scholarly journals Trypanosoma cruziPosttranscriptionally Up-Regulates and Exploits Cellular FLIP for Inhibition of Death-inducing Signal

2005 ◽  
Vol 16 (8) ◽  
pp. 3521-3528 ◽  
Author(s):  
Muneaki Hashimoto ◽  
Junko Nakajima-Shimada ◽  
Takashi Aoki
Keyword(s):  
Death Receptor ◽  
Protozoan Parasite ◽  
Host Cells ◽  
Mouse Heart ◽  
Inhibitory Protein ◽  
Protozoan Infection ◽  
Heart Muscle Cells ◽  
Infected Cells ◽  
Interfering Rna

Intracellular persistence of the protozoan parasite, Trypanosoma cruzi, is an aggravating cause of Chagas' disease, involving that the protozoan infection specifically inhibits death receptor-mediated apoptosis of host cells. Here we demonstrate that the parasite dramatically up-regulates cellular FLICE inhibitory protein (c-FLIP), the only known mammalian inhibitor specific for death receptor signaling, in infected cells by an unusual, posttranscriptional stabilization of the short-lived protein. We also show that c-FLIP is accumulated in T. cruzi–infected mouse heart muscle cells in vivo. Stimulation of death receptor Fas in infected cells induces recruitment of c-FLIP to block the procaspase-8 activation at the most upstream caspase cascade. c-FLIP knock-down with a small interfering RNA significantly restores Fas-mediated apoptosis in infected cells. Taken together, our findings indicate that T. cruzi posttranscriptionally up-regulates and exploits host c-FLIP for the inhibition of death-inducing signal, a mechanism that may allow parasites to persist in host cells.

scholarly journals Efficient Killing of Multidrug-Resistant Intracellular Bacteria by AIEgens in Vivo

2020 ◽  
Author(s):  
Ying Li ◽  
Fei Liu ◽  
Jiangjiang Zhang ◽  
Xiaoye Liu ◽  
Peihong Xiao ◽  
...  
Keyword(s):  
Membrane Damage ◽  
Multidrug Resistant ◽  
Host Cells ◽  
Comparable Efficacy ◽  
Intracellular Bacteria ◽  
New Agents ◽  
Immune Clearance ◽  
Infected Cells ◽  
Dose Levels

<p><a>Bacteria infected cells acting as “Trojan horses” not only protect bacteria from antibiotic therapies and immune clearance, but also increase the dissemination of pathogens from the initial sites of infection. Antibiotics are hard and insufficient to treat such hidden intracellular bacteria, especially the multidrug</a>-resistant (MDR) bacteria. Herein, aggregation-induced emission luminogens (AIEgens) such as TBPs showed potent broad-spectrum bactericidal activity against both <a></a><a>extracellular and intracellular</a> Gram-positive pathogens at low-dose levels. TBPs triggered reactive oxygen species (ROS)-mediated membrane damage to kill bacteria, regardless of light irradiation. Additionally, such AIEgens activated mitochondria dependent autophagy to eliminate intracellular bacteria in host cells. Compared to the routinely used vancomycin in clinics, TBPs showed comparable efficacy against methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) <i>in vivo</i>. Our studies demonstrate that AIEgens are promising new agents for the treatment of MDR bacteria associated infections.</p>

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

1984 ◽  
Vol 70 (1) ◽  
pp. 73-81
Author(s):  
K. Tanabe ◽  
K. Murakami
Keyword(s):  
Membrane Potential ◽  
Toxoplasma Gondii ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Protozoan Parasite ◽  
Fluorescent Dye ◽  
Host Cells ◽  
Intracellular Parasites ◽  
Neutral Compounds ◽  
Infected 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.

scholarly journals The ketolide antibiotics HMR 3647 and HMR 3004 are active against Toxoplasma gondii in vitro and in murine models of infection.

