Membrane sterol depletion impairs miltefosine action in wild-type and miltefosine-resistant Leishmania donovani promastigotes

The involvement of a carrier for sinefungin (SF) uptake in Leishmania donovani promastigotes is indicated by saturation kinetics, competition studies and SF accumulation against a 270-fold concentration gradient across the cell membrane. Whether SF uptake occurs via nucleoside- or AdoMet-carrier systems was investigated by competition experiments and comparison of the uptake of various molecules in wild-type and SF-resistant cells. Results show that SF did not inhibit purine or pyrimidine uptake whereas it competitively inhibited AdoMet uptake. Furthermore, the uptake of nucleosides in SF-resistant cells is similar to that in wild-type cells, whereas uptake of SF and AdoMet is lower.

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Characterization of the Proliferating Cell Nuclear Antigen of Leishmania donovani Clinical Isolates and Its Association with Antimony Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01847-13 ◽

2014 ◽

Vol 58 (6) ◽

pp. 2997-3007 ◽

Cited By ~ 9

Author(s):

Rati Tandon ◽

Sharat Chandra ◽

Rajendra Kumar Baharia ◽

Sanchita Das ◽

Pragya Misra ◽

...

Keyword(s):

Clinical Isolate ◽

Proliferating Cell Nuclear Antigen ◽

Leishmania Donovani ◽

Clinical Isolates ◽

Resistant Isolate ◽

Nuclear Antigen ◽

Wild Type ◽

Content Type ◽

Sensitive Isolate ◽

Proliferating Cell

ABSTRACTPreviously, through a proteomic analysis, proliferating cell nuclear antigen (PCNA) was found to be overexpressed in the sodium antimony gluconate (SAG)-resistant clinical isolate compared to that in the SAG-sensitive clinical isolate ofLeishmania donovani. The present study was designed to explore the potential role of the PCNA protein in SAG resistance inL. donovani. For this purpose, the protein was cloned, overexpressed, purified, and modeled. Western blot (WB) and real-time PCR (RT-PCR) analyses confirmed that PCNA was overexpressed by ≥3-fold in the log phase, stationary phase, and peanut agglutinin isolated procyclic and metacyclic stages of the promastigote form and by ∼5-fold in the amastigote form of the SAG-resistant isolate compared to that in the SAG-sensitive isolate.L. donovaniPCNA (LdPCNA) was overexpressed as a green fluorescent protein (GFP) fusion protein in a SAG-sensitive clinical isolate ofL. donovani, and modulation of the sensitivities of the transfectants to pentavalent antimonial (SbV) and trivalent antimonial (SbIII) drugs was assessedin vitroagainst promastigotes and intracellular (J774A.1 cell line) amastigotes, respectively. Overexpression of LdPCNA in the SAG-sensitive isolate resulted in an increase in the 50% inhibitory concentrations (IC50) of SbV(from 41.2 ± 0.6 μg/ml to 66.5 ± 3.9 μg/ml) and SbIII(from 24.0 ± 0.3 μg/ml to 43.4 ± 1.8 μg/ml). Moreover, PCNA-overexpressing promastigote transfectants exhibited less DNA fragmentation compared to that of wild-type SAG-sensitive parasites upon SbIIItreatment. In addition, SAG-induced nitric oxide (NO) production was found to be significantly inhibited in the macrophages infected with the transfectants compared with that in wild-type SAG-sensitive parasites. Consequently, we infer that LdPCNA has a significant role in SAG resistance inL. donovaniclinical isolates, which warrants detailed investigations regarding its mechanism.

