Reduction in the mitochondrial membrane potential of Toxoplasma gondii after invasion of host cells

The membrane potential of Toxoplasma gondii, an obligatory intracellular protozoan parasite, was monitored with the cationic permeant fluorescent dye rhodamine 123 (R123). Fluorescence microscopy revealed R123 to be partitioned predominantly in a restricted part of the parasite, which consisted of twisted or branched tubules, or of granular bodies. These structures were frequently connected to each other. The dye retention by these structures was markedly reduced by treating R123-labelled parasites with the proton ionophore, carbonylcyanide m-chlorophenylhydrazone, the potassium ionophore, valinomycin and the inhibitor of electron transport, antimycin A. Thus, these structures are regarded as the parasite mitochondria. Another cationic fluorescent dye, rhodamine 6G, stained the parasite mitochondria, whereas a negatively charged fluorescent dye, fluorescein, and the neutral compounds, rhodamine 110 and rhodamine B, did not. This fact indicates that R123 monitored the parasite mitochondrial membrane potential. T. gondii-infected 3T3 cells were also stained with R123. In contrast to the mitochondria of extracellular parasites, those of intracellular parasites failed to take up the dye. The absence of fluorescence in intracellular parasites persisted until the infected host cells ruptured and liberated daughter parasites 1 day after infection. Parasites, liberated from the host cells, either spontaneously or artificially by passing the infected cells through a 27G needle, regained the ability to take up the dye. After direct microinjection of R123 into the vacuole in which the parasite grows and multiples, the dye appeared in the host-cell mitochondria but not in the parasite's mitochondria. Thus, we conclude that the mitochondrial membrane potential of T. gondii was reduced after invasion of host cells by the parasite.

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Leishmanicidal Activity of Betulin Derivatives in Leishmania amazonensis; Effect on Plasma and Mitochondrial Membrane Potential, and Macrophage Nitric Oxide and Superoxide Production

Microorganisms ◽

10.3390/microorganisms9020320 ◽

2021 ◽

Vol 9 (2) ◽

pp. 320

Author(s):

Wilmer Alcazar ◽

Sami Alakurtti ◽

Maritza Padrón-Nieves ◽

Maija Liisa Tuononen ◽

Noris Rodríguez ◽

...

Keyword(s):

Nitric Oxide ◽

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Leishmania Amazonensis ◽

Superoxide Production ◽

In Vitro Susceptibility ◽

Intracellular Parasites ◽

Betulin Derivatives

Herein, we evaluated in vitro the anti-leishmanial activity of betulin derivatives in Venezuelan isolates of Leishmania amazonensis, isolated from patients with therapeutic failure. Methods: We analyzed promastigote in vitro susceptibility as well as the cytotoxicity and selectivity of the evaluated compounds. Additionally, the activity of selected compounds was determined in intracellular amastigotes. Finally, to gain hints on their potential mechanism of action, the effect of the most promising compounds on plasma and mitochondrial membrane potential, and nitric oxide and superoxide production by infected macrophages was determined. Results: From the tested 28 compounds, those numbered 18 and 22 were chosen for additional studies. Both 18 and 22 were active (GI50 ≤ 2 µM, cytotoxic CC50 > 45 µM, SI > 20) for the reference strain LTB0016 and for patient isolates. The results suggest that 18 significantly depolarized the plasma membrane potential (p < 0.05) and the mitochondrial membrane potential (p < 0.05) when compared to untreated cells. Although neither 18 nor 22 induced nitric oxide production in infected macrophages, 18 induced superoxide production in infected macrophages. Conclusion: Our results suggest that due to their efficacy and selectivity against intracellular parasites and the potential mechanisms underlying their leishmanicidal effect, the compounds 18 and 22 could be used as tools for designing new chemotherapies against leishmaniasis.

