scholarly journals Disuccinyl Betulin Triggers Metacaspase-Dependent Endonuclease G-Mediated Cell Death in Unicellular Protozoan Parasite Leishmania donovani

2014 ◽  
Vol 58 (4) ◽  
pp. 2186-2201 ◽  
Author(s):  
Sayan Chowdhury ◽  
Tulika Mukherjee ◽  
Somenath Roy Chowdhury ◽  
Souvik Sengupta ◽  
Sibabrata Mukhopadhyay ◽  
...  
Keyword(s):  
Cell Death ◽  
Leishmania Donovani ◽  
Protozoan Parasite ◽  
Degradation Process ◽  
Dna Lesions ◽  
Death Process ◽  
Unicellular Organism ◽  
Content Type ◽  
Atp Production ◽  
Endonuclease G

ABSTRACTThe unicellular organismLeishmaniaundergoes apoptosis-like cell death in response to external stress or exposure to antileishmanial agents. Here, we showed that 3-O,28-O-disuccinyl betulin (DiSB), a potent topoisomerase type IB inhibitor, induced parasitic cell death by generating oxidative stress. The characteristic feature of the death process resembled the programmed cell death (PCD) seen in higher eukaryotes. In the current study, the generation of reactive oxygen species (ROS), followed by the depolarization of mitochondrial membrane potential (ΔΨm), caused a loss in ATP production inLeishmaniaparasites. This further gave positive feedback to produce a large amount of ROS, which in turn caused oxidative DNA lesions and genomic DNA fragmentation. The treatment of promastigotes with DiSB induced high expression levels of metacaspase protein that led to cell death in this unicellular organism. The PCD was insensitive to benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk), suggesting that the death process was not associated with the activation of caspases. DiSB treatment translocatedLeishmania donovaniendonuclease G (LdEndoG) from mitochondria to the nucleus, which was responsible for the DNA degradation process. Conditional antisense knockdown ofL. donovanimetacaspase (LdMC), as well as EndoG, -subverted death of the parasite and rescued cell cycle arrest in G1phase. The present study on the effector molecules associated with the PCD pathway of the parasite should help to manifest the mechanisms of PCD and also might be exploited in antileishmanial chemotherapy.

scholarly journals Characterization of Metacaspases with Trypsin-Like Activity and Their Putative Role in Programmed Cell Death in the Protozoan Parasite Leishmania

2007 ◽  
Vol 6 (10) ◽  
pp. 1745-1757 ◽  
Author(s):  
Nancy Lee ◽  
Sreenivas Gannavaram ◽  
Angamuthu Selvapandiyan ◽  
Alain Debrabant
Keyword(s):  
Hydrogen Peroxide ◽  
Cell Death ◽  
Programmed Cell Death ◽  
Leishmania Donovani ◽  
Protozoan Parasite ◽  
Protein Band ◽  
Mrna Levels ◽  
Enzymatic Assays ◽  
Rich Domain ◽  
Axenic Amastigotes

ABSTRACT In this report, we have characterized two metacaspases of Leishmania donovani, L. donovani metacaspase-1 (LdMC1) and LdMC2. These two proteins show 98% homology with each other, and both contain a characteristic C-terminal proline-rich domain. Both genes are transcribed in promastigotes and axenic amastigotes of L. donovani; however, LdMC1 shows increased mRNA levels in axenic amastigotes. An anti-LdMC antibody was obtained and showed reactivity with a single ∼42-kDa protein band in both promastigote and axenic amastigote parasite whole-cell lysates by Western blotting. Pulse-chase experiments suggest that LdMCs are not synthesized as proenzymes, and immunofluorescence studies show that LdMCs are associated with the acidocalcisome compartments of L. donovani. Enzymatic assays of immunoprecipitated LdMCs show that native LdMCs efficiently cleave trypsin substrates and are unable to cleave caspase-specific substrates. Consistently, LdMC activity is insensitive to caspase inhibitors and is efficiently inhibited by trypsin inhibitors, such as leupeptin, antipain, and N α-tosyl-l-lysine-chloromethyl ketone (TLCK). In addition, our results show that LdMC activity was induced in parasites treated with hydrogen peroxide, a known trigger of programmed cell death (PCD) in Leishmania and that parasites overexpressing metacaspases are more sensitive to hydrogen peroxide-induced PCD. These findings suggest that Leishmania metacaspases are not responsible for the caspase-like activities reported in this organism and suggest a possible role for LdMCs as effector molecules in Leishmania PCD.

