluconate (SAG) has become a major problem in the treatment of kala azar (visceral leishmaniasis) in India. The mechanism of resistance is unclear in these clinical isolates, although work has been conducted with Leishmania species mutants selected in vitro by stepwise increase of drug concentration, using antimony-related metal arsenic and, more recently, SAG. In the present study, we investigated the molecular aspect of drug resistance in clinically confirmed SAG-resistant field isolates. Our results show that the mechanisms of resistance postulated for laboratory mutants of Leishmania species are not operating in field isolates of Leishmania donovani. Instead, we identified a novel gene amplified in these drug-resistant parasites whose locus is on chromosome 9. The significant finding was that this isolated fragment confers antimony resistance to wild-type Leishmania species after transfection. We speculate that protein phosphorylation may play a role in signal transduction pathway in the parasite after exposure to drug-conferring resistance.

Flow cytometric determination of intracellular non-protein thiols in Leishmania promastigotes using 5-chloromethyl fluorescein diacetate.

Leishmania parasites lack catalase and therefore, their anti-oxidant system hinges primarily upon non-protein thiols; accordingly, depletion of thiols could potentially serve as an effective drug target. We have developed a flow cytometry based assay using 5-chloromethyl fluorescein diacetate based upon its selective staining of non-protein thiols. Its specificity was confirmed using buthionine sulphoximine (a gamma-glutamyl cysteine synthetase inhibitor), diamide (an oxidizing agent of intracellular thiols) and N-ethylmaleimide (a covalent modifier of cysteine residues) as evidenced by reduction in fluorescence; furthermore, restoration of fluorescence by N-acetyl cysteine corroborated specificity of 5-chloromethyl fluorescein diacetate to measure non-protein thiols. Differences in basal level of thiols in antimony sensitive and antimony resistant Leishmania field isolates were detected. The depletion of non-protein thiols by conventional anti-leishmanial drugs e.g. antimony and miltefosine was demonstrated. Furthermore, fluorescence was unaffected by depletion of ATP in majority of the strains studied, indicating that 5-chloromethyl fluorescein diacetate is not a substrate for the pump operative in most Leishmania donovani strains. Taken together, measurement of 5-chloromethyl fluorescein diacetate fluorescence is an effective method for monitoring non-protein thiols in Leishmania promastigotes.

Green Synthesis of Silver and Titanium Dioxide Nanoparticles Using Euphorbia prostrata Extract Shows Shift from Apoptosis to G0/G1 Arrest followed by Necrotic Cell Death in Leishmania donovani

ABSTRACT The aim of the present study was to synthesize silver (Ag) and titanium dioxide (TiO2) 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 TiO2 NPs, synthesized Ag NPs were found to be most active against Leishmania parasites after 24 h exposure, with 50% inhibitory concentrations (IC50) of 14.94 μg/ml and 3.89 μg/ml in promastigotes and intracellular amastigotes, respectively. A significant increase in G0/G1 phase of the cell cycle with a subsequent decrease in S (synthesis) and G2/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 (G0/G1 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 cost-effective and safer alternative treatment against visceral leishmaniasis.

Molecular docking, structure–activity relationship and biological evaluation of the anticancer drug monastrol as a pteridine reductase inhibitor in a clinical isolate of Leishmania donovani

OBJECTIVES Using the pteridine reductase (PTR1) enzyme of Leishmania as the target, the objective of our study was to find a drug candidate that can enter the clinical development process after being evaluated for safety and efficacy in animals. METHODS Monastrol (R) and (S) enantiomers were docked using the QUANTUM program into the active site of a Leishmania donovani PTR1 (LdPTR1) homology model. A structure-activity relationship based on a homology model of a recombinant enzyme was substantiated by a recombinant enzyme inhibition assay. We adapted an L. donovani (transfected with green fluorescent protein) intramacrophage amastigote screening assay as a cellular model for leishmaniasis. Furthermore, since the clinicopathological features and immunopathological mechanisms of visceral leishmaniasis (VL) in a hamster model are remarkably similar to those of human disease, systemic infection of hamsters with L. donovani was utilized to collect in vivo data for monastrol. RESULTS Both monastrol (R) and (S) enantiomers fit well in the ligand-binding pocket of LdPTR1. Monastrol exhibits a K(i) value of 0.428 microM in the recombinant enzyme inhibition assay. We confirm monastrol as a potent inhibitor of PTR1 in Leishmania; it inhibits proliferation of amastigotes with an IC(50) (50% inhibitory concentration) of 10 microM in macrophage cultures infected with an L. donovani clinical isolate, with no host cytotoxicity. We also show that in experimental animals, oral administration of a 5 mg/kg dose of monastrol on two alternate days inhibits 50% of parasite growth, giving therapeutic backing to the use of monastrol as a potent antileishmanial in human VL cases. CONCLUSIONS To our knowledge, this is the first report presenting monastrol as a potent oral antileishmanial.

Diagnostic PCR with Leishmania donovani specificity using sequences from the variable region of kinetoplast minicircle DNA.

