Electrophoretic Patterns of Toxoplasma gondii Excreted/Secreted Antigens and Their Role in Induction of the Humoral Immune Response

Jundishapur J Microbiol. 2014 Apr;7(4):e9525. doi: 10.5812/jjm.9525. Epub 2014 Apr 1

 

Daryani A¹, Sharif M¹, Kalani H¹, Rafiei A², Kalani F², Ahmadpour E¹.

BACKGROUND:

Toxoplasma gondii is an obligatory intracellular protozoan parasite on which studies are pending regarding production of vaccine. To date, the production of human vaccine has not been successful where approximately one third of the world's population is thought to be infected with T. gondii.

OBJECTIVES:

The present study was designed to compare the electrophoretic patterns of T. gondii excreted/secreted antigens (ESAs) and determine their role in the stimulation of the humoral immune response.

MATERIALS AND METHODS:

T. gondii ESAs were prepared from cell cultures (albino rat fibroblast) and cell-free mediums (RPMI-1640). Next, the SDS-PAGE technique was used for comparing molecular weights of the antigens. Forty C57BL/6 mice were divided randomly into four groups (n = 10). Immunization was performed subcutaneously at an interval of 2 weeks in two groups by injecting 100 µg of each of the above-mentioned antigens. Two groups, as negative control, also received fibroblast lysate proteins or adjuvant separately. All of the groups were then challenged with the T. gondii RH strain. Serum samples were collected from all mice and measured by immunoblotting technique for detection of immunogenic antigens.

RESULTS:

The electrophoretic mobility of the prepared antigens/proteins from cell culture, cell-free media, Fibroblast Lysate Proteins and Toxoplasma Lysate Antigens (TLA) showed 13, 12, 8 and 8 bands, respectively. The case groups, in challenge with T. gondii (RH strain), showed more survival prolongation than the control groups. Furthermore, the survival period was identical for both case groups with a tendency for slightly higher survival of mice receiving ESA from cell-free medium. Analysis of sera by immunoblotting also revealed one band of 65 KDa in sera from both case groups.

CONCLUSIONS:

We suggest that this band be extracted and its amino acids sequence determined to produce Synthetic Polypeptide for immunization studies.

KEYWORDS:

Immunization; Immunoblotting; Toxoplasma

PMID:

25147706

[PubMed]

Internalization and TLR-dependent type I interferon production by monocytes in response to Toxoplasma gondii

Immunol Cell Biol. 2014 Aug 26. doi: 10.1038/icb.2014.70. [Epub ahead of print]

 

Han SJ¹, Melichar HJ¹, Coombes JL¹, Chan SW¹, Koshy AA², Boothroyd JC³, Barton GM¹, Robey EA¹.

 

The classic anti-viral cytokine interferon (IFN)-β can be induced during parasitic infection, but relatively little is know about the cell types and signaling pathways involved. Here we show that inflammatory monocytes (IMs), but not neutrophils, produce IFN-β in response to T. gondii infection. This difference correlated with the mode of parasite entry into host cells, with phagocytic uptake predominating in IMs and active invasion predominating in neutrophils. We also show that expression of IFN-β requires phagocytic uptake of the parasite by IMs, and signaling through Toll-like receptors (TLRs) and MyD88. Finally, we show that IMs are major producers of IFN-β in mesenteric lymph nodes following in vivo oral infection of mice, and mice lacking the receptor for type I IFN-1 show higher parasite loads and reduced survival. Our data reveal a TLR and internalization-dependent pathway in IMs for IFN-β induction to a non-viral pathogen.Immunology and Cell Biology advance online publication, 26 August 2014; doi:10.1038/icb.2014.70.

PMID:

25155465

[PubMed - as supplied by publisher]

Nuclear Glycolytic Enzyme Enolase of Toxoplasma gondii Functions as a Transcriptional Regulator

PLoS One. 2014 Aug 25;9(8):e105820. doi: 10.1371/journal.pone.0105820. eCollection 2014.

 

Mouveaux T¹, Oria G¹, Werkmeister E¹, Slomianny C², Fox BA³, Bzik DJ³, Tomavo S¹.