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Role of Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase in Visceral Organ Infection by Leishmania donovani

Eukaryotic Cell ◽

10.1128/ec.00263-12 ◽

2012 ◽

Vol 12 (1) ◽

pp. 70-77 ◽

Cited By ~ 13

Author(s):

Wen-Wei Zhang ◽

Laura-Isobel McCall ◽

Greg Matlashewski

Keyword(s):

Leishmania Donovani ◽

Null Mutant ◽

Wild Type ◽

Visceral Organ ◽

Content Type ◽

Visceral Organs ◽

Deficient Medium ◽

Visceral Disease ◽

Organ Infection

ABSTRACTThe initial 7 steps of the glycolytic pathway from glucose to 3-phosphoglycerate are localized in the glycosomes inLeishmania, including step 6, catalyzed by the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). InL. donovaniandL. mexicana, there exists a second GAPDH enzyme present in the cytosol that is absent inL. braziliensisand that has become a pseudogene inL. major.To investigate the role of the cytosolic GAPDH (cGAPDH), anL. donovanicGAPDH-null mutant was generated, and conversely, the functionalL. donovanicGAPDH was introduced intoL. majorand the resulting engineered parasites were characterized. TheL. donovanicGAPDH-null mutant was able to proliferate at the same rate as the wild-type parasite in glucose-deficient medium. However, in the presence of glucose, theL. donovanicGAPDH-null mutant consumed less glucose and proliferated more slowly than the wild-type parasite and displayed reduced infectivity in visceral organs of experimentally infected mice. This demonstrates that cGAPDH is functional inL. donovaniand is required for survival in visceral organs. Restoration of cGAPDH activity inL. major, in contrast, had an adverse effect onL. majorproliferation in glucose-containing medium, providing a possible explanation of why it has evolved into a pseudogene inL. major. This study indicates that there is a difference in glucose metabolism betweenL. donovaniandL. major, and this may represent an important factor in the ability ofL. donovanito cause visceral disease.

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Genomic and Metabolomic Polymorphism among Experimentally Selected Paromomycin-Resistant Leishmania donovani Strains

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00904-19 ◽

2019 ◽

Vol 64 (1) ◽

Cited By ~ 4

Author(s):

C. D. Shaw ◽

H. Imamura ◽

T. Downing ◽

G. Blackburn ◽

G. D. Westrop ◽

...

Keyword(s):

Leishmania Donovani ◽

Wild Type ◽

Genetic Changes ◽

Content Type ◽

Novel Treatments ◽

Branched Chain Amino Acid ◽

Amastigote Stage ◽

Branched Chain ◽

Intracellular Amastigote ◽

Selection Of

ABSTRACT Understanding the mechanism(s) underpinning drug resistance could lead to novel treatments to reverse the increased tolerance of a pathogen. In this study, paromomycin (PMM) resistance (PMMr) was induced in three Nepalese clinical strains of Leishmania donovani with different inherent susceptibilities to antimony (Sb) drugs by stepwise exposure of promastigotes to PMM. Exposure to PMM resulted in the production of mixed populations of parasites, even though a single cloned population was used at the start of selection. PMM 50% inhibitory concentration (IC50) values for PMMr parasites varied between 104 and 481 μM at the promastigote stage and 32 and 195 μM at the intracellular amastigote stage. PMM resistance was associated with increased resistance to nitric oxide at the amastigote stage but not the promastigote stage (P < 0.05). This effect was most marked in the Sb-resistant (Sbr) PMMr clone, in which PMM resistance was associated with a significant upregulation of glutathione compared to that in its wild type (P < 0.05), although there was no change in the regulation of trypanothione (detected in its oxidized form). Interestingly, PMMr strains showed an increase in either the keto acid derivative of isoleucine (Sb intermediate PMMr) or the 2-hydroxy acids derived from arginine and tyrosine (Sb susceptible PMMr and Sbr PMMr). These results are consistent with the recent finding that the upregulation of the branched-chain amino acid aminotransferase and d-lactate dehydrogenase is linked to PMMr. In addition, we found that PMMr is associated with a significant increase in aneuploidy during PMM selection in all the strains, which could allow the rapid selection of genetic changes that confer a survival advantage.