scholarly journals Leishmania donovani Lipophosphoglycan Increases Macrophage-Dependent Chemotaxis of CXCR6-Expressing Cells via CXCL16 Induction

2019 ◽  
Vol 87 (5) ◽  
Author(s):  
Visnu Chaparro ◽  
Louis-Philippe Leroux ◽  
Aude Zimmermann ◽  
Armando Jardim ◽  
Brent Johnston ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Leishmania Donovani ◽  
Molecular Mechanisms ◽  
Defense Mechanism ◽  
Protozoan Parasite ◽  
Parasitic Infections ◽  
Dependent Manner ◽  
Chemokine Production ◽  
Content Type

ABSTRACT CXCL16 is a multifunctional chemokine that is highly expressed by macrophages and other immune cells in response to bacterial and viral pathogens; however, little is known regarding the role of CXCL16 during parasitic infections. The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis. Even though chemokine production is a host defense mechanism during infection, subversion of the host chemokine system constitutes a survival strategy adopted by the parasite. Here, we report that L. donovani promastigotes upregulate CXCL16 synthesis and secretion by bone marrow-derived macrophages (BMDM). In contrast to wild-type parasites, a strain deficient in the virulence factor lipophosphoglycan (LPG) failed to induce CXCL16 production. Consistent with this, cell treatment with purified L. donovani LPG augmented CXCL16 expression and secretion. Notably, the ability of BMDM to promote migration of cells expressing CXCR6, the cognate receptor of CXCL16, was augmented upon L. donovani infection in a CXCL16- and LPG-dependent manner. Mechanistically, CXCL16 induction by L. donovani required the activity of AKT and the mechanistic target of rapamycin (mTOR) but was independent of Toll-like receptor signaling. Collectively, these data provide evidence that CXCL16 is part of the inflammatory response elicited by L. donovani LPG in vitro. Further investigation using CXCL16 knockout mice is required to determine whether this chemokine contributes to the pathogenesis of visceral leishmaniasis and to elucidate the underlying molecular mechanisms.

scholarly journals Perifosine Mechanisms of Action in Leishmania Species

2017 ◽  
Vol 61 (4) ◽  
Author(s):  
Atteneri López-Arencibia ◽  
Carmen Martín-Navarro ◽  
Ines Sifaoui ◽  
María Reyes-Batlle ◽  
Carolina Wagner ◽  
...  
Keyword(s):  
Cell Death ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Leishmania Donovani ◽  
Leishmania Species ◽  
Mechanisms Of Action ◽  
Content Type ◽  
Phosphatidylserine Externalization ◽  
Potential Use

ABSTRACT Here the mechanism by which perifosine induced cell death in Leishmania donovani and Leishmania amazonensis is described. The drug reduced Leishmania mitochondrial membrane potential and decreased cellular ATP levels while increasing phosphatidylserine externalization. Perifosine did not increase membrane permeabilization. We also found that the drug inhibited the phosphorylation of Akt in the parasites. These results highlight the potential use of perifosine as an alternative to miltefosine against Leishmania.

scholarly journals Proteomic-Based Approach To Gain Insight into Reprogramming of THP-1 Cells Exposed to Leishmania donovani over an Early Temporal Window

