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

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.

Download Full-text

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

Journal of Cell Science ◽

10.1242/jcs.70.1.73 ◽

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.

Download Full-text

Equilibrative Nucleoside Transporters Mediate the Import of Nicotinamide Riboside and Nicotinic Acid Riboside into Human Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22031391 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1391

Author(s):

Andrey Kropotov ◽

Veronika Kulikova ◽

Kirill Nerinovski ◽

Alexander Yakimov ◽

Maria Svetlova ◽

...

Keyword(s):

Nicotinic Acid ◽

Mammalian Cells ◽

Experimental Models ◽

The Body ◽

Nucleoside Transporters ◽

Hek293 Cells ◽

Nucleoside Transporter ◽

Vitamin B3 ◽

Animal Cells ◽

Nicotinamide Riboside

Nicotinamide riboside (NR), a new form of vitamin B3, is an effective precursor of nicotinamide adenine dinucleotide (NAD+) in human and animal cells. The introduction of NR into the body effectively increases the level of intracellular NAD+ and thereby restores physiological functions that are weakened or lost in experimental models of aging and various pathologies. Despite the active use of NR in applied biomedicine, the mechanism of its transport into mammalian cells is currently not understood. In this study, we used overexpression of proteins in HEK293 cells, and metabolite detection by NMR, to show that extracellular NR can be imported into cells by members of the equilibrative nucleoside transporter (ENT) family ENT1, ENT2, and ENT4. After being imported into cells, NR is readily metabolized resulting in Nam generation. Moreover, the same ENT-dependent mechanism can be used to import the deamidated form of NR, nicotinic acid riboside (NAR). However, NAR uptake into HEK293 cells required the stimulation of its active utilization in the cytosol such as phosphorylation by NR kinase. On the other hand, we did not detect any NR uptake mediated by the concentrative nucleoside transporters (CNT) CNT1, CNT2, or CNT3, while overexpression of CNT3, but not CNT1 or CNT2, moderately stimulated NAR utilization by HEK293 cells.

Download Full-text

Diverse Functions of Polyamines in Virus Infection

Biomolecules ◽

10.3390/biom10040628 ◽

2020 ◽

Vol 10 (4) ◽

pp. 628 ◽

Cited By ~ 3

Author(s):

Mason R. Firpo ◽

Bryan C. Mounce

Keyword(s):

Virus Infection ◽

Mammalian Cells ◽

Building Blocks ◽

Host Cells ◽

Genome Packaging ◽

Bacteriophage Infection ◽

Intracellular Parasites ◽

Initial Work ◽

Critical Resource ◽

Positively Charged

As obligate intracellular parasites, viruses rely on host cells for the building blocks of progeny viruses. Metabolites such as amino acids, nucleotides, and lipids are central to viral proteins, genomes, and envelopes, and the availability of these molecules can restrict or promote infection. Polyamines, comprised of putrescine, spermidine, and spermine in mammalian cells, are also critical for virus infection. Polyamines are small, positively charged molecules that function in transcription, translation, and cell cycling. Initial work on the function of polyamines in bacteriophage infection illuminated these molecules as critical to virus infection. In the decades since early virus-polyamine descriptions, work on diverse viruses continues to highlight a role for polyamines in viral processes, including genome packaging and viral enzymatic activity. On the host side, polyamines function in the response to virus infection. Thus, viruses and hosts compete for polyamines, which are a critical resource for both. Pharmacologically targeting polyamines, tipping the balance to favor the host and restrict virus replication, holds significant promise as a broad-spectrum antiviral strategy.

Download Full-text

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

Parasites & Vectors ◽

10.1186/s13071-020-04324-7 ◽

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.

Download Full-text

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

mSphere ◽

10.1128/msphere.00858-19 ◽

2020 ◽

Vol 5 (1) ◽

Cited By ~ 9

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.

Download Full-text

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

Journal of Experimental Medicine ◽

10.1084/jem.20082108 ◽

2009 ◽

Vol 206 (2) ◽

pp. 399-410 ◽

Cited By ~ 115

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.