 

Apicomplexan parasites including Toxoplasma gondii have complex life cycles within different hosts and their infectivity relies on their capacity to regulate gene expression. However, little is known about the nuclear factors that regulate gene expression in these pathogens. Here, we report that T. gondii enolase TgENO2 is targeted to the nucleus of actively replicating parasites, where it specifically binds to nuclear chromatin in vivo. Using a ChIP-Seq technique, we provide evidence for TgENO2 enrichment at the 5' untranslated gene regions containing the putative promoters of 241 nuclear genes. Ectopic expression of HA-tagged TgENO1 or TgENO2 led to changes in transcript levels of numerous gene targets. Targeted disruption of TgENO1 gene results in a decrease in brain cyst burden of chronically infected mice and in changes in transcript levels of several nuclear genes. Complementation of this knockout mutant with ectopic TgENO1-HA fully restored normal transcript levels. Our findings reveal that enolase functions extend beyond glycolytic activity and include a direct role in coordinating gene regulation in T. gondii.

PMID:

25153525

[PubMed - in process]

Protozoan parasites and type I interferons: a cold case reopened

Trends Parasitol. 2014 Aug 18. pii: S1471-4922(14)00128-7. doi: 10.1016/j.pt.2014.07.007. [Epub ahead of print

 

Beiting DP.

 

Protozoan parasites, such as Plasmodium, Toxoplasma, Cryptosporidium, trypanosomes, and Leishmania, are a major cause of disease in both humans and other animals, highlighting the need to understand the full spectrum of strategies used by the host immune system to sense and respond to parasite infection. Although type II interferon (IFN-γ) has long been recognized as an essential antiparasite immune effector, much less is known about the role of type I interferons (IFN-α and -β) in host defense, particularly in vivo. Recent studies are reviewed which collectively highlight that type I IFN can be induced in response to parasite infection and influence the outcome of infection.
Copyright © 2014 Elsevier Ltd. All rights reserved.

KEYWORDS:

immunity; infection; intracellular parasite; protozoan; type I interferon

PMID:

25153940

[PubMed - as supplied by publisher]

Nucleotide-Oligomerization-Domain-2 Affects Commensal Gut Microbiota Composition and Intracerebral Immunopathology in Acute Toxoplasma gondii Induced Murine Ileitis

PLoS One. 2014 Aug 20;9(8):e105120. doi: 10.1371/journal.pone.0105120. eCollection 2014.

 

Heimesaat MM¹, Dunay IR², Alutis M¹, Fischer A¹, Möhle L², Göbel UB¹, Kühl AA³, Bereswill S¹.

BACKGROUND:

Within one week following peroral high dose infection with Toxoplasma (T.) gondii, susceptible mice develop non-selflimiting acute ileitis due to an underlying Th1-type immunopathology. The role of the innate immune receptor nucleotide-oligomerization-domain-2 (NOD2) in mediating potential extra-intestinal inflammatory sequelae including the brain, however, has not been investigated so far.

METHODOLOGY/PRINCIPAL FINDINGS:

Following peroral infection with 100 cysts of T. gondii strain ME49, NOD2-/- mice displayed more severe ileitis and higher small intestinal parasitic loads as compared to wildtype (WT) mice. However, systemic (i.e. splenic) levels of pro-inflammatory cytokines such as TNF-α and IFN-γ were lower in NOD2-/- mice versus WT controls at day 7 p.i. Given that the immunopathological outcome might be influenced by the intestinal microbiota composition, which is shaped by NOD2, we performed a quantitative survey of main intestinal bacterial groups by 16S rRNA analysis. Interestingly, Bifidobacteria were virtually absent in NOD2-/- but not WT mice, whereas differences in remaining bacterial species were rather subtle. Interestingly, more distinct intestinal inflammation was accompanied by higher bacterial translocation rates to extra-intestinal tissue sites such as liver, spleen, and kidneys in T. gondii infected NOD2-/- mice. Strikingly, intracerebral inflammatory foci could be observed as early as seven days following T. gondii infection irrespective of the genotype of animals, whereas NOD2-/- mice exhibited higher intracerebral parasitic loads, higher F4/80 positive macrophage and microglia numbers as well as higher IFN-γ mRNA expression levels as compared to WT control animals.

CONCLUSION/SIGNIFICANCE:

NOD2 signaling is involved in protection of mice from T. gondii induced acute ileitis. The parasite-induced Th1-type immunopathology at intestinal as well as extra-intestinal sites including the brain is modulated in a NOD2-dependent manner.