2015 ◽  
Vol 83 (5) ◽  
pp. 1853-1868 ◽  
Author(s):  
Alok Kumar Singh ◽  
Rajeev Kumar Pandey ◽  
Jair Lage Siqueira-Neto ◽  
Yong-Jun Kwon ◽  
Lucio H. Freitas-Junior ◽  
...  
Keyword(s):  
Host Cell ◽  
Rna Splicing ◽  
Leishmania Donovani ◽  
Expression Profiles ◽  
Cell Metabolism ◽  
Protozoan Parasite ◽  
Human Monocyte ◽  
Absolute Quantification ◽  
Acid Oxidation ◽  
Content Type

Leishmania donovani, a protozoan parasite, is the causative agent of visceral leishmaniasis. It lives and multiplies within the harsh environment of macrophages. In order to investigate how intracellular parasite manipulate the host cell environment, we undertook a quantitative proteomic study of human monocyte-derived macrophages (THP-1) following infection withL. donovani. We used the isobaric tags for relative and absolute quantification (iTRAQ) method and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to compare expression profiles of noninfected andL. donovani-infected THP-1 cells. We detected modifications of protein expression in key metabolic pathways, including glycolysis and fatty acid oxidation, suggesting a global reprogramming of cell metabolism by the parasite. An increased abundance of proteins involved in gene transcription, RNA splicing (heterogeneous nuclear ribonucleoproteins [hnRNPs]), histones, and DNA repair and replication was observed at 24 h postinfection. Proteins involved in cell survival and signal transduction were more abundant at 24 h postinfection. Several of the differentially expressed proteins had not been previously implicated in response to the parasite, while the others support the previously identified proteins. Selected proteomics results were validated by real-time PCR and immunoblot analyses. Similar changes were observed inL. donovani-infected human monocyte-derived primary macrophages. The effect of RNA interference (RNAi)-mediated gene knockdown of proteins validated the relevance of the host quantitative proteomic screen. Our findings indicate that the host cell proteome is modulated afterL. donovaniinfection, provide evidence for global reprogramming of cell metabolism, and demonstrate the complex relations between the host and parasite at the molecular level.

scholarly journals Liposomal Cholesterol Delivery Activates the Macrophage Innate Immune Arm To Facilitate Intracellular Leishmania donovani Killing

2013 ◽  
Vol 82 (2) ◽  
pp. 607-617 ◽  
Author(s):  
June Ghosh ◽  
Rajan Guha ◽  
Shantanabha Das ◽  
Syamal Roy
Keyword(s):  
Antigen Presentation ◽  
Leishmania Donovani ◽  
Protozoan Parasite ◽  
Innate Immune ◽  
Dependent Manner ◽  
Necrosis Factor Alpha ◽  
Content Type ◽  
Mitogen Activated Protein ◽  
Reactive Nitrogen Intermediates ◽  
Stress Dependent

ABSTRACTLeishmania donovanicauses visceral leishmaniasis (VL) by infecting the monocyte/macrophage lineage and residing inside specialized structures known as parasitophorous vacuoles. The protozoan parasite has adopted several means of escaping the host immune response, with one of the major methods being deactivation of host macrophages. Previous reports highlight dampened macrophage signaling, defective antigen presentation due to increased membrane fluidity, and the downregulation of several genes associated withL. donovaniinfection. We have reported previously that the defective antigen presentation in infected hamsters could be corrected by a single injection of a cholesterol-containing liposome. Here we show that cholesterol in the form of a liposomal formulation can stimulate the innate immune arm and reactivate macrophage function. Augmented levels of reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI), along with proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), corroborate intracellular parasite killing. Cholesterol incorporation kinetics is favored in infected macrophages more than in normal macrophages. Such an enhanced cholesterol uptake is associated with preferential apoptosis of infected macrophages in an endoplasmic reticulum (ER) stress-dependent manner. All these events are coupled with mitogen-activated protein (MAP) kinase activation, while inhibition of such pathways resulted in increased parasite loads. Hence, liposomal cholesterol is a potential facilitator of the macrophage effector function in favor of the host, independently of the T-cell arm.

scholarly journals Ascorbate Peroxidase, a Key Molecule Regulating Amphotericin B Resistance in Clinical Isolates of Leishmania donovani