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SEARCH FOR NEW ANTILEISHMANIAL CHEMOTHERAPEUTICS

International Journal of Pharmacy and Pharmaceutical Sciences ◽

10.22159/ijpps.2018v10i1.20859 ◽

2018 ◽

Vol 10 (1) ◽

pp. 46

Author(s):

Nabanita Kar ◽

Santanu Ghosh ◽

Leena Kumari ◽

Shreyasi Chakraborty ◽

Tanmoy Bera

Keyword(s):

Betulinic Acid ◽

Leishmania Donovani ◽

Antileishmanial Activity ◽

Gas Chromatography Mass Spectrometry ◽

Drug Resistant ◽

Wild Type ◽

Leaf Oil ◽

Resistant Strains ◽

Type Strains

Objective: The objective of this work was to screen a number of compounds for their antileishmanial efficacy and cytotoxicity profiling.Methods: Curry leaf oil, cypress oil and spikenard oil were identified by gas chromatography-mass spectrometry (GC/MS) analysis. Betulinic acid, spikenard oil, cypress oil and curry leaf oil were evaluated for their in vitro antileishmanial activity against Leishmania donovani AG83 wild-type, sodium stibogluconate resistant (SSG-resistant), paromomycin (PMM-resistant) and GE1 field type strains on axenic and cellular amastigote model and compared the results with standard drugs used to treat leishmaniasis.Results: Betulinic acid showed strong antileishmanial activity against wild-type (SI= 192.8), SSG-resistant (SI= 19.3) and GE1 strains (SI= 100), whereas cypress oil has produced highest antileishmanial activity against PMM-resistant strains (SI= 15.09) among all the tested drugs. The data obtained also revealed that cypress oil had the maximum CC50 value of 452.9 μl among all standard and tested drugs.Conclusion: All tested drugs had antileishmanial property but among them, betulinic acid possess strong antileishmanial activity in case of both wild-type and drug-resistant leishmaniasis.

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Studies on the Antileishmanial Mechanism of Action of the Arylimidamide DB766: Azole Interactions and Role of CYP5122A1

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02405-14 ◽

2014 ◽

Vol 58 (8) ◽

pp. 4682-4689 ◽

Cited By ~ 6

Author(s):

Trupti Pandharkar ◽

Xiaohua Zhu ◽

Radhika Mathur ◽

Jinmai Jiang ◽

Thomas D. Schmittgen ◽

...

Keyword(s):

Cytochrome P450 ◽

Mechanism Of Action ◽

Leishmania Donovani ◽

Synergistic Activity ◽

Wild Type ◽

Content Type ◽

Axenic Amastigotes ◽

Intracellular Parasites

ABSTRACTArylimidamides (AIAs) are inspired by diamidine antimicrobials but show superior activity against intracellular parasites. The AIA DB766 {2,5-bis[2-(2-i-propoxy)-4-(2-pyridylimino)aminophenyl]furan hydrochloride} displays outstanding potency against intracellularLeishmaniaparasites and is effective in murine and hamster models of visceral leishmaniasis when given orally, but its mechanism of action is unknown. In this study, through the use of continuous DB766 pressure, we raisedLeishmania donovaniaxenic amastigotes that displayed 12-fold resistance to this compound. These DB766-resistant (DB766R) parasites were 2-fold more sensitive to miltefosine than wild-type organisms and were hypersensitive to the sterol 14α-demethylase (CYP51) inhibitors ketoconazole and posaconazole (2,000-fold more sensitive and over 12,000-fold more sensitive than the wild type, respectively). Western blot analysis of DB766R parasites indicated that while expression of CYP51 is slightly increased in these organisms, expression of CYP5122A1, a recently identified cytochrome P450 associated with ergosterol metabolism inLeishmania, is dramatically reduced in DB766R parasites.In vitrosusceptibility assays demonstrated that CYP5122A1 half-knockoutL. donovanipromastigotes were significantly less susceptible to DB766 and more susceptible to ketoconazole than their wild-type counterparts, consistent with observations in DB766R parasites. Further, DB766-posaconazole combinations displayed synergistic activity in both axenic and intracellularL. donovaniamastigotes. Taken together, these studies implicate CYP5122A1 in the antileishmanial action of the AIAs and suggest that DB766-azole combinations are potential candidates for the development of synergistic antileishmanial therapy.