PMID:

25141224

[PubMed - in process]

Design, synthesis and biological characterization of thiazolidin-4-one derivatives as promising inhibitors of Toxoplasma gondii

Eur J Med Chem. 2014 Aug 15;86C:17-30. doi: 10.1016/j.ejmech.2014.08.046. [Epub ahead of print]

 

D'Ascenzio M¹, Bizzarri B², De Monte C¹, Carradori S¹, Bolasco A¹, Secci D¹, Rivanera D³, Faulhaber N⁴, Bordón C⁴, Jones-Brando L⁴.

 

We designed and synthesized a large number of novel thiazolidin-4-one derivatives for the evaluation of their anti-Toxoplasma gondii activity. This scaffold was functionalized at the N1-hydrazine portion with aliphatic, cycloaliphatic and (hetero)aromatic moieties. Then, a benzyl pendant was introduced at the lactamic NH of the core nucleus to evaluate the influence of this chemical modification on biological activity. The compounds were subjected to several in vitro assays to assess their anti-parasitic efficacy, cytotoxicity on fibroblasts, inhibition of tachyzoite invasion/attachment and replication after treatment. Results showed that fourteen of these thiazole-based compounds compare favorably to control compound trimethoprim in terms of parasite growth inhibition.
Copyright © 2014. Published by Elsevier Masson SAS.

KEYWORDS:

Cytotoxicity; Host cell invasion; Parasite growth inhibition; Thiazolidin-4-one; Toxoplasma

PMID:

25140751

[PubMed - as supplied by publisher]

Toxoplasma gondii infection reduces predator aversion in rats through epigenetic modulation in the host medial amygdala

Mol Ecol. 2014 Aug 20. doi: 10.1111/mec.12888. [Epub ahead of print]

 

Hari Dass SA¹, Vyas A.

Abstract

Male rats (Rattus novergicus) infected with protozoan Toxoplasma gondii relinquish their innate aversion to the cat odors. This behavioral change is postulated to increase transmission of the parasite to its definitive felid hosts. Here, we show that the Toxoplasma gondii infection institutes an epigenetic change in the DNA methylation of the arginine vasopressin promoter in the medial amygdala of male rats. Infected animals exhibit hypomethylation of arginine vasopressin promoter, leading to greater expression of this nonapeptide. The infection also results in the greater activation of the vasopressinergic neurons after exposure to the cat odor. Furthermore, we show that loss of fear in the infected animals can be rescued by the systemic hypermethylation, and recapitulated by directed hypomethylation in the medial amygdala. These results demonstrate an epigenetic proximate mechanism underlying the extended phenotype in the Rattus novergicus - Toxoplasma gondii association. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.

KEYWORDS:

Arginine vasopressin; Behavioral manipulation; DNA methylation; Extended phenotype; Fear; Testosterone

PMID:

25142402

[PubMed - as supplied by publisher]

Effects of soluble extracts from Leishmania infantum promastigotes, Toxoplasma gondii tachyzoites on TGF-β mediated pathways in activated CD4+ T lymphocytes

Microbes Infect. 2014 Aug 12. pii: S1286-4579(14)00102-6. doi: 10.1016/j.micinf.2014.08.002. [Epub ahead of print]

 

Clemente AM¹, Severini C², Castronovo G³, Tanturli M³, Perissi E³, Cozzolino F³, Torcia MG⁴.

 

Interference with transforming growth factor-β-mediated pathways helps several parasites to survive for long periods in immunocompetent hosts. Macrophages and dendritic cells infected by Toxoplasma, Leishmania and Plasmodium spp. produce large amounts of transforming growth factor-β and induce the differentiation of antigen-specific T-regulatory cells. Mechanisms not mediated by antigen-presentation could also account for the expansion of T-regulatory cells in parasitic diseases and they also might be mediated through transforming growth factor-β-receptor activated pathways. We explored the properties of soluble extracts from Leishmania infantum promastigotes, Toxoplasma gondii tachyzoites, Trichinella spiralis muscle larvae to expand the pool of T-regulatory cells in a population of polyclonally activated T cells in the absence of accessory cells, and compared their effects to those induced by Plasmodium falciparum extracts. Similarly to Plasmodium falciparum, Leishmania infantum extracts activate the latent soluble form of transforming growth factor-β and that bound to the membrane of activated T lymphocytes. The interaction of the active cytokine with transforming growth factor-β receptor induces Foxp3 expression by activated lymphocytes, favouring their conversion through the T-regulatory phenotype. Both Toxoplasma gondii and Leishmania infantum extracts are able to induce transforming growth factor-β production by activated T cells in the absence of accessory cells.
Copyright © 2014. Published by Elsevier Masson SAS.

