scholarly journals p47 GTPases Regulate Toxoplasma gondii Survival in Activated Macrophages

2005 ◽  
Vol 73 (6) ◽  
pp. 3278-3286 ◽  
Author(s):  
Barbara A. Butcher ◽  
Robert I. Greene ◽  
Stanley C. Henry ◽  
Kimberly L. Annecharico ◽  
J. Brice Weinberg ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Acute Infection ◽  
Host Cells ◽  
Intracellular Growth ◽  
Latex Beads ◽  
Cellular Factors ◽  
Ifn Γ ◽  
Normal Inhibition

ABSTRACT The cytokine gamma interferon (IFN-γ) is critical for resistance to Toxoplasma gondii. IFN-γ strongly activates macrophages and nonphagocytic host cells to limit intracellular growth of T. gondii; however, the cellular factors that are required for this effect are largely unknown. We have shown previously that IGTP and LRG-47, members of the IFN-γ-regulated family of p47 GTPases, are required for resistance to acute T. gondii infections in vivo. In contrast, IRG-47, another member of this family, is not required. In the present work, we addressed whether these GTPases are required for IFN-γ-induced suppression of T. gondii growth in macrophages in vitro. Bone marrow macrophages that lacked IGTP or LRG-47 displayed greatly attenuated IFN-γ-induced inhibition of T. gondii growth, while macrophages that lacked IRG-47 displayed normal inhibition. Thus, the ability of the p47 GTPases to limit acute infection in vivo correlated with their ability to suppress intracellular growth in macrophages in vitro. Using confocal microscopy and sucrose density fractionation, we demonstrated that IGTP largely colocalizes with endoplasmic reticulum markers, while LRG-47 was mainly restricted to the Golgi. Although both IGTP and LRG-47 localized to vacuoles containing latex beads, neither protein localized to vacuoles containing live T. gondii. These results suggest that IGTP and LRG-47 are able to regulate host resistance to acute T. gondii infections through their ability to inhibit parasite growth within the macrophage.

scholarly journals Inducible Nitric Oxide Is Essential for Host Control of Persistent but Not Acute Infection with the Intracellular Pathogen Toxoplasma gondii

1997 ◽  
Vol 185 (7) ◽  
pp. 1261-1274 ◽  
Author(s):  
Tanya M. Scharton-Kersten ◽  
George Yap ◽  
Jeanne Magram ◽  
Alan Sher
Keyword(s):  
Nitric Oxide ◽  
Toxoplasma Gondii ◽  
Acute Infection ◽  
Protective Role ◽  
Major Mechanism ◽  
Reactive Nitrogen Intermediates ◽  
Ifn Γ ◽  
Inos Knockout Mice

The induction by IFN-γ of reactive nitrogen intermediates has been postulated as a major mechanism of host resistance to intracellular pathogens. To formally test this hypothesis in vivo, the course of Toxoplasma gondii infection was assessed in nitric oxide synthase (iNOS)−/− mice. As expected, macrophages from these animals displayed defective microbicidal activity against the parasite in vitro. Nevertheless, in contrast to IFN-γ−/− or IL-12 p40−/− animals, iNOSdeficient mice survived acute infection and controlled parasite growth at the site of inoculation. This early resistance was ablated by neutralization of IFN-γ or IL-12 in vivo and markedly diminished by depletion of neutrophils, demonstrating the existence of previously unappreciated NO independent mechanisms operating against the parasite during early infection. By 3-4 wk post infection, however, iNOS knockout mice did succumb to T. gondii. At that stage parasite expansion and pathology were evident in the central nervous system but not the periphery suggesting that the protective role of nitric oxide against this intracellular infection is tissue specific rather than systemic.