1997 ◽  
Vol 41 (10) ◽  
pp. 2137-2140 ◽  
Author(s):  
F G Araujo ◽  
A A Khan ◽  
T L Slifer ◽  
A Bryskier ◽  
J S Remington
Keyword(s):  
Toxoplasma Gondii ◽  
Protozoan Parasite ◽  
Host Cells ◽  
Resistant Bacteria ◽  
New Class ◽  
Human Foreskin Fibroblasts ◽  
Significant Toxicity ◽  
Different Strains

Ketolides are a new class of macrolide antibiotics that have been shown to be active against a variety of bacteria including macrolide-resistant bacteria and mycobacteria. We examined two ketolides, HMR 3647 and HMR 3004, for their in vitro and in vivo activities against the protozoan parasite Toxoplasma gondii. In vitro, both ketolides at concentrations as low as 0.05 microg/ml markedly inhibited replication of tachyzoites of the RH strain within human foreskin fibroblasts. HMR 3004 demonstrated some toxicity for host cells after they were exposed to 5 microg of the drug per ml for 72 h. In contrast, HMR 3647 did not show any significant toxicity even at concentrations as high as 25 microg/ml. In vivo, both ketolides provided remarkable protection against death in mice lethally infected intraperitoneally with tachyzoites of the RH strain or orally with tissue cysts of the C56 strain of T. gondii. A dosage of 100 mg of HMR 3647 per kg of body weight per day administered for 10 days protected 50% of mice infected with tachyzoites. The same dosage of HMR 3004 protected 100% of the mice. In mice infected with cysts, a dosage of 30 mg of HMR 3647 per kg per day protected 100% of the mice, whereas a dosage of 40 mg of HMR 3004 per kg per day protected 75% of the mice. These results demonstrate that HMR 3647 and HMR 3004 possess excellent activities against two different strains of T. gondii and may be useful for the treatment of toxoplasmosis in humans.

scholarly journals In vivo analysis of influenza A mRNA secondary structures identifies critical regulatory motifs

2019 ◽  
Vol 47 (13) ◽  
pp. 7003-7017 ◽  
Author(s):  
Lisa Marie Simon ◽  
Edoardo Morandi ◽  
Anna Luganini ◽  
Giorgio Gribaudo ◽  
Luis Martinez-Sobrido ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Influenza A ◽  
Host Cells ◽  
Messenger Rnas ◽  
Infected Cells ◽  
In Vivo Analysis ◽  
First Time ◽  
Negative Sense

AbstractThe influenza A virus (IAV) is a continuous health threat to humans as well as animals due to its recurring epidemics and pandemics. The IAV genome is segmented and the eight negative-sense viral RNAs (vRNAs) are transcribed into positive sense complementary RNAs (cRNAs) and viral messenger RNAs (mRNAs) inside infected host cells. A role for the secondary structure of IAV mRNAs has been hypothesized and debated for many years, but knowledge on the structure mRNAs adopt in vivo is currently missing. Here we solve, for the first time, the in vivo secondary structure of IAV mRNAs in living infected cells. We demonstrate that, compared to the in vitro refolded structure, in vivo IAV mRNAs are less structured but exhibit specific locally stable elements. Moreover, we show that the targeted disruption of these high-confidence structured domains results in an extraordinary attenuation of IAV replicative capacity. Collectively, our data provide the first comprehensive map of the in vivo structural landscape of IAV mRNAs, hence providing the means for the development of new RNA-targeted antivirals.

scholarly journals Compartmentalization of the Broad-Range Phospholipase C Activity to the Spreading Vacuole Is Critical for Listeria monocytogenes Virulence

2006 ◽  
Vol 75 (1) ◽  
pp. 44-51 ◽  
Author(s):  
P. S. Marie Yeung ◽  
Yoojin Na ◽  
Amanda J. Kreuder ◽  
Hélène Marquis
Keyword(s):  
Listeria Monocytogenes ◽  
Phospholipase C ◽  
Membrane Damage ◽  
Bacterial Pathogen ◽  
Host Cells ◽  
Virulence Attenuation ◽  
Infected Cells ◽  
Host Cell Membranes ◽  
Cell Spread