KEYWORDS:

Leishmania infantum; T-regulatory cells; Toxoplasma gondii; transforming growth factor

PMID:

25130316

[PubMed - as supplied by publisher]

Withdrawal of skeletal muscle cells from cell cycle progression triggers differentiation of Toxoplasma gondii towards the bradyzoite stage

Cell Microbiol. 2014 Aug 14. doi: 10.1111/cmi.12342. [Epub ahead of print]

 

Swierzy IJ¹, Lüder CG.

 

Toxoplasma gondii is a widespread intracellular parasite of mammals and birds and an important opportunistic pathogen of humans. Following primary infection, fast replicating tachyzoites disseminate within the host and are subsequently either eliminated by the immune system or transform to latent bradyzoites which preferentially persist in brain and muscle tissues. The factors which determine the parasites' tissue distribution during chronic toxoplasmosis are unknown. Here we show that mouse skeletal muscle cells (SkMCs) after differentiation to mature, myosin heavy chain-positive, polynucleated myotubes, significantly restrict tachyzoite replication and facilitate expression of bradyzoite-specific antigens and tissue cyst formation. In contrast, proliferating mononuclear myoblasts and control fibroblasts enable vigorous T. gondii replication but do not sustain bradyzoite or tissue cyst formation. Bradyzoite formation correlates with up-regulation of testis-specific Y-encoded-like protein-2 gene expression (Tspyl2) and p21^Waf1/Cip1 as well as down-regulation of cyclin B1 and absence of DNA synthesis, i.e. a cell cycle arrest of syncytial myotubes. Following infection with T. gondii, myotubes but not myoblasts or fibroblasts further up-regulate the negative cell cycle regulator Tspyl2. Importantly, RNA interference-mediated knockdown of Tspyl2 abrogates differentiation of SkMCs to myotubes and enables T. gondii to replicate vigorously but abolishes bradyzoite-specific gene expression and tissue cyst formation. Together, these data indicate that Tspyl2-mediated host cell cycle withdrawal is a physiological trigger of Toxoplasma stage conversion in mature SkMCs. This finding might explain the preferred distribution of T. gondii tissue cysts in vivo.
This article is protected by copyright. All rights reserved.

PMID:

25131712

[PubMed - as supplied by publisher]

One minute ultraviolet exposure inhibits Toxoplasma gondii tachyzoite replication and cyst conversion without diminishing host humoral-mediated immune response

Exp Parasitol. 2014 Aug 14. pii: S0014-4894(14)00191-X. doi: 10.1016/j.exppara.2014.08.001. [Epub ahead of print

 

Kannan G¹, Prandovszky E², Steinfeldt CB², Gressitt KL², Yang C³, Yolken RH², Severance EG², Jones-Brando L², Pletnikov MV⁴

 

We developed a protocol to inactivate Toxoplasma gondii (T. gondii) tachyzoites employing 1 minute of ultraviolet (UV) exposure. We show that this treatment completely inhibited parasite replication and cyst formation in vitro and in vivo but did not affect the induction of a robust IgG response in mice. We propose that our protocol can be used to study the contribution of the humoral immune response to rodent behavioral alterations following T. gondii infection.
Copyright © 2014. Published by Elsevier Inc.

KEYWORDS:

Animal models; Behavior; Immune response; Inactivation, Cysts; Toxoplasma

PMID:

25131777

[PubMed - as supplied by publisher]

Toxoplasma gondii and anxiety disorders in a community-based sample

Brain Behav Immun. 2014 Aug 12. pii: S0889-1591(14)00418-8. doi: 10.1016/j.bbi.2014.08.001. [Epub ahead of print]

 

Markovitz A¹, Simanek AM², Yolken R³, Galea S⁴, Koenen KC⁴, Chen S⁵, Aiello AE⁶.