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The U95 Protein of Human Herpesvirus 6B Interacts with Human GRIM-19: Silencing of U95 Expression Reduces Viral Load and Abrogates Loss of Mitochondrial Membrane Potential

Journal of Virology ◽

10.1128/jvi.01156-07 ◽

2007 ◽

Vol 82 (2) ◽

pp. 1011-1020 ◽

Cited By ~ 23

Author(s):

W. M. Yeo ◽

Yuji Isegawa ◽

Vincent T. K. Chow

Keyword(s):

Cell Death ◽

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Human Herpesvirus ◽

Ultrastructural Changes ◽

Gene Encoding ◽

Interacting Protein ◽

Functional Aspects ◽

Infected Cells

ABSTRACT To better understand the pathogenesis of human herpesvirus 6 (HHV-6), it is important to elucidate the functional aspects of immediate-early (IE) genes at the initial phase of the infection. To study the functional role of the HHV-6B IE gene encoding U95, we generated a U95-Myc fusion protein and screened a pretransformed bone marrow cDNA library for U95-interacting proteins, using yeast-two hybrid analysis. The most frequently appearing U95-interacting protein identified was GRIM-19, which belongs to the family of genes associated with retinoid-interferon mortality and serves as an essential component of the oxidative phosphorylation system. This interaction was verified by both coimmunoprecipitation and confocal microscopic coimmunolocalization. Short-term HHV-6B infection of MT-4 T-lymphocytic cells induced syncytial formation, resulted in decreased mitochondrial membrane potential, and led to progressively pronounced ultrastructural changes, such as mitochondrial swelling, myelin-like figures, and a loss of cristae. Compared to controls, RNA interference against U95 effectively reduced the U95 mRNA copy number and abrogated the loss of mitochondrial membrane potential. Our results indicate that the high affinity between U95 early viral protein and GRIM-19 may be closely linked to the detrimental effect of HHV-6B infection on mitochondria. These findings may explain the alternative cell death mechanism of expiration, as opposed to apoptosis, observed in certain productively HHV-6B-infected cells. The interaction between U95 and GRIM-19 is thus functionally and metabolically significant in HHV-6B-infected cells and may be a means through which HHV-6B modulates cell death signals by interferon and retinoic acid.

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The Interferon Stimulator Mitochondrial Antiviral Signaling Protein Facilitates Cell Death by Disrupting the Mitochondrial Membrane Potential and by Activating Caspases

Journal of Virology ◽

10.1128/jvi.02174-09 ◽

2009 ◽

Vol 84 (5) ◽

pp. 2421-2431 ◽

Cited By ~ 41

Author(s):

Chia-Yi Yu ◽

Ruei-Lin Chiang ◽

Tsung-Hsien Chang ◽

Ching-Len Liao ◽

Yi-Ling Lin

Keyword(s):

Cell Death ◽

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Caspase Cleavage ◽

Infected Cells ◽

Mitochondrial Antiviral Signaling ◽

Antiviral Innate Immunity ◽

Viral Components

ABSTRACT Interferon (IFN) signaling is initiated by the recognition of viral components by host pattern recognition receptors. Dengue virus (DEN) triggers IFN-β induction through a molecular mechanism involving the cellular RIG-I/MAVS signaling pathway. Here we report that the MAVS protein level is reduced in DEN-infected cells and that caspase-1 and caspase-3 cleave MAVS at residue D429. In addition to its well-known function in IFN induction, MAVS is also a proapoptotic molecule that triggers disruption of the mitochondrial membrane potential and activation of caspases. Although different domains are required for the induction of cytotoxicity and IFN, caspase cleavage at residue 429 abolished both functions of MAVS. The apoptotic role of MAVS in viral infection and double-stranded RNA (dsRNA) stimulation was demonstrated in cells with reduced endogenous MAVS expression induced by RNA interference. Even though IFN-β promoter activation was largely suppressed, DEN production was not affected greatly in MAVS knockdown cells. Instead, DEN- and dsRNA-induced cell death and caspase activation were delayed and attenuated in the cells with reduced levels of MAVS. These results reveal a new role of MAVS in the regulation of cell death beyond its well-known function of IFN induction in antiviral innate immunity.