ABSTRACT Listeria monocytogenes is a bacterial pathogen that multiplies in the cytosol of host cells and spreads directly from cell to cell by using an actin-based mechanism of motility. The broad-range phospholipase C (PC-PLC) of L. monocytogenes contributes to bacterial escape from vacuoles formed upon cell-to-cell spread. PC-PLC is made as an inactive proenzyme whose activation requires cleavage of an N-terminal propeptide. During infection, PC-PLC is activated specifically in acidified vacuoles. To assess the importance of compartmentalizing PC-PLC activity during infection, we created a mutant that makes constitutively active PC-PLC (the plcBΔpro mutant). Results from intracellular growth and cell-to-cell spread assays showed that the plcBΔpro mutant was sensitive to gentamicin, suggesting that unregulated PC-PLC activity causes damage to host cell membranes. This was confirmed by the observation of a twofold increase in staining of live infected cells by a non-membrane-permeant DNA fluorescent dye. However, membrane damage was not sufficient to cause cell lysis and was dependent on bacterial cell-to-cell spread, suggesting that damage was localized to bacterium-containing filopodia. Using an in vivo competitive infection assay, we observed that the plcBΔpro mutant was outcompeted up to 200-fold by the wild-type strain in BALB/c mice. Virulence attenuation was greater when mice were infected orally than when they were infected intravenously, presumably because the plcBΔpro mutant was initially outcompeted in the intestines, reducing the number of mutant bacteria reaching the liver and spleen. Together, these results emphasize the importance for L. monocytogenes virulence of compartmentalizing the activity of PC-PLC during infection.

scholarly journals Synergistic interactions between interferon-γ and TRAIL modulate c-FLIP in endothelial cells, mediating their lineage-specific sensitivity to thrombotic thrombocytopenic purpura plasma–associated apoptosis

Blood ◽  
2008 ◽  
Vol 112 (2) ◽  
pp. 340-349 ◽  
Author(s):  
Radu Stefanescu ◽  
Dustin Bassett ◽  
Rozbeh Modarresi ◽  
Francisco Santiago ◽  
Mohamad Fakruddin ◽  
...  
Keyword(s):  
Thrombotic Thrombocytopenic Purpura ◽  
Interferon Γ ◽  
Microvascular Endothelial Cell ◽  
Caspase 8 ◽  
Inhibitory Protein ◽  
Thrombocytopenic Purpura ◽  
Ifn Γ ◽  
Interfering Rna

Abstract Microvascular endothelial cell (MVEC) injury coupled to progression of platelet microthrombi facilitated by ADAMTS13 deficiency is characteristic of idiopathic and HIV-linked thrombotic thrombocytopenic purpura (TTP). Cytokines capable of inducing MVEC apoptosis in vitro are up-regulated in both TTP and HIV infection. However, the concentrations of these cytokines required to elicit EC apoptosis in vitro are 2- to 3-log–fold greater than present in patient plasmas. We report that clinically relevant levels of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) and interferon (IFN)–γ act in synergy to induce apoptosis in dermal MVECs, but have no effect on large-vessel ECs or pulmonary MVECs. This reflects the tissue distribution of TTP lesions in vivo. Sensitivity to TTP plasma or TRAIL plus IFN-γ is paralleled by enhanced ubiquitination of the caspase-8 regulator cellular FLICE-like inhibitory protein (c-FLIP), targeting it for proteasome degradation. c-FLIP silencing with anti-FLIP short interfering RNA (siRNA) in pulmonary MVECs rendered them susceptible to TTP plasma– and cytokine-mediated apoptosis, while up-regulation of c-FLIP by gene transfer partially protected dermal MVECs from such injury. TTP plasma–mediated apoptosis appears to involve cytokine-induced acceleration of c-FLIP degradation, sensitizing cells to TRAIL-mediated caspase-8 activation and cell death. Suppression of TRAIL or modulation of immunoproteasome activity may have therapeutic relevance in TTP.