Abstract

A growing body of literature suggests that exposure to the neurotropic parasite Toxoplasma gondii (T. gondii) is associated with increased risk of mental disorders, particularly schizophrenia. However, a potential association between T. gondii exposure and anxiety disorders has not been rigorously explored. Here, we examine the association of T. gondii infection with both anxiety and mood disorders. Participants (n=484) were drawn from the Detroit Neighborhood Health Study, a population-representative sample of Detroit residents. Logistic regression was used to examine the associations between T. gondii exposure (defined by seropositivity and IgG antibody levels) and three mental disorders: generalized anxiety disorder (GAD), posttraumatic stress disorder (PTSD) and depression. We found that T. gondii seropositivity was associated with a 2 times greater odds of GAD (odds ratio (OR), 2.25; 95% confidence interval (CI), 1.11-4.53) after adjusting for age, gender, race, income, marital status, and medication. Individuals in the highest antibody level category had more than 3 times higher odds of GAD (OR, 3.35; 95% CI, 1.41-7.97). Neither T. gondii seropositivity nor IgG antibody levels was significantly associated with PTSD or depression. Our findings indicate that T. gondii infection is strongly and significantly associated with GAD. While prospective confirmation is needed, T. gondii infection may play a role in the development of GAD.
Copyright © 2014. Published by Elsevier Inc.

KEYWORDS:

Depression; Generalized anxiety disorder; Infection; Mental health; PTSD; Toxoplasma gondii

PMID:

25124709

[PubMed - as supplied by publisher]

Sequence Variation in Toxoplasma gondii rop17 Gene among Strains from Different Hosts and Geographical Locations

ScientificWorldJournal. 2014;2014:349325. doi: 10.1155/2014/349325. Epub 2014 Jul 10.

 

Zhang NZ¹, Xu Y², Huang SY¹, Zhou DH¹, Wang RA³, Zhu XQ¹.

Abstract

Genetic diversity of T. gondii is a concern of many studies, due to the biological and epidemiological diversity of this parasite. The present study examined sequence variation in rhoptry protein 17 (ROP17) gene among T. gondii isolates from different hosts and geographical regions. The rop17 gene was amplified and sequenced from 10 T. gondii strains, and phylogenetic relationship among these T. gondii strains was reconstructed using maximum parsimony (MP), neighbor-joining (NJ), and maximum likelihood (ML) analyses. The partial rop17 gene sequences were 1375 bp in length and A+T contents varied from 49.45% to 50.11% among all examined T. gondii strains. Sequence analysis identified 33 variable nucleotide positions (2.1%), 16 of which were identified as transitions. Phylogeny reconstruction based on rop17 gene data revealed two major clusters which could readily distinguish Type I and Type II strains. Analyses of sequence variations in nucleotides and amino acids among these strains revealed high ratio of nonsynonymous to synonymous polymorphisms (>1), indicating that rop17 shows signs of positive selection. This study demonstrated the existence of slightly high sequence variability in the rop17 gene sequences among T. gondii strains from different hosts and geographical regions, suggesting that rop17 gene may represent a new genetic marker for population genetic studies of T. gondii isolates.

PMID:

25126596

[PubMed - in process]

PMCID:

PMC4121216

Activated microglia contribute to neuronal apoptosis in Toxoplasmic Encephalitis

Parasit Vectors. 2014 Aug 15;7(1):372. [Epub ahead of print]

 

Zhang YH, Chen H, Chen Y, Wang L, Cai YH, Li M, Wen HQ, Du J, An R, Luo QL, Wang XL, Lun ZR, Xu YH, Shen JL.

Abstract

BACKGROUND:

A plethora of evidence shows that activated microglia play a critical role in the pathogenesis of the central nervous system (CNS). Toxoplasmic encephalitis (TE) frequently occurs in HIV/AIDS patients. However, knowledge remains limited on the contributions of activated microglia to the pathogenesis of TE.

METHODS:

A murine model of reactivated encephalitis was generated in a latent infection with Toxoplasma gondii induced by cyclophosphamide. The neuronal apoptosis in the CNS and the profile of pro-inflammatory cytokines were assayed in both in vitro and in vivo experiments.

RESULTS:

Microglial cells were found to be activated in the cortex and hippocampus in the brain tissues of mice. The in vivo expression of interleukin-6 (IL-6), interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and inducible nitric oxide synthase (iNOS) were up-regulated in TE mice, and accordingly, the neuronal apoptosis was significantly increased. The results were positively correlated with those of the in vitro experiments. Additionally,apoptosis of the mouse neuroblastoma type Neuro2a (N2a) remarkably increased when the N2a was co-cultured in transwell with microglial cells and Toxoplasma tachyzoites. Both in vivo and in vitro experiments showed that minocycline (a microglia inhibitor) treatment notably reduced microglial activation and neuronal apoptosis.