Download Full-text

Functional characterization of a recombinant sodium-dependent nucleoside transporter with selectivity for pyrimidine nucleosides (cNT1rat) by transient expression in cultured mammalian cells

Biochemical Journal ◽

10.1042/bj3170457 ◽

1996 ◽

Vol 317 (2) ◽

pp. 457-465 ◽

Cited By ~ 49

Author(s):

Xiao FANG ◽

Fiona E. PARKINSON ◽

Delores A. MOWLES ◽

James D. YOUNG ◽

Carol E. CASS

Keyword(s):

Mammalian Cells ◽

Expression System ◽

Functional Characterization ◽

Transport Processes ◽

Nucleoside Transport ◽

Single Type ◽

Nucleoside Transporter ◽

Pyrimidine Nucleosides ◽

Transporter Protein ◽

Sodium Dependent

We have demonstrated that monkey kidney (COS-1) cells have a single type of nucleoside transport process, which, because it was equilibrative, sodium-independent and could be inhibited by nitrobenzylthioinosine (NBMPR), was identified as the ‘equilibrative sensitive’ or ‘es’ transporter. Using NBMPR or dilazep to inhibit the endogenous nucleoside transport activity, we have transiently expressed a cDNA that encodes an inhibitor-insensitive, concentrative nucleoside transporter protein (cNT1rat) of rat intestine in COS-1 cells. The production of recombinant cNT1rat was examined by immunoblotting using an epitope-tagged construct and by analysis of inward fluxes of 3H-labelled nucleosides. Recombinant cNT1rat was sodium-dependent and selective for pyrimidine nucleosides, with approximate Km values of 21 μM, 12.5 μM and 15 μM for uridine, thymidine and adenosine, respectively. Although adenosine exhibited high affinity for the recombinant transporter, its Vmax value was low. A variety of anti-viral and anti-cancer nucleoside drugs inhibited cNT1rat-mediated uptake of uridine by transfected COS-1 cells although to different extents (Floxidine > Idoxuridine > Zidovudine > Zalcitabine > Cytarabine > Gemcitabine), suggesting that the concentrative pyrimidine-selective nucleoside transporters, of which cNT1rat is a representative, may play a role in cellular uptake of these drugs. The cNT1rat/COS-1 expression system is a useful tool for analysis of cNT1rat-mediated transport processes.

Download Full-text

Revisiting the role of Toxoplasma gondii ERK7 in the maintenance and stability of the apical complex

10.1101/2021.03.22.436543 ◽

2021 ◽

Author(s):

Dominique Soldati-Favre ◽

Nicolas Dos Santos Pacheco ◽

Nicolò Tosetti ◽

Aarti Krishnan ◽

Romuald Haase

Keyword(s):

Toxoplasma Gondii ◽

Comparative Proteomics ◽

Host Cells ◽

Wild Type ◽

Proteomics Analysis ◽

Polar Ring ◽

Intracellular Parasites ◽

Apical Complex ◽

Microneme Proteins

Toxoplasma gondii ERK7 is known to contribute to the integrity of the apical complex and to be involved only in the final step of the conoid biogenesis. In the absence of ERK7, mature parasites lose their conoid complex and are unable to glide, invade or egress from host cells. In contrast to a previous report, we show here that depletion of ERK7 phenocopies the depletion of the apical cap proteins AC9 or AC10. The absence of ERK7 leads to the loss of the apical polar ring, the disorganization of the basket of subpellicular microtubules and an impairment in micronemes secretion. Ultra-expansion microscopy (U-ExM) coupled to NHS-Ester staining on intracellular parasites offers an unprecedented level of resolution and highlights the disorganization of the rhoptries as well as the dilated plasma membrane at the apical pole in the absence of ERK7. Comparative proteomics analysis of wild-type and ERK7 or AC9 depleted parasites led to the disappearance of known, predicted, as well as putative novel components of the apical complex. In contrast, the absence of ERK7 led to an accumulation of microneme proteins, resulting from the defect in exocytosis of the organelles.