scholarly journals Influenza virus infection modulates the death receptor pathway during early stages of infection in human bronchial epithelial cells

2018 ◽  
Vol 50 (9) ◽  
pp. 770-779 ◽  
Author(s):  
Sreekumar Othumpangat ◽  
Donald H. Beezhold ◽  
John D. Noti
Keyword(s):  
Epithelial Cells ◽  
Early Stage ◽  
Death Receptor ◽  
Bronchial Epithelial Cells ◽  
Host Cells ◽  
Pcr Array ◽  
Human Bronchial Epithelial Cells ◽  
Bronchial Epithelial ◽  
Early Stages ◽  
Infected Cells

Host-viral interaction occurring throughout the infection process between the influenza A virus (IAV) and bronchial cells determines the success of infection. Our previous studies showed that the apoptotic pathway triggered by the host cells was repressed by IAV facilitating prolonged survival of infected cells. A detailed understanding on the role of IAV in altering the cell death pathway during early-stage infection of human bronchial epithelial cells (HBEpCs) is still unclear. We investigated the gene expression profiles of IAV-infected vs. mock-infected cells at the early stage of infection with a PCR array for death receptor (DR) pathway. At early stages infection (2 h) with IAV significantly upregulated DR pathway genes in HBEpCs, whereas 6 h exposure to IAV resulted in downregulation of the same genes. IAV replication in HBEpCs decreased the levels of DR pathway genes including TNF-receptor superfamily 1, Fas-associated death domain, caspase-8, and caspase-3 by 6 h, resulting in increased survival of cells. The apoptotic cell population decreased in 6 h compared with the 2 h exposure to IAV. The PCR array data were imported into Ingenuity Pathway Analysis software, resulting in confirmation of the model showing significant modulation of the DR pathway. Our data indicate that a significant transcriptional regulation of apoptotic, necrotic, and DR genes occur at early and late hours of infection that are vital in modulating the survival of host cells and replication of IAV. These data may have provided a likely roadmap for translational approaches targeting the DR pathway to enhance apoptosis and inhibit replication of the virus.

scholarly journals Deubiquitinating Function of Adenovirus Proteinase

2002 ◽  
Vol 76 (12) ◽  
pp. 6323-6331 ◽  
Author(s):  
Maxim Y. Balakirev ◽  
Michel Jaquinod ◽  
Arthur L. Haas ◽  
Jadwiga Chroboczek
Keyword(s):  
Structural Model ◽  
Proteolytic Processing ◽  
Host Cells ◽  
Cellular Proteins ◽  
Viral Gene ◽  
Cellular Processes ◽  
Infected Cells ◽  
Substrate Binding Sites

ABSTRACT The invasion strategy of many viruses involves the synthesis of viral gene products that mimic the functions of the cellular proteins and thus interfere with the key cellular processes. Here we show that adenovirus infection is accompanied by an increased ubiquitin-cleaving (deubiquitinating) activity in the host cells. Affinity chromatography on ubiquitin aldehyde (Ubal), which was designed to identify the deubiquitinating proteases, revealed the presence of adenovirus L3 23K proteinase (Avp) in the eluate from adenovirus-infected cells. This proteinase is known to be necessary for the processing of viral precursor proteins during virion maturation. We show here that in vivo Avp deubiquitinates a number of cellular proteins. Analysis of the substrate specificity of Avp in vitro demonstrated that the protein deubiquitination by this enzyme could be as efficient as proteolytic processing of viral proteins. The structural model of the Ubal-Avp interaction revealed some similarity between S1-S4 substrate binding sites of Avp and ubiquitin hydrolases. These results may reflect the acquisition of an advantageous property by adenovirus and may indicate the importance of ubiquitin pathways in viral infection.

scholarly journals In vivo delivery of caspase-8 or Fas siRNA improves the survival of septic mice