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Intracellular distribution of the fluorescent dye nonyl acridine orange responds to the mitochondrial membrane potential: implications for assays of cardiolipin and mitochondrial mass

Journal of Neurochemistry ◽

10.1046/j.1471-4159.2002.00945.x ◽

2002 ◽

Vol 82 (2) ◽

pp. 224-233 ◽

Cited By ~ 79

Author(s):

Jake Jacobson ◽

Michael R. Duchen ◽

Simon J. R. Heales

Keyword(s):

Membrane Potential ◽

Acridine Orange ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Intracellular Distribution ◽

Fluorescent Dye ◽

Mitochondrial Mass

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Vaccinia virus induces apoptosis of infected macrophages

Journal of General Virology ◽

10.1099/0022-1317-83-11-2821 ◽

2002 ◽

Vol 83 (11) ◽

pp. 2821-2832 ◽

Cited By ~ 37

Author(s):

Zuzana Humlová ◽

Martin Vokurka ◽

Mariano Esteban ◽

Zora Mělková

Keyword(s):

Gene Expression ◽

Nitric Oxide ◽

Membrane Potential ◽

Vaccinia Virus ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

Host Cells ◽

Early Gene ◽

Early Gene Expression ◽

Induction Of Apoptosis

Vaccinia virus (VV) infects a broad range of host cells, and while it usually causes their lysis (i.e. necrosis), the nature of the cell-death phenomenon is not well understood. In this study, we show that VV induces apoptosis of cells of the murine macrophage line J774.G8, as revealed by morphological signs, DNA ladder formation, changes of mitochondrial membrane potential and annexin-V positivity. Apoptosis occurred in both untreated and IFN-γ-pretreated macrophages, and could not be inhibited by aminoguanidine, a relatively specific inhibitor of inducible nitric oxide synthase. Inhibition of VV DNA synthesis and late gene expression by cytosine arabinoside also did not prevent apoptosis, while heat- or psoralen/UV-inactivated VV did not cause any apoptosis. Thus, VV early gene expression seems to be required for induction of apoptosis. At the cellular level, infection with VV induced a decrease in the levels of Bcl-xL, an anti-apoptotic member of the Bcl-2 family. The importance of loss of Bcl-xL was demonstrated by prevention of VV-mediated apoptosis on expression of Bcl-2, a functional homologue of Bcl-xL. Our findings provide evidence that induction of apoptosis by VV in macrophages requires virus early gene expression, does not involve nitric oxide, induces a decrease in mitochondrial membrane potential and is associated with altered levels of Bcl-xL.

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Benzodiazepine-423, an Inhibitor of Mitochondrial Respiration, Causes Selective Apoptosis of Activated Lymphocytes and Reverses Experimental GVHD While Preserving GVL Effects.

Blood ◽

10.1182/blood.v110.11.68.68 ◽

2007 ◽

Vol 110 (11) ◽

pp. 68-68

Author(s):

Erin Gatza ◽

Shawn G. Clouthier ◽

Pavan Reddy ◽

Chen Liu ◽

Anthony W. Opipari ◽

...

Keyword(s):