Analogs of marinopyrrole A show enhancement to observed in vitro potency against acute Toxoplasma gondii infection

2021 ◽  
Author(s):  
Matthew C. Martens ◽  
Yan Liu ◽  
Austin G. Sanford ◽  
Alexander I. Wallick ◽  
Rosalie C. Warner ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Acute Infection ◽  
Standard Of Care ◽  
Host Cells ◽  
Infection Model ◽  
Current Standard ◽  
Clinical Consequences ◽  
Toxoplasma Gondii Infection

The apicomplexan parasite Toxoplasma gondii is the causative agent of toxoplasmosis, a globally distributed infection with severe clinical consequences for immunocompromised individuals and developing fetuses. There are few available treatments, and these are associated with potentially severe adverse effects. Marinopyrrole A, a compound discovered in a marine Streptomyces species, has previously been found to exhibit potent antimicrobial activity, prompting our interest in exploring efficacy against Toxoplasma gondii . We found that marinopyrrole A was a highly potent anti- Toxoplasma molecule, with an in vitro 50% maximal inhibitory concentration (IC 50 ) of 0.31 μM corresponding to a higher potency than that of the current standard of care (pyrimethamine); however, addition of 20% serum led to abrogation of potency, and toxicity to human cell lines was observed. Yet, application of marinopyrrole A to an in vivo lethal acute infection model facilitated significantly enhanced survival at doses of 5, 10, and 20 mg/kg. We then tested a series of marinopyrrole A analogs—RL002, RL003, and RL125—demonstrating significantly increased potency in vitro , with IC 50 values ranging from 0.09-0.17 μM (3.6-6.8X increase relative to pyrimethamine). No detectable cytotoxicity was observed up to 50 μM in human foreskin fibroblasts, with cytotoxicity in HepG2 cells ranging from ∼28-50 μM, corresponding to >200X selectivity for parasites over host cells. All analogs additionally showed reduced sensitivity to serum. Further, RL003 potently inhibited in vitro -generated bradyzoites at 0.245 μM. Taken together, these data support further development of marinopyrrole A analogs as promising anti- Toxoplasma molecules to further combat this prevalent infection.

scholarly journals High throughput screen identifies IFN-γ-dependent inhibitors of Toxoplasma gondii growth

2018 ◽  
Author(s):  
Joshua B. Radke ◽  
Kimberly L. Carey ◽  
Subrata Shaw ◽  
Shailesh R. Metkar ◽  
Carol Mulrooney ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
High Throughput ◽  
Severe Disease ◽  
Acute Infection ◽  
Congenital Infection ◽  
Host Cells ◽  
High Throughput Screen ◽  
Chronic Infections ◽  
Ifn Γ

AbstractToxoplasma gondii is an obligate intracellular parasite capable of causing severe disease due to congenital infection and in patients with compromised immune systems. Control of infection is dependent on a robust Th1 type immune response including production of interferon gamma (IFN-γ), which is essential for control. IFN-γ activates a variety of anti-microbial mechanisms in host cells, which are then able to control intracellular parasites such as T. gondii. Despite the effectiveness of these pathways in controlling acute infection, the immune system is unable to eradicate chronic infections that can persist for life. Similarly, while antibiotic treatment can control acute infection, it is unable to eliminate chronic infection. To identify compounds that would act synergistically with IFN-γ, we performed a high-throughput screen of diverse small molecule libraries to identify inhibitors of T. gondii. We identified a number of compounds that inhibited parasite growth in vitro at low μM concentrations and that demonstrated enhanced potency in the presence of low level of IFN-γ. A subset of these compounds act by enhancing the recruitment of LC3 to the parasite-containing vacuole, suggesting they work by an autophagy-related process, while others were independent of this pathway. The pattern of IFN-γ-dependence was shared among the majority of analogs from 6 priority scaffolds and analysis of structure activity relationships for one such class revealed specific stereochemistry associated with this feature. Identification of these IFN-γ-dependent leads may lead to development of improved therapeutics due to their synergistic interactions with immune responses.

scholarly journals Activity of Gatifloxacin Alone or in Combination with Pyrimethamine or Gamma Interferon against Toxoplasma gondii

2001 ◽  
Vol 45 (1) ◽  
pp. 48-51 ◽  
Author(s):  
Anis A. Khan ◽  
Teri R. Slifer ◽  
Fausto G. Araujo ◽  
Jack S. Remington
Keyword(s):  
Toxoplasma Gondii ◽  
Inhibitory Concentration ◽  
Day Treatment ◽  
Gamma Interferon ◽  
Host Cells ◽  
Time To Death ◽  
Significant Survival ◽  
Ifn Γ