Blood ◽  
2005 ◽  
Vol 106 (7) ◽  
pp. 2295-2301 ◽  
Author(s):  
Doreen E. Wesche-Soldato ◽  
Chun-Shiang Chung ◽  
Joanne Lomas-Neira ◽  
Lesley A. Doughty ◽  
Stephen H. Gregory ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Cecal Ligation ◽  
Death Receptor ◽  
Lymphoid Cells ◽  
Caspase 8 ◽  
Gfp Fluorescence ◽  
Green Fluorescent ◽  
Interfering Rna ◽  
In Vivo Administration

Abstract Although studies have shown increased evidence of death receptor-driven apoptosis in intestinal lymphoid cells, splenocytes, and the liver following the onset of polymicrobial sepsis, little is known about the mediators controlling this process or their pathologic contribution. We therefore attempted to test the hypothesis that the hydrodynamic administration of small interfering RNA (siRNA) against the death receptor, Fas or caspase-8, should attenuate the onset of morbidity and mortality seen in sepsis, as produced by cecal ligation and puncture (CLP). We initially show that in vivo administration of green fluorescent protein (GFP) siRNA in GFP transgenic mice results in a decrease in GFP fluorescence in most tissues. Subsequently, we also found that treating septic nontransgenic mice with siRNA targeting Fas or caspase-8 but not GFP (used as a control here) decreased the mRNA, in a sustained fashion up to 10 days, and protein expression of Fas and caspase-8, respectively. In addition, transferase-mediated dUTP (deoxyuridine triphosphate) nick end labeling (TUNEL) and active caspase-3 analyses revealed a decrease in apoptosis in the liver and spleen but not the thymus following siRNA treatment. Indices of liver damage were also decreased. Finally, the injection of Fas or caspase-8 given not only 30 minutes but up to 12 hours after CLP significantly improved the survival of septic mice.

scholarly journals Regulation of Cardiac Stress Signaling by Protein Kinase D1

2006 ◽  
Vol 26 (10) ◽  
pp. 3875-3888 ◽  
Author(s):  
Brooke C. Harrison ◽  
Mi-Sung Kim ◽  
Eva van Rooij ◽  
Craig F. Plato ◽  
Philip J. Papst ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cardiac Remodeling ◽  
Rho Gtpases ◽  
Nuclear Export ◽  
Mouse Heart ◽  
Stress Signaling ◽  
Protein Kinase D1 ◽  
Downstream Effector ◽  
Interfering Rna

ABSTRACT In response to pathological stresses such as hypertension or myocardial infarction, the heart undergoes a remodeling process that is associated with myocyte hypertrophy, myocyte death, and fibrosis. Histone deacetylase 5 (HDAC5) is a transcriptional repressor of cardiac remodeling that is subject to phosphorylation-dependent neutralization in response to stress signaling. Recent studies have suggested a role for protein kinase C (PKC) and its downstream effector, protein kinase D1 (PKD1), in the control of HDAC5 phosphorylation. While PKCs are well-documented regulators of cardiac signaling, the function of PKD1 in heart muscle remains unclear. Here, we demonstrate that PKD1 catalytic activity is stimulated in cardiac myocytes by diverse hypertrophic agonists that signal through G protein-coupled receptors (GPCRs) and Rho GTPases. PKD1 activation in cardiomyocytes occurs through PKC-dependent and -independent mechanisms. In vivo, cardiac PKD1 is activated in multiple rodent models of pathological cardiac remodeling. PKD1 activation correlates with phosphorylation-dependent nuclear export of HDAC5, and reduction of endogenous PKD1 expression with small interfering RNA suppresses HDAC5 shuttling and associated cardiomyocyte growth. Conversely, ectopic overexpression of constitutively active PKD1 in mouse heart leads to dilated cardiomyopathy. These findings support a role for PKD1 in the control of pathological remodeling of the heart via its ability to phosphorylate and neutralize HDAC5.

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