CONCLUSIONS:

Activated microglia contribute to neuronal apoptosis in TE and inhibition of microglia activation might represent a novel therapeutic strategy of TE.

PMID:

25128410

[PubMed - as supplied by publisher]

The potential of quinoline derivatives for the treatment of Toxoplasma gondii Infection

Exp Parasitol. 2014 Aug 13. pii: S0014-4894(14)00198-2. doi: 10.1016/j.exppara.2014.08.008. [Epub ahead of print]

 

Kadri D¹, Crater AK¹, Lee H², Raja Solomon V³, Ananvoranich S⁴.

Abstract

Here we reported our investigation, as part of our drug repositioning effort, on anti-Toxoplasma properties of newly synthesized quinoline compounds. A collection of 4-aminoquinoline and 4-piperazinylquinoline analogs have recently been synthesized for use in cancer chemotherapy. Some analogs were able to outperform chloroquine, a quinoline derivative drug which is commonly used in the treatment of malaria and other parasitic infections. Herein 58 compounds containing one or two quinoline rings were examined for their effectiveness as potential anti-Toxoplasma compounds. Of these 58 compounds, 32 were efficient at inhibiting Toxoplasma growth (IC₅₀ < 100 μM). Five compounds with single and simple quinoline rings exhibited similar cLogP values of ∼2 and IC₅₀ values between 5 - 6 μM, with one exception of 8-hydroxyquinoline whose IC₅₀ value was 213 nM. The addition of one hydroxyl group at position 8 caused a 40-fold increase in the inhibitory effect of quinoline. A significant improvement in anti-Toxoplasma effect among quinoline derivatives was detected in B11, B12, B23, and B24, whose structures carry two quinoline rings, and their resultant cLogP values are ⩾7. Among these compounds, B23 was the most effective compound with IC₅₀ value of 425 ± 35 nM, and TI value of 4.9. It was also noted that compounds with at least one quinoline ring, displaying anti-Toxoplasma effects were capable of causing the disappearance of the apicoplast, a plastid-like organelle. When treated with quinoline, 8-hydroxyquinoline or B23, 40 - 45% of the parasites lost their apicoplasts. Our findings recapitulate the properties of quinoline derivatives in diminishing apicoplast. This could aid further investigations of anti-parasitic treatments specific to Apicomplexan. More importantly, B12 and B23 which harbor superior anti-cancer properties than chloroquine, have effective anti-Toxoplasma activity. These compounds therefore have significant potential for future development of chemotherapeutic agents for patients suffering from breast cancers and parasitic infection.
Copyright © 2014. Published by Elsevier Inc.

KEYWORDS:

Apicoplast; Quinoline derivatives; Toxoplasma gondii

PMID:

25128801

[PubMed - as supplied by publisher]

The Toxoplasma pseudokinase ROP5 is an allosteric inhibitor of the immunity-related GTPases

J Biol Chem. 2014 Aug 12. pii: jbc.M114.567057. [Epub ahead of print]

Reese ML1, Shah N2, Boothroyd JC3.

Author information

Abstract

The Red Queen hypothesis proposes that there is an evolutionary arms race between host and pathogen. One possible example of such a phenomenon could be the recently discovered interaction between host defense proteins known as immunity-related GTPases (IRGs) and a family of rhoptry pseudokinases (ROP5) expressed by the protozoan parasite, Toxoplasma gondii. Mouse IRGs are encoded by an extensive and rapidly evolving family of over 20 genes. Similarly, the ROP5 family is highly polymorphic and consists of 4-10 genes, depending on the strain of Toxoplasma. IRGs are known to be avidly bound and functionally inactivated by ROP5 proteins but the molecular basis of this interaction/inactivation has not previously been known. Here we show that ROP5 uses a highly polymorphic surface to bind adjacent to the nucleotide-binding domain of an IRG and that this produces a profound allosteric change in the IRG structure. This has two dramatic effects: 1) it prevents oligomerization of the IRG and 2) it alters the orientation of two threonine residues that are targeted by the Toxoplasma Ser/Thr kinases, ROP17 and ROP18. ROP5s are highly specific in the IRGs they will bind and the fact that it is the most highly polymorphic surface of ROP5 that binds the IRG strongly supports the notion that these two protein families are co-evolving in a way predicted by the Red Queen hypothesis.