T Cells ◽

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Respiration ◽

Mitochondrial Membrane ◽

Ex Vivo ◽

Host Cells ◽

Activated Lymphocytes ◽

Damage Indices ◽

Ifn Γ

Abstract Benzodiazepine (Bz)-423 is a benzodiazepine derivative that targets activated lymphocytes through the mitochondrial F1F0-ATPase, causing loss of mitochondrial membrane potential and apoptosis without affecting resting lymphocytes. We tested Bz-423 in a non-irradiated B6-Ly5.2 → B6D2F1 model of graft-versus-host disease (GVHD) where donor cells were labeled with CFSE to discriminate 3 days after injection between activated (CFSElo) and unactivated (CFSEhi) Ly5.2+ donor or host Ly5.1+ lymphocytes. Compared to controls, Bz-423 caused loss of mitochondrial membrane polarization within 6h of delivery as measured by 3,3’-dihexyloxacarbocyanine iodide (DiOC6(3)) staining in both activated donor CD4+ (12.6% vs 3.3%, p=0.002) and activated CD8+ (12.9% vs 3.0%, p<0.001) T cells but not in unactivated donor or host cells. Loss of mitochondrial membrane potential was followed by selective apoptosis (Annexin-V+) of donor CD4+ (34.9% vs 16.5%, p=0.04) and CD8+ (29.8% vs 12.2%, p=0.03) T cells. Intraperitoneal injection of 60mg/kg Bz-423 3 times weekly beginning 7d after GVHD induction significantly reduced mortality (50% vs 100%, p<0.02). We next used Bz-423 in a miHA-disparate, CD8+ T cell-mediated model of GVHD (C3H.SW → B6) in which B6 hosts received 9 Gy of TBI followed by injection of 5x106 C3H.SW BM cells and 4x106 T cells. We initiated Bz-423 injections 7d after BMT, when GVHD was already established. The drug significantly reduced GVHD clinical scores and improved survival compared to controls (74% vs 29%, p≤0.02). Bz-423 also significantly reduced quantitative GVHD histologic damage indices in the liver (3.6 vs 11.2, p<0.03) and the GI tract (7.0 vs 15.8, p<0.02). Complete donor engraftment was observed in all animals. Bz-423 reduced IFN-γ, a known mediator of GVHD, in the serum (8.4 vs 21.7 pg/ml, p<0.03) and decreased IFN-γ+CD8+ effector spleen T cells (0.64x105 vs 2.2x105, p=0.008), but did not impair the lysis of tumor targets by CD8+ T cells ex vivo. We tested Bz-423 next in a graft-versus-leukemia (GVL) model where EL-4 lymphoma cells (4x103) that are syngeneic to B6 recipients were injected on the day of BMT. No recipients of syngeneic BMT survived EL-4 challenge (0/12) and no untreated allogeneic BMT survived GVHD (0/12) but 9/14 (64%) of Bz-423 treated allogeneic recipients were alive on day 50 without evidence of lymphoma (p=0.003, Fig 1a). We confirmed the effectiveness of Bz-423 in a third model of GVHD to MHC differences (Balb/c → B6) where again Bz-423 significantly reduced all clinical, biochemical and histologic GVHD parameters and improved day 60 survival (58% vs 8%, p<0.002). Bz-423 also preserved GVL effects in this model where 50% of recipients survived without evidence of EL-4 lymphoma compared to 0% of controls (p<0.04, Fig 1b). We conclude that Bz-423, a first-in-class compound that selectively inhibits mitochondrial respiration and causes apoptosis of activated lymphocytes, can reverse experimental GVHD while preserving beneficial GVL effects. Figure Figure

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Uptake of Nitrobenzylthioinosine and Purine β-l-Nucleosides by Intracellular Toxoplasma gondii

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.10.3247-3251.2003 ◽

2003 ◽

Vol 47 (10) ◽

pp. 3247-3251 ◽

Cited By ~ 23

Author(s):

Omar N. Al Safarjalani ◽

Fardos N. M. Naguib ◽

Mahmoud H. el Kouni

Keyword(s):