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Reduced Infectivity of a Leishmania donovani Biopterin Transporter Genetic Mutant and Its Use as an Attenuated Strain for Vaccination

Infection and Immunity ◽

10.1128/iai.70.1.62-68.2002 ◽

2002 ◽

Vol 70 (1) ◽

pp. 62-68 ◽

Cited By ~ 54

Author(s):

Barbara Papadopoulou ◽

Gaétan Roy ◽

Marie Breton ◽

Christoph Kündig ◽

Carole Dumas ◽

...

Keyword(s):

Homologous Recombination ◽

Recent Work ◽

Interferon Gamma ◽

Protective Immunity ◽

Gene Disruption ◽

Leishmania Donovani ◽

Vaccination Strategy ◽

Enzyme Linked Immunosorbent Assay ◽

Null Mutant ◽

Wild Type

ABSTRACT Pterins are essential for the growth of Leishmania species, and recent work has led to the isolation of the biopterin transporter BT1. In this study, we inactivated the Leishmania donovani biopterin transporter BT1 by gene disruption mediated by homologous recombination. No transport of biopterin was detected in this mutant. The L. donovani BT1 null mutant showed a much lesser capacity for inducing infection in mice than wild-type parasites and could elicit protective immunity in mice susceptible to infection against a L. donovani challenge. Splenocytes isolated from mice immunized with the BT1 null mutant parasites produced significant amounts of interferon gamma following stimulation with L. donovani promastigotes as measured by enzyme-linked immunosorbent assay and enzyme-linked immunospot assays. Overall, these results show that by genetically manipulating the pterin transport in L. donovani, it is possible to generate an attenuated organism that could be part of a vaccination strategy.

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Mechanism of Amphotericin B Resistance inLeishmania donovani Promastigotes

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.2.352 ◽

1998 ◽

Vol 42 (2) ◽

pp. 352-357 ◽

Cited By ~ 102

Author(s):

Nicolas Mbongo ◽

Philippe M. Loiseau ◽

Marie A. Billion ◽

Malka Robert-Gero

Keyword(s):

Amphotericin B ◽

Leishmania Donovani ◽

Saturated Fatty Acids ◽

Major Fatty Acid ◽

Fluorescence Measurement ◽

Wild Type ◽

Drug Pressure ◽

Resistant Cells

ABSTRACT Amphotericin B (AmB)-resistant Leishmania donovanipromastigotes were selected by increasing drug pressure, and their biological features were compared with those of the wild-type parent strain. The 50% inhibitory concentration for resistant cells was 20 times higher than that for the wild-type. Resistance was stable after more than 40 passages in drug-free medium, and resistant promastigotes were infective to macrophages in vitro but lost their virulence in vivo. They had 2.5 times longer generation time, decreased AmB uptake, and increased AmB efflux in comparison to the wild type. Fluorescence measurement with a specific plasma membrane probe, 1-[4-(trimethylammonio)-1,6-diphenylhexa]-1,3,5-triene, showed increased membrane fluidity in drug-resistant promastigotes. Analysis of lipid composition showed that in resistant cells saturated fatty acids were prevalent, with stearic acid as the major fatty acid, and the major sterol was an ergosterol precursor, the cholesta-5, 7, 24-trien-3β-ol and not ergosterol as in the AmB-sensitive strain.

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Multidrug resistance in Leishmania donovani is conferred by amplification of a gene homologous to the mammalian mdr1 gene.

Molecular and Cellular Biology ◽

10.1128/mcb.12.6.2855 ◽

1992 ◽

Vol 12 (6) ◽

pp. 2855-2865 ◽

Cited By ~ 106

Author(s):

D M Henderson ◽

C D Sifri ◽

M Rodgers ◽

D F Wirth ◽

N Hendrickson ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Drug Resistance ◽