2014 ◽  
Vol 58 (10) ◽  
pp. 6172-6184 ◽  
Author(s):  
Ashish Kumar ◽  
Sushmita Das ◽  
Bidyut Purkait ◽  
Abul Hasan Sardar ◽  
Ayan Kumar Ghosh ◽  
...  
Keyword(s):  
Cell Death ◽  
Amphotericin B ◽  
Ascorbate Peroxidase ◽  
Resistant Strain ◽  
Leishmania Donovani ◽  
Cytosolic Calcium ◽  
Resistant Isolate ◽  
Secondary Resistance ◽  
Content Type ◽  
Nuclear Alterations

ABSTRACTAmphotericin B (AmB), a polyene macrolide, is now a first-line treatment of visceral leishmaniasis cases refractory to antimonials in India. AmB relapse cases and the emergence of secondary resistance have now been reported. To understand the mechanism of AmB, differentially expressed genes in AmB resistance strains were identified by a DNA microarray and real-time reverse transcriptase PCR (RT-PCR) approach. Of the many genes functionally overexpressed in the presence of AmB, the ascorbate peroxidase gene from a resistantLeishmania donovanistrain (LdAPx gene) was selected because the gene is present only inLeishmania, not in humans. Apoptosis-like cell death after exposure to AmB was investigated in a wild-type (WT) strain in which the LdAPx gene was overexpressed and in AmB-sensitive and -resistant strains. A higher percentage of apoptosis-like cell death after AmB treatment was noticed in the sensitive strain than in both the resistant isolate and the strain sensitive to LdAPx overexpression. This event is preceded by AmB-induced formation of reactive oxygen species and elevation of the cytosolic calcium level. Enhanced cytosolic calcium was found to be responsible for depolarization of the mitochondrial membrane potential and the release of cytochromec(Cytc) into the cytosol. The redox behavior of Cytcshowed that it has a role in the regulation of apoptosis-like cell death by activating metacaspase- and caspase-like proteins and causing concomitant nuclear alterations, as determined by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and DNA fragmentation in the resistant strain. The present study suggests that constitutive overexpression of LdAPx in theL. donovaniAmB-resistant strain prevents cells from the deleterious effect of oxidative stress, i.e., mitochondrial dysfunction and cellular death induced by AmB.

scholarly journals The Aspergillus nidulansnucAEndoGHomologue Is Not Involved in Cell Death

2010 ◽  
Vol 10 (2) ◽  
pp. 276-283 ◽  
Author(s):  
Bárbara de Castro Pimentel Figueiredo ◽  
Patrícia Alves de Castro ◽  
Taísa Magnani Dinamarco ◽  
Maria Helena S. Goldman ◽  
Gustavo Henrique Goldman
Keyword(s):  
Cell Death ◽  
Aspergillus Nidulans ◽  
Dna Fragmentation ◽  
Filamentous Fungi ◽  
Large Scale ◽  
Apoptosis Induction ◽  
Content Type ◽  
Endonuclease G ◽  
First Time

ABSTRACTUpon apoptosis induction, translocation of mammalian mitochondrial endonuclease G (EndoG) to the nucleus coincides with large-scale DNA fragmentation. Here, we describe for the first time a homologue of EndoG in filamentous fungi by investigating if theAspergillus nidulanshomologue of the EndoG gene, namednucAEndoG, is being activated during farnesol-induced cell death. Our results suggest that NucA is not involved in cell death, but it plays a role in the DNA-damaging response inA. nidulans.

scholarly journals SrrAB Modulates Staphylococcus aureus Cell Death through Regulation ofcidABCTranscription

2016 ◽  
Vol 198 (7) ◽  
pp. 1114-1122 ◽  
Author(s):  
Ian H. Windham ◽  
Sujata S. Chaudhari ◽  
Jeffrey L. Bose ◽  
Vinai C. Thomas ◽  
Kenneth W. Bayles
Keyword(s):  
Reactive Oxygen Species ◽  
Staphylococcus Aureus ◽  
Cell Death ◽  
Reactive Oxygen ◽  
Extracellular Dna ◽  
Death Process ◽  
Oxygen Species ◽  
Content Type ◽  
Biofilm Development

