scholarly journals Metabolism and Selective Toxicity of 6-Nitrobenzylthioinosine in Toxoplasma gondii

1999 ◽  
Vol 43 (10) ◽  
pp. 2437-2443 ◽  
Author(s):  
Mahmoud H. el Kouni ◽  
Vincenzo Guarcello ◽  
Omar N. Al Safarjalani ◽  
Fardos N. M. Naguib
Keyword(s):  
Toxoplasma Gondii ◽  
Human Fibroblasts ◽  
Adenosine Kinase ◽  
Host Cells ◽  
Dependent Manner ◽  
Selective Toxicity ◽  
Link Type ◽  
Infected Cells ◽  
Dose Dependent ◽  
Uninfected Host

ABSTRACT The purine nucleoside analogue NBMPR {nitrobenzylthioinosine or 6-[(4-nitrobenzyl)thio]-9-β-d-ribofuranosylpurine} was selectively phosphorylated to its nucleoside 5′-monophosphate byToxoplasma gondii but not mammalian adenosine kinase (EC2.7.1.20). NBMPR was also cleaved in toxoplasma to its nucleobase, nitrobenzylmercaptopurine. However, nitrobenzylmercaptopurine was not a substrate for either adenosine kinase or hypoxanthine-guanine-xanthine phosphoribosyltransferase (EC 2.4.2.8). Because of this unique and previously unknown metabolism of NBMPR by the parasite, the effect of NBMPR as an antitoxoplasmic agent was tested. NBMPR killed T. gondii grown in human fibroblasts in a dose-dependent manner, with a 50% inhibitory concentration of approximately 10 μM and without apparent toxicity to host cells. Doses of up to 100 μM had no significant toxic effect on uninfected host cells. The promising antitoxoplasmic effect of NBMPR led to the testing of other 6-substituted 9-β-d-ribofuranosylpurines, which were shown to be good ligands of the parasite adenosine kinase (M. H. Iltzsch, S. S. Uber, K. O. Tankersley, and M. H. el Kouni, Biochem. Pharmacol. 49:1501–1512, 1995), as antitoxoplasmic agents. Among the analogues tested, 6-benzylthioinosine,p-nitrobenzyl-6-selenopurine riboside,N 6-(p-azidobenzyl)adenosine, andN 6-(p-nitrobenzyl)adenosine, like NBMPR, were selectively toxic to parasite-infected cells. Thus, it appears that the unique characteristics of purine metabolism inT. gondii render certain 6-substituted 9-β-d-ribofuranosylpurines promising antitoxoplasmic drugs.

scholarly journals 1,3,4-Thiadiazoles Effectively Inhibit Proliferation of Toxoplasma gondii

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1053
Author(s):  
Lidia Węglińska ◽  
Adrian Bekier ◽  
Katarzyna Dzitko ◽  
Barbara Pacholczyk-Sienicka ◽  
Łukasz Albrecht ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Cell Invasion ◽  
Direct Action ◽  
Human Fibroblasts ◽  
Imidazole Ring ◽  
Host Cells ◽  
In Vitro Assays ◽  
Host Cell Invasion

Congenital and acquired toxoplasmosis caused by the food- and water-born parasite Toxoplasma gondii (T. gondii) is one of the most prevalent zoonotic infection of global importance. T. gondii is an obligate intracellular parasite with limited capacity for extracellular survival, thus a successful, efficient and robust host cell invasion process is crucial for its survival, proliferation and transmission. In this study, we screened a series of novel 1,3,4-thiadiazole-2-halophenylamines functionalized at the C5 position with the imidazole ring (1b–12b) for their effects on T. gondii host cell invasion and proliferation. To achieve this goal, these compounds were initially subjected to in vitro assays to assess their cytotoxicity on human fibroblasts and then antiparasitic efficacy. Results showed that all of them compare favorably to control drugs sulfadiazine and trimethoprim in terms of T. gondii growth inhibition (IC50) and selectivity toward the parasite, expressed as selectivity index (SI). Subsequently, the most potent of them with meta-fluoro 2b, meta-chloro 5b, meta-bromo 8b, meta-iodo 11b and para-iodo 12b substitution were tested for their efficacy in inhibition of tachyzoites invasion and subsequent proliferation by direct action on established intracellular infection. All the compounds significantly inhibited the parasite invasion and intracellular proliferation via direct action on both tachyzoites and parasitophorous vacuoles formation. The most effective was para-iodo derivative 12b that caused reduction in the percentage of infected host cells by 44% and number of tachyzoites per vacuole by 93% compared to non-treated host cells. Collectively, these studies indicate that 1,3,4-thiadiazoles 1b–12b, especially 12b with IC50 of 4.70 µg/mL and SI of 20.89, could be considered as early hit compounds for future design and synthesis of anti-Toxoplasma agents that effectively and selectively block the invasion and subsequent proliferation of T. gondii into host cells.