Multidrug Resistance ◽

Gene Amplification ◽

Leishmania Donovani ◽

Wild Type ◽

Chain Reaction ◽

Parasitic Protozoa ◽

Polymerase Chain ◽

Mdr Genes

Drug resistance is a major impediment to the effective treatment of parasitic diseases. The role of multidrug resistance (mdr) genes and their products in this drug resistance phenomenon, however, remains controversial. In order to determine whether mdr gene amplification and overexpression can be connected to a multidrug resistance phenotype in parasitic protozoa, a mutant strain of Leishmania donovani was generated by virtue of its ability to proliferate in medium containing increasing concentrations of vinblastine. The vinblastine-resistant strain, VINB1000, displayed a cross-resistance to puromycin and the anthracyclines, a growth phenotype that could be attributed to an impaired ability to accumulate the toxic drugs. By using the polymerase chain reaction, two different DNA fragments, LEMDR06 and LEMDRF2, were amplified from leishmanial genomic DNA, and each amplified fragment encoded a product that was significantly homologous to parts of the mammalian P-glycoprotein. In the VINB1000 strain, the mdr gene recognized by the LEMDR06 probe was amplified approximately 50-fold in copy number, whereas the mdr genes that hybridized to LEMDRF2 or to a fragment of the previously characterized ltpgpA gene were not amplified. Moreover, the VINB1000 cell line expressed a LEMDR06 gene transcript of 12.5 kb in size that was not detected in the parental wild-type strain. To furnish a functional test for mdr gene amplification and expression in L. donovani, the L. donovani gene recognized by the LEMDR06 polymerase chain reaction product, ldmdr1, was isolated from a genomic library, transfected into wild-type cells, and amplified over 500-fold by selection in 0.5 mg of G418 per ml. The resulting transfectants were resistant to all drugs to which VINB1000 cells were resistant and sensitive to all drugs to which VINB1000 cells were sensitive. These studies demonstrate that amplification of the ldmdr1 gene either by direct selection or subsequent to transfection can confer a drug-resistant phenotype in parasitic protozoa similar to that observed for MDR mammalian cells.

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A Novel Spirooxindole Derivative Inhibits the Growth of Leishmania donovani Parasites bothIn VitroandIn Vivoby Targeting Type IB Topoisomerase

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00352-16 ◽

2016 ◽

Vol 60 (10) ◽

pp. 6281-6293 ◽

Cited By ~ 20

Author(s):

Sourav Saha ◽

Chiranjit Acharya ◽

Uttam Pal ◽

Somenath Roy Chowdhury ◽

Kahini Sarkar ◽

...

Keyword(s):

Leishmania Donovani ◽

Nuclear Dna ◽

Large Subunit ◽

Peritoneal Macrophages ◽

Small Subunit ◽

Drug Resistant ◽

Wild Type ◽

Content Type ◽

Fluorescence Studies ◽

Topoisomerase Ib

ABSTRACTVisceral leishmaniasis is a fatal parasitic disease, and there is an emergent need for development of effective drugs against this neglected tropical disease. We report here the development of a novel spirooxindole derivative,N-benzyl-2,2′α-3,3′,5′,6′,7′,7α,α′-octahydro-2methoxycarbonyl-spiro[indole-3,3′-pyrrolizidine]-2-one (compound 4c), which inhibitsLeishmania donovanitopoisomerase IB (LdTopIB) and kills the wild type as well as drug-resistant parasite strains. This compound inhibits catalytic activity of LdTopIB in a competitive manner. Unlike camptothecin (CPT), the compound does not stabilize the DNA-topoisomerase IB cleavage complex; rather, it hinders drug-DNA-enzyme covalent complex formation. Fluorescence studies show that the stoichiometry of this compound binding to LdTopIB is 2:1 (mole/mole), with a dissociation constant of 6.65 μM. Molecular docking with LdTopIB using the stereoisomers of compound 4c produced two probable hits for the binding site, one in the small subunit and the other in the hinge region of the large subunit of LdTopIB. This spirooxindole is highly cytotoxic to promastigotes ofL. donovaniand also induces apoptosis-like cell death in the parasite. Treatment with compound 4c causes depolarization of mitochondrial membrane potential, formation of reactive oxygen species inside parasites, and ultimately fragmentation of nuclear DNA. Compound 4c also effectively clears amastigote forms of wild-type and drug-resistant parasites from infected mouse peritoneal macrophages but has less of an effect on host macrophages. Moreover, compound 4c showed strong antileishmanial efficacies in the BALB/c mouse model of leishmaniasis. This compound potentially can be used as a lead for developing excellent antileishmanial agents against emerging drug-resistant strains of the parasite.

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