ABSTRACT The activity of gatifloxacin against Toxoplasma gondii, either alone or in combination with pyrimethamine or gamma interferon (IFN-γ), was examined in vitro and in vivo. In vitro, gatifloxacin significantly inhibited intracellular replication of tachyzoites of the RH strain with a 50% inhibitory concentration of 0.21 μg/ml at 48 h after addition of the drug to the cultures. Toxicity for host cells was not observed at this concentration. A synergistic effect (combination indices < 0.5) was demonstrated in vitro following 48 h of treatment with the combination of gatifloxacin and pyrimethamine (1:1 ratio). Doses of gatifloxacin of 100 and 200 mg/kg of body weight/day administered orally to mice for 10 days resulted in significant (P values of 0.056 and <0.0001, respectively) prolongation in time to death following infection with a lethal inoculum of tachyzoites. A dose of 400 mg/kg resulted in 20% survival (P = 0.0001). Mortality was 100% in untreated control mice and in mice treated with 25 or 50 mg/kg/day. Treatment of infected mice with a combination of gatifloxacin at 200 mg/kg/day and pyrimethamine at 12.5 mg/kg/day resulted in 85% survival, whereas 100 and 80% of mice treated with gatifloxacin alone or pyrimethamine alone, respectively, died (P < 0.0001). Moreover, a gatifloxacin dose of 200 mg/kg/day administered orally for 10 days plus 2 μg of recombinant murine IFN-γ/day administered intraperitoneally for 10 days resulted in significant survival compared with IFN-γ alone (P < 0.0001) or gatifloxacin alone (P < 0.007).

scholarly journals The Function of Gamma Interferon-Inducible GTP-Binding Protein IGTP in Host Resistance to Toxoplasma gondii Is Stat1 Dependent and Requires Expression in Both Hematopoietic and Nonhematopoietic Cellular Compartments

2002 ◽  
Vol 70 (12) ◽  
pp. 6933-6939 ◽  
Author(s):  
Carmen M. Collazo ◽  
George S. Yap ◽  
Sara Hieny ◽  
Patricia Caspar ◽  
Carl G. Feng ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Toxoplasma Gondii ◽  
Host Resistance ◽  
Chronic Infection ◽  
Acute Infection ◽  
Gamma Interferon ◽  
Ifn Γ ◽  
Bone Marrow Chimeras

ABSTRACT IGTP is a member of the 47-kDa family of gamma interferon (IFN-γ)-induced GTPases. We have previously shown that IGTP is critical for host resistance to Toxoplasma gondii infection. In the present study, we demonstrate that T. gondii-induced IGTP expression in vivo and IFN-γ-driven synthesis of the protein in vitro are dependent on Stat1. Consistent with this observation, Stat1-deficient animals succumbed to T. gondii infection with the same rapid kinetics as IGTP−/− mice. To ascertain the cellular levels at which IGTP functions in host control of acute infection, we constructed reciprocal bone marrow chimeras between IGTP-deficient and wild-type mice. Resistance to infection was observed only when IGTP was present in both hematopoietic and nonhematopoietic compartments. To assess the possible contribution of IGTP to the maintenance of parasite latency, partial chemotherapy was used to allow the establishment of chronic infection in IGTP-deficient animals. Upon cessation of drug treatment, these animals showed delayed mortality compared with similarly infected and treated IFN-γ-deficient or inducible nitric oxide synthase-deficient mice, which succumbed rapidly. Parallel experiments performed with drug-treated bone marrow chimeras supported a role for the hematopoietic compartment in this NO-dependent, IGTP-independent control of chronic infection. Taken together, our findings demonstrate that host resistance mediated by IGTP is a Stat1-induced function which in the case of T. gondii acts predominantly to restrict acute as opposed to chronic infection. This effector mechanism requires expression of IGTP in cells of both hematopoietic and nonhematopoietic origin. In contrast, in latent infection, hematopoietically derived cells mediate resistance by means of a largely NO-dependent pathway.

scholarly journals ROP18-Mediated Transcriptional Reprogramming of HEK293T Cell Reveals New Roles of ROP18 in the Interplay Between Toxoplasma gondii and the Host Cell