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.

Membrane Nanoparticles Derived from ACE2-rich Cells Block SARS-CoV-2 Infection

2020 ◽  
Author(s):  
Cheng Wang ◽  
Shaobo Wang ◽  
Yin Chen ◽  
Jianqi Zhao ◽  
Songling Han ◽  
...  
Keyword(s):  
Viral Entry ◽  
Inhibitory Concentration ◽  
Cell Metabolism ◽  
Host Cells ◽  
Dependent Manner ◽  
Human Embryonic Kidney ◽  
Proximal Tubular Cells ◽  
Tubular Cells ◽  
Proximal Tubular ◽  
Dose Dependent

ABSTRACTThe ongoing COVID-19 epidemic worldwide necessitates the development of novel effective agents against SARS-CoV-2. ACE2 is the main receptor of SARS-CoV-2 S1 protein and mediates viral entry into host cells. Herein, the membrane nanoparticles prepared from ACE2-rich cells are discovered with potent capacity to block SARS-CoV-2 infection. The membrane of human embryonic kidney-239T cell highly expressing ACE2 is screened to prepare nanoparticles. The nanomaterial termed HEK-293T-hACE2 NPs contains 265.1 ng mg−1 of ACE2 on the surface and acts as a bait to trap SARS-CoV-2 S1 in a dose-dependent manner, resulting in reduced recruitment of the viral ligand to host cells. Interestingly, SARS-CoV-2 S1 can translocate to the cytoplasm and affect the cell metabolism, which is also inhibited by HEK-293T-hACE2 NPs. Further studies reveal that HEK-293T-hACE2 NPs can efficiently suppress SARS-CoV-2 S pseudovirions entry into human proximal tubular cells and block viral infection with a low half maximal inhibitory concentration. Additionally, this biocompatible membrane nanomaterial is sufficient to block the adherence of SARS-CoV-2 D614G-S1 mutant to sensitive cells. Our study demonstrates a easy-to-acheive memrbane nano-antagonist for curbing SARS-CoV-2, which enriches the existing antiviral arsenal and provides new possibilities to treat COVID-19. Graphical Table of Contents

scholarly journals DNA double-strand breaks in the Toxoplasma gondii-infected cells by the action of reactive oxygen species

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Haohan Zhuang ◽  
Chaoqun Yao ◽  
Xianfeng Zhao ◽  
Xueqiu Chen ◽  
Yimin Yang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Toxoplasma Gondii ◽  
Double Strand Breaks ◽  
Host Cells ◽  
Oxygen Species ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response ◽  
Infected Cells

