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

2016 ◽  
Vol 60 (10) ◽  
pp. 6281-6293 ◽  
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.

Amino acids 39–456 of the large subunit and 210–262 of the small subunit constitute the minimal functionally interacting fragments of the unusual heterodimeric topoisomerase IB of Leishmania

2007 ◽  
Vol 409 (2) ◽  
pp. 481-489 ◽  
Author(s):  
Somdeb Bosedasgupta ◽  
Benu Brata Das ◽  
Souvik Sengupta ◽  
Agneyo Ganguly ◽  
Amit Roy ◽  
...  
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Large Subunit ◽  
Small Subunit ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Subunit Interaction ◽  
Unusual Structure ◽  
Topoisomerase Ib ◽  
Antisense Constructs

The unusual, heterodimeric topoisomerase IB of Leishmania shows functional activity upon reconstitution of the DNA-binding large subunit (LdTOPIL; or L) and the catalytic small subunit (LdTOPIS; or S). In the present study, we generated N- and C-terminal-truncated deletion constructs of either subunit and identified proteins LdTOPIL39–456 (lacking amino acids 1–39 and 457–635) and LdTOPIS210–262 (lacking amino acids 1–210) as the minimal interacting fragments. The interacting region of LdTOPIL lies between residues 40–99 and 435–456, while for LdTOPIS it lies between residues 210–215 and 245–262. The heterodimerization between the two fragments is weak and therefore co-purified fragments showed reduced DNA binding, cleavage and relaxation properties compared with the wild-type enzyme. The minimal fragments could complement their respective wild-type subunits inside parasites when the respective subunits were down-regulated by transfection with conditional antisense constructs. Site-directed mutagenesis studies identify Lys455 of LdTOPIL and Asp261 of LdTOPIS as two residues involved in subunit interaction. Taken together, the present study provides crucial insights into the mechanistic details for understanding the unusual structure and inter-subunit co-operativity of this heterodimeric enzyme.

scholarly journals Antileishmanial Mechanism of Diamidines Involves Targeting Kinetoplasts

2016 ◽  
Vol 60 (11) ◽  
pp. 6828-6836 ◽  
Author(s):  
Gyongseon Yang ◽  
Gahee Choi ◽  
Joo Hwan No
Keyword(s):  
Leishmania Donovani ◽  
Nuclear Dna ◽  
Binding Properties ◽  
Content Type ◽  
Topoisomerase Ib ◽  
Strong Localization ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Dna Binding Properties ◽  
Dose Dependent

ABSTRACTLeishmaniasis is a disease caused by pathogenicLeishmaniaparasites; current treatments are toxic and expensive, and drug resistance has emerged. While pentamidine, a diamidine-type compound, is one of the treatments, its antileishmanial mechanism of action has not been investigated in depth. Here we tested several diamidines, including pentamidine and its analog DB75, againstLeishmania donovaniand elucidated their antileishmanial mechanisms. We identified three promising new antileishmanial diamidine compounds with 50% effective concentrations (EC50s) of 3.2, 3.4, and 4.5 μM, while pentamidine and DB75 exhibited EC50s of 1.46 and 20 μM, respectively. The most potent antileishmanial inhibitor, compound 1, showed strong DNA binding properties, with a shift in the melting temperature (ΔTm) of 24.2°C, whereas pentamidine had a ΔTmvalue of 2.1°C, and DB75 had a ΔTmvalue of 7.7°C. Additionally, DB75 localized inL. donovanikinetoplast DNA (kDNA) and mitochondria but not in nuclear DNA (nDNA). For 2 new diamidines, strong localization signals were observed in kDNA at 1 μM, and at higher concentrations, the signals also appeared in nuclei. All tested diamidines showed selective and dose-dependent inhibition of kDNA, but not nDNA, replication, likely by inhibitingL. donovanitopoisomerase IB. Overall, these results suggest that diamidine antileishmanial compounds exert activity by accumulating toward and blocking replication of parasite kDNA.

scholarly journals Molecular Diversity of the Syndinean Genus Euduboscquella Based on Single-Cell PCR Analysis

2011 ◽  
Vol 78 (2) ◽  
pp. 334-345 ◽  
Author(s):  
Tsvetan R. Bachvaroff ◽  
Sunju Kim ◽  
Laure Guillou ◽  
Charles F. Delwiche ◽  
D. Wayne Coats
Keyword(s):  
Large Subunit ◽  
Small Subunit ◽  
Base Change ◽  
Pcr Analysis ◽  
Ssu Rrna ◽  
Compensatory Base Change ◽  
Content Type ◽  
Single Cell Pcr ◽  
Group I ◽  
Lsu Rrna