ABSTRACTThe death and lysis of a subpopulation inStaphylococcus aureusbiofilm cells are thought to benefit the surviving population by releasing extracellular DNA, a critical component of the biofilm extracellular matrix. Although the means by whichS. aureuscontrols cell death and lysis is not understood, studies implicate the role of thecidABCandlrgABoperons in this process. Recently, disruption of thesrrABregulatory locus was found to cause increased cell death during biofilm development, likely as a result of the sensitivity of this mutant to hypoxic growth. In the current study, we extended these findings by demonstrating that cell death in the ΔsrrABmutant is dependent on expression of thecidABCoperon. The effect ofcidABCexpression resulted in the generation of increased reactive oxygen species (ROS) accumulation and was independent of acetate production. Interestingly, consistently with previous studies,cidC-encoded pyruvate oxidase was found to be important for the generation of acetic acid, which initiates the cell death process. However, these studies also revealed for the first time an important role of thecidBgene in cell death, as disruption ofcidBin the ΔsrrABmutant background decreased ROS generation and cell death in acidC-independent manner. ThecidBmutation also caused decreased sensitivity to hydrogen peroxide, which suggests a complex role for this system in ROS metabolism. Overall, the results of this study provide further insight into the function of thecidABCoperon in cell death and reveal its contribution to the oxidative stress response.IMPORTANCEThe manuscript focuses on cell death mechanisms inStaphylococcus aureusand provides important new insights into the genes involved in this ill-defined process. By exploring the cause of increased stationary-phase death in anS. aureusΔsrrABregulatory mutant, we found that the decreased viability of this mutant was a consequence of the overexpression of thecidABCoperon, previously shown to be a key mediator of cell death. These investigations highlight the role of thecidBgene in the death process and the accumulation of reactive oxygen species. Overall, the results of this study are the first to demonstrate a positive role for CidB in cell death and to provide an important paradigm for understanding this process in all bacteria.

scholarly journals Apoptotic Marker Expression in the Absence of Cell Death in Staurosporine-Treated Leishmania donovani

2012 ◽  
Vol 57 (3) ◽  
pp. 1252-1261 ◽  
Author(s):  
Aude L. Foucher ◽  
Najma Rachidi ◽  
Sarah Gharbi ◽  
Thierry Blisnick ◽  
Philippe Bastin ◽  
...  
Keyword(s):  
Cell Death ◽  
Protein Kinases ◽  
Leishmania Donovani ◽  
Environmental Changes ◽  
Specific Inhibitor ◽  
Surface Binding ◽  
Casein Kinase 1 ◽  
Content Type ◽  
Apoptotic Marker ◽  
Marker Expression

ABSTRACTThe protozoan parasiteLeishmania donovaniundergoes several developmental transitions in its insect and vertebrate hosts that are induced by environmental changes. The roles of protein kinases in these adaptive differentiation steps and their potential as targets for antiparasitic intervention are only poorly characterized. Here, we used the generic protein kinase inhibitor staurosporine to gain insight into how interference with phosphotransferase activities affects the viability, growth, and motility ofL. donovanipromastigotesin vitro. Unlike the nonkinase drugs miltefosine and amphotericin B, staurosporine strongly reduced parasite biosynthetic activity and had a cytostatic rather than a cytotoxic effect. Despite the induction of a number of classical apoptotic markers, including caspase-like activity and surface binding of annexin V, we determined that, on the basis of cellular integrity, staurosporine did not cause cell death but caused cell cycle arrest and abrogated parasite motility. In contrast, targeted inhibition of the parasite casein kinase 1 (CK1) protein family by use of the CK1-specific inhibitor D4476 resulted in cell death. Thus, pleiotropic inhibition ofL. donovaniprotein kinases and possibly other ATP-binding proteins by staurosporine dissociates apoptotic marker expression from cell death, which underscores the relevance of specific rather than broad kinase inhibitors for antiparasitic drug development.

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