Abstract Background Toxoplasma gondii is an obligate parasite of all warm-blooded animals around the globe. Once infecting a cell, it manipulates the host’s DNA damage response that is yet to be elucidated. The objectives of the present study were three-fold: (i) to assess DNA damages in T. gondii-infected cells in vitro; (ii) to ascertain causes of DNA damage in T. gondii-infected cells; and (iii) to investigate activation of DNA damage responses during T. gondii infection. Methods HeLa, Vero and HEK293 cells were infected with T. gondii at a multiplicity of infection (MOI) of 10:1. Infected cells were analyzed for a biomarker of DNA double-strand breaks (DSBs) γH2AX at 10 h, 20 h or 30 h post-infection using both western blot and immunofluorescence assay. Reactive oxygen species (ROS) levels were measured using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), and ROS-induced DNA damage was inhibited by a ROS inhibitor N-acetylcysteine (NAC). Lastly, DNA damage responses were evaluated by detecting the active form of ataxia telangiectasia mutated/checkpoint kinase 2 (ATM/CHK2) by western blot. Results γH2AX levels in the infected HeLa cells were significantly increased over time during T. gondii infection compared to uninfected cells. NAC treatment greatly reduced ROS and concomitantly diminished γH2AX in host cells. The phosphorylated ATM/CHK2 were elevated in T. gondii-infected cells. Conclusions Toxoplasma gondii infection triggered DNA DSBs with ROS as a major player in host cells in vitro. It also activated DNA damage response pathway ATM/CHK2. Toxoplasma gondii manages to keep a balance between survival and apoptosis of its host cells for the benefit of its own survival.

scholarly journals Coimmunoprecipitation with MYR1 Identifies Three Additional Proteins within the Toxoplasma gondii Parasitophorous Vacuole Required for Translocation of Dense Granule Effectors into Host Cells

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Alicja M. Cygan ◽  
Terence C. Theisen ◽  
Alma G. Mendoza ◽  
Nicole D. Marino ◽  
Michael W. Panas ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Protein Translocation ◽  
Affinity Purification ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Effector Proteins ◽  
Host Cells ◽  
Content Type ◽  
Cyclin E1 ◽  
Infected Cells

ABSTRACT Toxoplasma gondii is a ubiquitous, intracellular protozoan that extensively modifies infected host cells through secreted effector proteins. Many such effectors must be translocated across the parasitophorous vacuole (PV), in which the parasites replicate, ultimately ending up in the host cytosol or nucleus. This translocation has previously been shown to be dependent on five parasite proteins: MYR1, MYR2, MYR3, ROP17, and ASP5. We report here the identification of several MYR1-interacting and novel PV-localized proteins via affinity purification of MYR1, including TGGT1_211460 (dubbed MYR4), TGGT1_204340 (dubbed GRA54), and TGGT1_270320 (PPM3C). Further, we show that three of the MYR1-interacting proteins, GRA44, GRA45, and MYR4, are essential for the translocation of the Toxoplasma effector protein GRA16 and for the upregulation of human c-Myc and cyclin E1 in infected cells. GRA44 and GRA45 contain ASP5 processing motifs, but like MYR1, processing at these sites appears to be nonessential for their role in protein translocation. These results expand our understanding of the mechanism of effector translocation in Toxoplasma and indicate that the process is highly complex and dependent on at least eight discrete proteins. IMPORTANCE Toxoplasma is an extremely successful intracellular parasite and important human pathogen. Upon infection of a new cell, Toxoplasma establishes a replicative vacuole and translocates parasite effectors across this vacuole to function from the host cytosol and nucleus. These effectors play a key role in parasite virulence. The work reported here newly identifies three parasite proteins that are necessary for protein translocation into the host cell. These results significantly increase our knowledge of the molecular players involved in protein translocation in Toxoplasma-infected cells and provide additional potential drug targets.

scholarly journals Adenoviruses Use Lactoferrin as a Bridge for CAR-Independent Binding to and Infection of Epithelial Cells

2006 ◽  
Vol 81 (2) ◽  
pp. 954-963 ◽  
Author(s):  
Cecilia Johansson ◽  
Mari Jonsson ◽  
Marko Marttila ◽  
David Persson ◽  
Xiao-Long Fan ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Innate Immune System ◽  
Innate Immune ◽  
Host Cells ◽  
Tear Fluid ◽  
Adenovirus Infection ◽  
Dependent Manner ◽  
Polarized Cells ◽  
Adenovirus Receptor ◽  
Dose Dependent