2020 ◽  
Vol 10 ◽  
Author(s):  
Jie-Xi Li ◽  
Jun-Jun He ◽  
Hany M. Elsheikha ◽  
Jun Ma ◽  
Xiao-Pei Xu ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Hek293t Cell ◽  
Effector Proteins ◽  
Host Cells ◽  
Pathway Enrichment Analysis ◽  
Type I ◽  
Human Embryonic Kidney Cells ◽  
Pathway Enrichment

Toxoplasma gondii secretes a number of virulence-related effector proteins, such as the rhoptry protein 18 (ROP18). To further broaden our understanding of the molecular functions of ROP18, we examined the transcriptional response of human embryonic kidney cells (HEK293T) to ROP18 of type I T. gondii RH strain. Using RNA-sequencing, we compared the transcriptome of ROP18-expressing HEK293T cells to control HEK293T cells. Our analysis revealed that ROP18 altered the expression of 750 genes (467 upregulated genes and 283 downregulated genes) in HEK293T cells. Gene ontology (GO) and pathway enrichment analyses showed that differentially expressed genes (DEGs) were significantly enriched in extracellular matrix– and immune–related GO terms and pathways. KEGG pathway enrichment analysis revealed that DEGs were involved in several disease-related pathways, such as nervous system diseases and eye disease. ROP18 significantly increased the alternative splicing pattern “retained intron” and altered the expression of 144 transcription factors (TFs). These results provide new insight into how ROP18 may influence biological processes in the host cells via altering the expression of genes, TFs, and pathways. More in vitro and in vivo studies are required to substantiate these findings.

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 The inhibitory effect of cromolyn sodium and ketotifen on Toxoplasma gondii entrance into host cells in vitro and in vivo

2014 ◽  
Vol 40 (3) ◽  
pp. 1001-1005 ◽  
Author(s):  
Fatemeh Rezaei ◽  
Mohammad Ali Ebrahimzadeh ◽  
Ahmad Daryani ◽  
Mehdi Sharif ◽  
Ehsan Ahmadpour ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Inhibitory Effect ◽  
Cromolyn Sodium ◽  
Host Cells

1,25(OH)2D3 inhibits in vitro and in vivo intracellular growth of apicomplexan parasite Toxoplasma gondii

2007 ◽  
Vol 103 (3-5) ◽  
pp. 811-814 ◽  
Author(s):  
Rohan Rajapakse ◽  
Béatrice Uring-Lambert ◽  
Kumari L. Andarawewa ◽  
R.P. Rajapakse ◽  
Ahmed Abou-Bacar ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Apicomplexan Parasite ◽  
Intracellular Growth

scholarly journals Actin depolymerizing factor controls actin turnover and gliding motility in Toxoplasma gondii

2011 ◽  
Vol 22 (8) ◽  
pp. 1290-1299 ◽  
Author(s):  
Simren Mehta ◽  
L. David Sibley
Keyword(s):  
Toxoplasma Gondii ◽  
Actin Filaments ◽  
Gliding Motility ◽  
Conditional Knockout ◽  
Host Cells ◽  
Actin Binding ◽  
Actin Depolymerizing Factor

Apicomplexan parasites rely on actin-based gliding motility to move across the substratum, cross biological barriers, and invade their host cells. Gliding motility depends on polymerization of parasite actin filaments, yet ∼98% of actin is nonfilamentous in resting parasites. Previous studies suggest that the lack of actin filaments in the parasite is due to inherent instability, leaving uncertain the role of actin-binding proteins in controlling dynamics. We have previously shown that the single allele of Toxoplasma gondii actin depolymerizing factor (TgADF) has strong actin monomer–sequestering and weak filament-severing activities in vitro. Here we used a conditional knockout strategy to investigate the role of TgADF in vivo. Suppression of TgADF led to accumulation of actin-rich filaments that were detected by immunofluorescence and electron microscopy. Parasites deficient in TgADF showed reduced speed of motility, increased aberrant patterns of motion, and inhibition of sustained helical gliding. Lack of TgADF also led to severe defects in entry and egress from host cells, thus blocking infection in vitro. These studies establish that the absence of stable actin structures in the parasite are not simply the result of intrinsic instability, but that TgADF is required for the rapid turnover of parasite actin filaments, gliding motility, and cell invasion.

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