Download Full-text

Nucleoside transport and proliferative rate in human thymocytes and lymphocytes

Blood ◽

10.1182/blood.v74.6.2038.bloodjournal7462038 ◽

1989 ◽

Vol 74 (6) ◽

pp. 2038-2042 ◽

Cited By ~ 1

Author(s):

CL Smith ◽

LM Pilarski ◽

ML Egerton ◽

JS Wiley

Keyword(s):

Cytosine Arabinoside ◽

Binding Sites ◽

Flow Cytometric Analysis ◽

Fold Increase ◽

S Phase ◽

Nucleoside Transporters ◽

Labeling Index ◽

Nucleoside Transport ◽

Nucleoside Transporter ◽

Equilibrative Nucleoside Transporter

The thymus is a site of active T-lymphoid cell proliferation and DNA synthesis. In this study, the capacity of human thymocytes for nucleoside transport was assessed both by cytosine arabinoside influx and by equilibrium binding of nitrobenzylmercaptopurine riboside (NBMPR), a specific ligand for the equilibrative nucleoside transporter of leukocytes. The proportion of freshly isolated thymocytes synthesizing DNA was 8.6% +/- 2.1% (n = 12) by 3H-thymidine labeling index and 7.8% +/- 2.9% (n = 4) S-phase cells by flow cytometric analysis of DNA content. In comparison, both methods gave proliferation S-phase values less than 1% for peripheral blood lymphocytes (PBLs). Thymocytes expressed a high density of specific NBMPR binding sites (26,068 +/- 8,776 sites per cell, n = 12) as compared with PBLs (1,123 +/- 553 sites per cell, n = 8). The initial influx of cytosine arabinoside into thymocytes was 14-fold greater than into PBLs, and in both cell types the influx of nucleoside was totally inhibited by 0.5 mumol/L NBMPR, which is known to inhibit the major equilibrative nucleoside transporter in white blood cells. Depletion of mature CD3+ cells from the thymocyte preparation by anti-CD3 antibody left a residual population with both increased labeling index and up to twofold greater density of NBMPR binding sites. When PBLs were cultured for 48 hours with the T-cell mitogen phytohemagglutinin, a 40-fold increase in labeling index was observed, together with a 30-fold increase in the density of specific NBMPR binding sites. Thus, fresh thymocytes from human thymus are actively proliferating and express high densities of a functional nucleoside transporter. The more immature cells in the thymocyte population which are proliferating more actively have a greater density of nucleoside transporters than the whole population. In contrast, mitotically inactive PBLs-have few nucleoside transporters, but after mitogenic stimulation PBLs express large numbers of this transmembrane molecule.

Download Full-text

Paclitaxel Arrests Growth of IntracellularToxoplasma gondii

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.8.2036 ◽

1998 ◽

Vol 42 (8) ◽

pp. 2036-2040 ◽

Cited By ~ 10

Author(s):

Randee Estes ◽

Nicolas Vogel ◽

Douglas Mack ◽

Rima McLeod

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Fresh Medium ◽

Host Cells ◽

Normal Morphology ◽

Intracellular Form ◽

Human Foreskin Fibroblasts ◽

The Future ◽

Infected Cells ◽

Parasite Multiplication

ABSTRACT Addition of paclitaxel (Taxol) at a concentration of 1 μM toToxoplasma gondii-infected human foreskin fibroblasts arrested parasite multiplication. Division of theT. gondii tachyzoite nucleus was inhibited, leading to syncytium-like parasite structures within the fibroblasts by 24 h after infection and treatment of the cultures. By 4 days after infection and treatment of the cultures with paclitaxel, this inhibition was irreversible, since the arrested intracellular form was incapable of leaving the host cell, infecting new cells, and initiating the growth of tachyzoites with normal morphology. Specifically, when paclitaxel was added to infected cells for 4 days and then removed by washing and the infected, paclitaxel-treated cells were cultured for 4 more days, there were no remaining T. gondii organisms with normal morphology. Syncytium-like structures in the cultures that were infected and treated with paclitaxel for 8 days were similar in appearance to those in preparations of infected paclitaxel-treated fibroblasts that had been cultured for 24 to 48 h. Pretreatment of the tachyzoites for 1 h with paclitaxel followed by the removal of the paclitaxel by repeatedly centrifuging and resuspending the parasites in fresh medium without paclitaxel and then adding fresh medium prior to culture of the parasites with fibroblasts did not prevent their invasion of fibroblasts but did affect their subsequent ability to replicate within fibroblasts. Pretreatment of the fibroblasts with paclitaxel also diminished subsequent replication ofT. gondii in such host cells after 8 days. Thus, paclitaxel alters the ability of T. gondii to replicate in host cells. Inhibition of parasite microtubules by such compounds at concentrations which do not interfere with the function of host cell microtubules may be useful for development of novel medicines to treat T. gondii infections in the future.

Download Full-text