Copyright © 2014, The American Society for Biochemistry and Molecular Biology.

KEYWORDS:

GTPase; crystal structure; innate immunity; parasitology; protein kinase

PMID:

 

25118287

 

[PubMed - as supplied by publisher] 

Toxoplasma gondii and the host cells

Ann Parasitol. 2014;60(2):83-8.

Długońska H.

Abstract

The protozoan Toxoplasma gondii, described by Nicolle and Manceaux in 1908, is a ubiquitous and cosmopolitan parasite that infects a wide range of mammal and bird species with high prevalence. The biological success of T. gondii is associated with the formation of a specific relationship between the parasite and host cells leading to the establishment of a latent, chronic infection. During primary infection, acquired mostly by the oral route, the quickly multiplying tachyzoites disseminate through the body crossing several structural-functional barriers as blood-brain or blood-retina, then they transform into dormant bradyzoites which, enclosed in tissue cysts, occupy preferentially the brain, skeletal muscle and eye. Although T. gondii is able to infect all kinds of nucleated cells, it uses strictly defined host cells, dependent on the life-cycle phase and infection stage. The article discusses selected aspects of the parasite passing via the host body barriers as well as particular role of dendritic cells and skeletal muscle cells, used by the parasite as an very effective vehicle to disseminate throughout the host body or the site of long-term T. gondii persistence, respectively.

PMID:

 

25115058

 

[PubMed - in process]

CCR5 Controls Immune and Metabolic Functions during Toxoplasma gondii Infection

PLoS One. 2014 Aug 13;9(8):e104736. doi: 10.1371/journal.pone.0104736. eCollection 2014.

Bonfá G1, Benevides L1, Souza Mdo C1, Fonseca DM1, Mineo TW2, Rossi MA3, Silva NM2, Silva JS1, de Barros Cardoso CR4.

Author information

Abstract

CCR5, an important receptor related to cell recruitment and inflammation, is expressed during experimental Toxoplasma gondii infection. However, its role in the immunopathology of toxoplasmosis is not clearly defined yet. Thus, we inoculated WT and CCR5-/- mice with a sub lethal dose of the parasite by oral route. CCR5-/- mice were extremely susceptible to infection, presenting higher parasite load and lower tissue expression of IL-12p40, IFN-γ, TNF, IL-6, iNOS, Foxp3, T-bet, GATA-3 and PPARα. Although both groups presented inflammation in the liver with prominent neutrophil infiltration, CCR5-/- mice had extensive tissue damage with hepatocyte vacuolization, steatosis, elevated serum triglycerides and transaminases. PPARα agonist Gemfibrozil improved the vacuolization but did not rescue CCR5-/- infected mice from high serum triglycerides levels and enhanced mortality. We also found intense inflammation in the ileum of CCR5-/- infected mice, with epithelial ulceration, augmented CD4 and decreased frequency of NK cells in the gut lamina propria. Most interestingly, these findings were accompanied by an outstanding accumulation of neutrophils in the ileum, which seemed to be involved in the gut immunopathology, once the depletion of these cells was accompanied by reduced local damage. Altogether, these data demonstrated that CCR5 is essential to the control of T. gondii infection and to maintain the metabolic, hepatic and intestinal integrity. These findings add novel information on the disease pathogenesis and may be relevant for directing future approaches to the treatment of multi-deregulated diseases.

PMID:

 

25119429

 

[PubMed - in process] 

Structure of Toxoplasma gondii coronin, an actin-binding protein that relocalizes to the posterior pole of invasive parasites and contributes to invasion and egress

See comment in PubMed Commons below

 

FASEB J. 2014 Aug 11. pii: fj.14-252569. [Epub ahead of print]

 

Salamun J¹, Kallio JP², Daher W¹, Soldati-Favre D³, Kursula I⁴.