Summary Kala azar or visceral leishmaniasis, a parasitic disease caused by Leishmania donovani, is presently causing an epidemic in the eastern region of India. Diagnosis of kala azar is often complicated. We developed a pair of oligonucleotides suitable as primers from the variable region of a predominant sequence class of minicircles of L. donovani. These primers were used in a nonisotopic polymerase chain reaction and found to be highly specific for the parasites of L. donovani complex. Using these primers, amplification of L. donovani kinetoplast DNA minicircle from the peripheral blood of kala azar patients results in a product of 204 bp. The patient group was comprised of individuals from a highly endemic region of India. We feel that PCR could assess the efficacy of new leishmanicidal drugs under investigation in these patients. PCR could also predict response to therapy which would be useful for both clinical and research applications.

SOLiD™ Sequencing of Genomes of Clinical Isolates of Leishmania donovani from India Confirm Leptomonas Co-Infection and Raise Some Key Questions

Background Known as ‘neglected disease’ because relatively little effort has been applied to finding cures, leishmaniasis kills more than 150,000 people every year and debilitates millions more. Visceral leishmaniasis (VL), also called Kala Azar (KA) or black fever in India, claims around 20,000 lives every year. Whole genome analysis presents an excellent means to identify new targets for drugs, vaccine and diagnostics development, and also provide an avenue into the biological basis of parasite virulence in the L. donovani complex prevalent in India. Methodology/Principal Findings In our presently described study, the next generation SOLiD™ platform was successfully utilized for the first time to carry out whole genome sequencing of L. donovani clinical isolates from India. We report the exceptional occurrence of insect trypanosomatids in clinical cases of visceral leishmaniasis (Kala Azar) patients in India. We confirm with whole genome sequencing analysis data that isolates which were sequenced from Kala Azar (visceral leishmaniasis) cases were genetically related to Leptomonas. The co-infection in splenic aspirate of these patients with a species of Leptomonas and how likely is it that the infection might be pathogenic, are key questions which need to be investigated. We discuss our results in the context of some important probable hypothesis in this article. Conclusions/Significance Our intriguing results of unusual cases of Kala Azar found to be most similar to Leptomonas species put forth important clinical implications for the treatment of Kala Azar in India. Leptomonas have been shown to be highly susceptible to several standard leishmaniacides in vitro. There is very little divergence among these two species viz. Leishmania sp. and L. seymouri, in terms of genomic sequence and organization. A more extensive perception of the phenomenon of co-infection needs to be addressed from molecular pathogenesis and eco-epidemiological standpoint.

Leishmania donovani pteridine reductase 1: Biochemical properties and structure-modeling studies

Pteridine reductase 1 (PTR1, EC 1.5.1.33) is a NADPH dependent short-chain reductase (SDR) responsible for the salvage of pterins in the protozoan parasite Leishmania. This enzyme acts as a metabolic bypass for drugs targeting dihydrofolate reductase, therefore, for successful antifolate chemotherapy to be developed against Leishmania, it must target both enzyme activities. Based on homology model drawn on recombinant pteridine reductase isolated from a clinical isolate of L. donovani, we carried out molecular modeling and docking studies with two compounds of dihydrofolate reductase specificity showing promising antileishmanial activity in vitro. Both the inhibitors appeared to fit well in the active pocket revealing the tight binding of the carboxylic acid ethyl ester group of pyridine moiety to pteridine reductase and identify the important interactions necessary to assist the structure based development of novel pteridine reductase inhibitors.

Molecular typing of HLA class I and class II antigens in Indian kala-azar patients

Summary HLA has been shown to be associated with many diseases. To find out whether host genetic factors like the HLA are involved in susceptibility to kala‐azar (visceral leishmaniasis) in India, we formulated an association study with genetically related controls. All samples were typed by PCR SSOP (sequence specific oligonucleotide probes) for HLA class I (A and B) and class II (DR) antigens. The test of association we used was the transmission disequilibrium test (TDT). No significant evidence for association with any of the three HLA loci was obtained.

An orally effective dihydropyrimidone (DHPM) analogue induces apoptosis-like cell death in clinical isolates of Leishmania donovani overexpressing pteridine reductase 1.

The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis. The enzyme pteridine reductase 1 (PTR1) of L. donovani acts as a metabolic bypass for drugs targeting dihydrofolate reductase (DHFR); therefore, for successful antifolate chemotherapy to be developed against Leishmania, it must target both enzyme activities. Leishmania cells overexpressing PTR1 tagged at the N-terminal with green fluorescent protein were established to screen for proprietary dihydropyrimidone (DHPM) derivatives of DHFR specificity synthesised in our laboratory. A cell-permeable molecule with impressive antileishmanial in vitro and in vivo oral activity was identified. Structure activity relationship based on homology model drawn on our recombinant enzyme established the highly selective inhibition of the enzyme by this analogue. It was seen that the leishmanicidal effect of this analogue is triggered by programmed cell death mediated by the loss of plasma membrane integrity as detected by binding of annexin V and propidium iodide (PI), loss of mitochondrial membrane potential culminating in cell cycle arrest at the sub-G0/G1 phase and oligonucleosomal DNA fragmentation. Hence, this DHPM analogue [(4-fluoro-phenyl)-6-methyl-2-thioxo-1, 2, 3, 4-tetrahydropyrimidine-5-carboxylic acid ethyl ester] is a potent antileishmanial agent that merits further pharmacological investigation.