ABSTRACTThe genusEuduboscquellais one of a few described genera within the syndinean dinoflagellates, an enigmatic lineage with abundant diversity in marine environmental clone libraries based on small subunit (SSU) rRNA. The region composed of the SSU through to the partial large subunit (LSU) rRNA was determined from 40 individual tintinnid ciliate loricae infected withEuduboscquellasampled from eight surface water sites in the Northern Hemisphere, producing seven distinct SSU sequences. The corresponding host SSU rRNA region was also amplified from eight host species. The SSU tree ofEuduboscquellaand syndinean group I sequences from environmental clones had seven well-supported clades and one poorly supported clade across data sets from 57 to 692 total sequences. The genusEuduboscquellaconsistently formed a supported monophyletic clade within a single subclade of group I sequences. For most parasites with identical SSU sequences, the more variable internal transcribed spacer (ITS) to LSU rRNA regions were polymorphic at 3 to 10 sites. However, inE. cachonithere was variation between ITS to LSU copies at up to 20 sites within an individual, while in a parasite ofTintinnopsisspp., variation between different individuals ranged up to 19 polymorphic sites. However, applying the compensatory base change model to the ITS2 sequences suggested no compensatory changes within or between individuals with the same SSU sequence, while one to four compensatory changes between individuals with similar but not identical SSU sequences were found. Comparisons between host and parasite phylogenies do not suggest a simple pattern of host or parasite specificity.

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

2014 ◽  
Vol 58 (6) ◽  
pp. 2997-3007 ◽  
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.

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

2012 ◽  
Vol 12 (1) ◽  
pp. 70-77 ◽  
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.

Modulation of procaspase-7 self-activation by PEST amino acid residues of the N-terminal prodomain and intersubunit linker

2017 ◽  
Vol 95 (6) ◽  
pp. 634-643
Author(s):  
Juliano Alves ◽  
Miguel Garay-Malpartida ◽  
João M. Occhiucci ◽  
José E. Belizário
Keyword(s):  
Amino Acid ◽  
Large Subunit ◽  
Small Subunit ◽  
Cleavage Sites ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Caspase 7 ◽  
Caspase Family ◽  
The Impact

Procaspase-7 zymogen polypeptide is composed of a short prodomain, a large subunit (p20), and a small subunit (p10) connected to an intersubunit linker. Caspase-7 is activated by an initiator caspase-8 and -9, or by autocatalysis after specific cleavage at IQAD198↓S located at the intersubunit linker. Previously, we identified that PEST regions made of amino acid residues Pro (P), Glu (E), Asp (D), Ser (S), Thr (T), Asn (N), and Gln (Q) are conserved flanking amino acid residues in the cleavage sites within a prodomain and intersubunit linker of all caspase family members. Here we tested the impact of alanine substitution of PEST amino acid residues on procaspase-7 proteolytic self-activation directly in Escherichia coli. The p20 and p10 subunit cleavage were significantly delayed in double caspase-7 mutants in the prodomain (N18A/P26A) and intersubunit linker (S199A/P201A), compared with the wild-type caspase-7. The S199A/P201A mutants effectively inhibited the p10 small subunit cleavage. However, the mutations did not change the kinetic parameters (kcat/KM) and optimal tetrapeptide specificity (DEVD) of the purified mutant enzymes. The results suggest a role of PEST-amino acid residues in the molecular mechanism for prodomain and intersubunit cleavage and caspase-7 self-activation.

scholarly journals SlyA Is a Transcriptional Regulator Involved in the Virulence of Enterococcus faecalis

2011 ◽  
Vol 79 (7) ◽  
pp. 2638-2645 ◽  
Author(s):  
Charlotte Michaux ◽  
Maurizio Sanguinetti ◽  
Fany Reffuveille ◽  
Yanick Auffray ◽  
Brunella Posteraro ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Galleria Mellonella ◽  
Peritoneal Macrophages ◽  
Bacterial Virulence ◽  
Transcriptional Analysis ◽  
Infection Model ◽  
Wild Type ◽  
Content Type ◽  
Wild Type Parent