Toxoplasma Gondii ◽

Mammalian Cells ◽

Nucleoside Transporters ◽

Host Cells ◽

Purine Nucleoside ◽

Nucleoside Transport ◽

Fibroblast Cells ◽

Nucleoside Transporter ◽

Intracellular Parasites ◽

Infected Cells

ABSTRACT Intracellular Toxoplasma gondii grown in human foreskin fibroblast cells transported nitrobenzylthioinosine {NBMPR; 6-[(4-nitrobenzyl)mercapto]-9-β-d-ribofuranosylpurine}, an inhibitor of nucleoside transport in mammalian cells, as well as the nonphysiological β-l-enantiomers of purine nucleosides, β-l-adenosine, β-l-deoxyadenosine, and β-l-guanosine. The β-l-pyrimidine nucleosides, β-l-uridine, β-l-cytidine, and β-l-thymidine, were not transported. The uptake of NBMPR and the nonphysiological purine nucleoside β-l-enantiomers by the intracellular parasites also implies that Toxoplasma-infected cells can transport these nucleosides. In sharp contrast, under the same conditions, uninfected fibroblast cells did not transport NBMPR or any of the unnatural β-l-nucleosides. β-d-Adenosine and dipyridamole, another inhibitor of nucleoside transport, inhibited the uptake of NBMPR and β-l-stereoisomers of the purine nucleosides by intracellular Toxoplasma and Toxoplasma-infected cells. Furthermore, infection with a Toxoplasma mutant deficient in parasite adenosine/purine nucleoside transport reduced or abolished the uptake of β-d-adenosine, NBMPR, and purine β-l-nucleosides. Hence, the presence of the Toxoplasma adenosine/purine nucleoside transporters is apparently essential for the uptake of NBMPR and purine β-l-nucleosides by intracellular Toxoplasma and Toxoplasma-infected cells. These results also demonstrate that, in contrast to the mammalian nucleoside transporters, the Toxoplasma adenosine/purine nucleoside transporter(s) lacks stereospecificity and substrate specificity in the transport of purine nucleosides. In addition, infection with T. gondii confers the properties of the parasite's purine nucleoside transport on the parasitized host cells and enables the infected cells to transport purine nucleosides that were not transported by uninfected cells. These unique characteristics of purine nucleoside transport in T. gondii may aid in the identification of new promising antitoxoplasmic drugs.

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Reverting the mode of action of the mitochondrial FOF1-ATPase by Legionella pneumophila preserves its replication niche

10.1101/2021.05.12.443790 ◽

2021 ◽

Author(s):

Pedro Escoll ◽

Lucien Platon ◽

Mariatou Drame ◽

Tobias Sahr ◽

Silke Schmidt ◽

...

Keyword(s):

Cell Death ◽

Membrane Potential ◽

Host Cell ◽

Mitochondrial Membrane Potential ◽

Legionella Pneumophila ◽

Mitochondrial Membrane ◽

Dependent Manner ◽

Infected Cells ◽

Bacterial Replication ◽

Fof1 Atpase

Legionella pneumophila, the causative agent of Legionnaires disease, a severe pneumonia, injects via a type-IV-secretion-system (T4SS) more than 300 proteins into macrophages, its main host cell in humans. Certain of these proteins are implicated in reprogramming the metabolism of infected cells by reducing mitochondrial oxidative phosphorylation (OXPHOS) early after infection. Here we show that despite reduced OXPHOS, the mitochondrial membrane potential is maintained during infection of primary human monocyte-derived macrophages (hMDMs). We reveal that L. pneumophila reverses the ATP-synthase activity of the mitochondrial FOF1-ATPase to ATP-hydrolase activity in a T4SS-dependent manner, which leads to a conservation of the mitochondrial membrane potential, preserves mitochondrial polarization and prevents macrophage cell death. Analyses of T4SS effectors known to target mitochondrial functions revealed that LpSpl is partially involved in conserving the mitochondrial membrane potential, but not LncP and MitF. The inhibition of the L. pneumophila-induced reverse mode of the FOF1-ATPase collapsed the mitochondrial membrane potential and caused cell death in infected cells. Single-cell analyses suggested that bacterial replication occurs preferentially in hMDMs that conserved the mitochondrial membrane potential and showed delayed cell death. This direct manipulation of the mode of activity of the FOF1-ATPase is a newly identified feature of L. pneumophila allowing to delay host cell death and thereby to preserve the bacterial replication niche during infection.

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