ABSTRACT Most adenoviruses bind to the coxsackie- and adenovirus receptor (CAR). Surprisingly, CAR is not expressed apically on polarized cells and is thus not easily available to viruses. Consequently, alternative mechanisms for entry of coxsackievirus and adenovirus into cells have been suggested. We have found that tear fluid promotes adenovirus infection, and we have identified human lactoferrin (HLf) as the tear fluid component responsible for this effect. HLf alone was found to promote binding of adenovirus to epithelial cells in a dose-dependent manner and also infection of epithelial cells by adenovirus. HLf was also found to promote gene delivery from an adenovirus-based vector. The mechanism takes place at the binding stage and functions independently of CAR. Thus, we have identified a novel binding mechanism whereby adenovirus hijacks HLf, a component of the innate immune system, and uses it as a bridge for attachment to host cells.

scholarly journals Host ER–parasitophorous vacuole interaction provides a route of entry for antigen cross-presentation in Toxoplasma gondii–infected dendritic cells

2009 ◽  
Vol 206 (2) ◽  
pp. 399-410 ◽  
Author(s):  
Romina S. Goldszmid ◽  
Isabelle Coppens ◽  
Avital Lev ◽  
Pat Caspar ◽  
Ira Mellman ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Toxoplasma Gondii ◽  
Mhc Class I ◽  
Cd8 T Cells ◽  
Parasitophorous Vacuole ◽  
Host Cells ◽  
Class I ◽  
Cross Presentation ◽  
Infected Cells

Toxoplasma gondii tachyzoites infect host cells by an active invasion process leading to the formation of a specialized compartment, the parasitophorous vacuole (PV). PVs resist fusion with host cell endosomes and lysosomes and are thus distinct from phagosomes. Because the parasite remains sequestered within the PV, it is unclear how T. gondii–derived antigens (Ag’s) access the major histocompatibility complex (MHC) class I pathway for presentation to CD8+ T cells. We demonstrate that recruitment of host endoplasmic reticulum (hER) to the PV in T. gondii–infected dendritic cells (DCs) directly correlates with cross-priming of CD8+ T cells. Furthermore, we document by immunoelectron microscopy the transfer of hER components into the PV, a process indicative of direct fusion between the two compartments. In strong contrast, no association between hER and phagosomes or Ag presentation activity was observed in DCs containing phagocytosed live or dead parasites. Importantly, cross-presentation of parasite-derived Ag in actively infected cells was blocked when hER retrotranslocation was inhibited, indicating that the hER serves as a conduit for the transport of Ag between the PV and host cytosol. Collectively, these findings demonstrate that pathogen-driven hER–PV interaction can serve as an important mechanism for Ag entry into the MHC class I pathway and CD8+ T cell cross-priming.

scholarly journals Identification of Diphenoxylate as an Antiviral Agent Against Severe Acute Respiratory Syndrome Coronavirus 2

2020 ◽  
Author(s):  
Jin Soo Shin ◽  
Eunhye Jung ◽  
Yejin Jang ◽  
Soo Bong Han ◽  
Meehyein Kim
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Antiviral Treatment ◽  
Antiviral Agent ◽  
Selectivity Index ◽  
Dependent Manner ◽  
S Protein ◽  
Chemical Derivatives ◽  
Highly Active ◽  
Infected Cells ◽  
Dose Dependent

This is the first report to show that diphenoxylate is highly active against SARS-CoV-2 with EC<sub>50</sub> of 1.4 μM, CC<sub>50</sub> over 100 μM and selectivity index over 71.4 after screening of 14 diphenyl derivatives. Our results with highly purified solid diphenoxylate confirmatively demonstrate that the viral S protein is reduced in the virus-infected cells in a dose-dependent manner. It could provide insights for antiviral treatment of COVID-19 with diphenoxylate or its chemical derivatives

scholarly journals Grass Carp Reovirus VP35 Degrades MAVS Through the Autophagy Pathway to Inhibit Fish Interferon Production