ABSTRACTPhylogenetic analysis of the crystal structure of theEnterococcus faecalisSlyA (EF_3002) transcriptional factor places it between the SlyA and MarR regulator subfamilies. Proteins of these families are often involved in the regulation of genes important for bacterial virulence and stress response. To gather evidence for the role of this putative regulator inE. faecalisbiology, we dissected the genetic organization of theslyA-EF_3001 locus and constructed aslyAdeletion mutant as well as complemented strains. Interestingly, compared to the wild-type parent, the ΔslyAmutant is more virulent in an insect infection model (Galleria mellonella), exhibits increased persistence in mouse kidneys and liver, and survives better inside peritoneal macrophages. In order to identify a possible SlyA regulon, global microarray transcriptional analysis was performed. This study revealed that theslyA-EF_3001 locus appears to be autoregulated and that 117 genes were differentially regulated in the ΔslyAmutant. In the mutant strain, 111 were underexpressed and 6 overexpressed, indicating that SlyA functions mainly as an activator of transcription.

scholarly journals Surface Lipoprotein PpiA of Streptococcus mutans Suppresses Scavenger Receptor MARCO-Dependent Phagocytosis by Macrophages

2011 ◽  
Vol 79 (12) ◽  
pp. 4933-4940 ◽  
Author(s):  
Tadashi Mukouhara ◽  
Takafumi Arimoto ◽  
Kasei Cho ◽  
Matsuo Yamamoto ◽  
Takeshi Igarashi
Keyword(s):  
Streptococcus Mutans ◽  
Scavenger Receptor ◽  
Peritoneal Macrophages ◽  
Wild Type ◽  
Gene Encoding ◽  
Content Type ◽  
Macrophage Response ◽  
Human Macrophages ◽  
Scavenger Receptor A ◽  
Collagenous Structure

ABSTRACTStreptococcus mutansis associated with the initiation and progression of human dental caries and is occasionally isolated from the blood of patients with bacteremia and infective endocarditis. For the pathogen to survive in the infected host, surface lipoproteins ofS. mutansare likely to play important roles in interactions with the innate immune system. To clarify the role that a putative lipoprotein, peptidyl-prolylcis/trans-isomerase (PpiA), ofS. mutansplays in the macrophage response, we investigated the response of THP-1-derived macrophages toS. mutanschallenge. The deletion of the gene encoding Lgt eliminated PpiA on the cell surface ofS. mutans, which implies that PpiA is a lipoprotein that is lipid anchored in the cell membrane by Lgt. Human and murine peritoneal macrophages both showed higher phagocytic activities for theppiAandlgtmutants than the wild type, which indicates that the presence of PpiA reducesS. mutansphagocytosis. In addition, infection withS. mutansmarkedly induced mRNAs of macrophage receptor with collagenous structure (MARCO) and scavenger receptor A (SR-A) in human macrophages. In particular, transcriptional and translational levels of MARCO in human macrophages infected with theppiAmutant were higher than those in macrophages infected with the wild type. Phagocytosis ofS. mutansby human macrophages markedly decreased after treatment with anti-MARCO IgG. These results demonstrate that theS. mutanslipoprotein PpiA contributes to suppression of MARCO-mediated phagocytosis of this bacterium by macrophages.

scholarly journals NUCLEAR MUTATION INCREASES STREPTOMYCIN AND SPECTINOMYCIN SENSITIVITY IN CHLAMYDOMONAS

Genetics ◽  
1978 ◽  
Vol 88 (4) ◽  
pp. 643-650
Author(s):  
Robert W Lee ◽  
Jan A Sapp
Keyword(s):  
Large Subunit ◽  
Small Subunit ◽  
Wild Type ◽  
Resistance Mutations ◽  
Erythromycin Resistance ◽  
Spectinomycin Resistance ◽  
Nuclear Mutation ◽  
Resistant Strains ◽  
Nuclear Locus ◽  
Type Strains

ABSTRACT A spontaneously arising nuclear mutation, ss-1, has been identified in Chlamydomonas reinhardtii that decreases both streptomycin and spectinomycin resistance levels about 10-fold after its introduction into all wild-type, streptomycin-resistant and spectinomycin-resistant strains examined. The mutations for resistance map to nuclear and uniparentally inherited (chloroplast) loci. In contrast, no modification of erythromycin resistance was detected after introducing ss-1 into wild-type strains or into strains carrying nuclear or uniparentally inherited erythromycin-resistance mutations. We suggest that ss-1 affects the small subunit of the chloroplast ribosome because others have shown that streptomycin and spectinomycin resistance in C. reinhardtii are associated with this subunit, whereas erythromycin resistance is associated with the large subunit. ss-1 shows no linkage with the nuclear locus for streptomycin resistance.

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

2019 ◽  
Vol 64 (1) ◽  
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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