2021 ◽  
Vol 12 ◽  
Author(s):  
Long-Feng Lu ◽  
Can Zhang ◽  
Zhuo-Cong Li ◽  
Xiao-Yu Zhou ◽  
Jing-Yu Jiang ◽  
...  
Keyword(s):  
Grass Carp ◽  
Host Cells ◽  
Poly I:C ◽  
Dependent Manner ◽  
Grass Carp Reovirus ◽  
Polycytidylic Acid ◽  
Autophagy Pathway ◽  
Mitochondrial Antiviral Signaling ◽  
Dose Dependent ◽  
35 Kda Protein

Fish interferon (IFN) is a crucial cytokine for a host to resist external pathogens, conferring cells with antiviral capacity. Meanwhile, grass carp reovirus (GCRV) is a strong pathogen that causes high mortality in grass carp. Therefore, it is necessary to study the strategy used by GCRV to evade the cellular IFN response. In this study, we found that GCRV 35-kDa protein (VP35) inhibited the host IFN production by degrading mitochondrial antiviral signaling (MAVS) protein through the autophagy pathway. First, the overexpression of VP35 inhibited the IFN activation induced by polyinosinic-polycytidylic acid (poly I:C) and MAVS, and the expression of downstream IFN-stimulated genes (ISGs) was also decreased by using VP35 under the stimulation. Second, VP35 interacted with MAVS; the experiments of truncated mutants of MAVS demonstrated that the caspase recruitment domain (CARD) and proline-rich (PRO) domains of MAVS were not necessary for this binding. Then, MAVS was degraded by using VP35 in a dose-dependent manner, and 3-MA (the autophagy pathway inhibitor) significantly blocked the degradation, meaning that MAVS was degraded by using VP35 in the autophagy pathway. The result of MAVS degradation suggested that the antiviral capacity of MAVS was remarkably depressed when interrupted by VP35. Finally, in the host cells, VP35 reduced ifn transcription and made the cells vulnerable to virus infection. In conclusion, our results reveal that GCRV VP35 impairs the host IFN response by degrading MAVS through the autophagy pathway, supplying evidence of a fish virus immune evasion strategy.

scholarly journals Circulating ACE2-expressing Exosomes Block SARS-CoV-2 Infection as an Innate Antiviral Mechanism

2020 ◽  
Author(s):  
Lamiaa El-Shennawy ◽  
Andrew D. Hoffmann ◽  
Nurmaa K. Dashzeveg ◽  
Paul J. Mehl ◽  
Zihao Yu ◽  
...  
Keyword(s):  
Viral Entry ◽  
Therapeutic Potential ◽  
Adaptive Immune Response ◽  
Host Cells ◽  
Dependent Manner ◽  
Angiotensin Converting Enzyme 2 ◽  
Healthy Donors ◽  
Dose Dependent ◽  
Entry Receptor ◽  
Antiviral Mechanism

AbstractThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19) with innate and adaptive immune response triggered in such patients by viral antigens. Both convalescent plasma and engineered high affinity human monoclonal antibodies have shown therapeutic potential to treat COVID-19. Whether additional antiviral soluble factors exist in peripheral blood remain understudied. Herein, we detected circulating exosomes that express the SARS-CoV-2 viral entry receptor angiotensin-converting enzyme 2 (ACE2) in plasma of both healthy donors and convalescent COVID-19 patients. We demonstrated that exosomal ACE2 competes with cellular ACE2 for neutralization of SARS-CoV-2 infection. ACE2-expressing (ACE2+) exosomes blocked the binding of the viral spike (S) protein RBD to ACE2+ cells in a dose dependent manner, which was 400- to 700-fold more potent than that of vesicle-free recombinant human ACE2 extracellular domain protein (rhACE2). As a consequence, exosomal ACE2 prevented SARS-CoV-2 pseudotype virus tethering and infection of human host cells at a 50-150 fold higher efficacy than rhACE2. A similar antiviral activity of exosomal ACE2 was further demonstrated to block wild-type live SARS-CoV-2 infection. Of note, depletion of ACE2+ exosomes from COVID-19 patient plasma impaired the ability to block SARS-CoV-2 RBD binding to host cells. Our data demonstrate that ACE2+ exosomes can serve as a decoy therapeutic and a possible innate antiviral mechanism to block SARS-CoV-2 infection.

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