 

Coronins are involved in the regulation of actin dynamics in a multifaceted way, participating in cell migration and vesicular trafficking. Apicomplexan parasites, which exhibit an actin-dependent gliding motility that is essential for traversal through tissues, as well as invasion of and egress from host cells, express only a single coronin, whereas higher eukaryotes possess several isoforms. We set out to characterize the 3-D structure, biochemical function, subcellular localization, and genetic ablation of Toxoplasma gondii coronin (TgCOR), to shed light on its biological role. A combination of X-ray crystallography, small-angle scattering of X-rays, and light scattering revealed the atomic structure of the conserved WD40 domain and the dimeric arrangement of the full-length protein. TgCOR binds to F-actin and increases the rate and extent of actin polymerization. In vivo, TgCOR relocalizes transiently to the posterior pole of motile and invading parasites, independent of actin dynamics, but concomitant to microneme secretory organelle discharge. TgCOR contributes to, but is not essential for, invasion and egress. Taken together, our data point toward a role for TgCOR in stabilizing newly formed, short filaments and F-actin cross-linking, as well as functions linked to endocytosis and recycling of membranes.-Salamun, J., Kallio, J. P., Daher, W., Soldati-Favre, D., Kursula, I. Structure of Toxoplasma gondii coronin, an actin-binding protein that relocalizes to the posterior pole of invasive parasites and contributes to invasion and egress.
© FASEB.

KEYWORDS:

WD40 domain; apicomplexan parasite; gliding motility

PMID:

25114175

[PubMed - as supplied by publisher]

ATG8 localization in apicomplexan parasites: Apicoplast and more?

Autophagy. 2014 Aug 4;10(9). [Epub ahead of print]

Mizushima N, Sahani MH.

Author information

Abstract

The ATG genes are highly conserved in eukaryotes including yeasts, plants, and mammals. However, these genes appear to be only partially present in most protists. Recent studies demonstrated that, in the apicomplexan parasites Plasmodium (malaria parasites) and Toxoplasma, ATG8 localizes to the apicoplast, a unique nonphotosynthetic plastid with 4 limiting membranes. In contrast to this established localization, it remains unclear whether these parasites can induce canonical macroautophagy and if ATG8 localizes to autophagosomes. Furthermore, the molecular function of ATG8 in its novel workplace, the apicoplast, is totally unknown. Here, we review recent studies on ATG8 in Plasmodium and Toxoplasma, summarize both consensus and controversial findings, and discuss its potential role in these parasites.

KEYWORDS:

ATG8; apicoplast; autophagy; plasmodium; toxoplasma

PMID:

 

25102412

 

[PubMed - as supplied by publisher]

A conserved apicomplexan microneme protein contributes to T. gondii invasion and virulence

Infect Immun. 2014 Aug 4. pii: IAI.01877-14. [Epub ahead of print]

Huynh MH1, Boulanger MJ2, Carruthers VB3.

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Abstract

The obligate intracellular parasite Toxoplasma gondii critically relies on host cell invasion during infection. Proteins secreted from the apical micronemes are central components for host cell recognition, invasion, egress and virulence. Although previous work established that SPATR (Sporozoite Protein with an Altered Thrombospondin Repeat) is a micronemal protein conserved in other apicomplexan parasites including Plasmodium, Neospora and Eimeria, no genetic evidence of its contribution to invasion has been reported. SPATR contains a predicted epidermal growth factor domain and two thrombospondin type 1 repeats, implying a role in host cell recognition. In this study we assess the contribution of TgSPATR to T. gondii invasion by genetically ablating it and restoring its expression by genetic complementation. Δspatr parasites were ∼50% reduced in invasion when compared to parental strains, a defect that was reversed in the complemented strain. In mouse virulence assays, Δspatr parasites were significantly attenuated, with ∼20% of mice surviving infection. Given the conservation of this protein among the Apicomplexa, we assessed whether the P. falciparum ortholog could complement the absence of the TgSPATR. Although PfSPATR showed correct micronemal localization, it did not reverse the invasion deficiency of Δspatr parasites because of an apparent failure in secretion. Overall the results suggest that TgSPATR contributes to invasion and virulence, findings that have implications for the many genera and life stages of apicomplexans that express SPATR.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

PMID:

 

25092910

 

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Mast cells are crucial in the resistance against Toxoplasma gondii oral infection

Eur J Immunol. 2014 Aug 4. doi: 10.1002/eji.201344185. [Epub ahead of print]

Cruz A1, Mendes EA, de Andrade MV, do Nascimento VC, Cartelle CT, Arantes RM, da Cunha Melo JR, Gazzinelli RT, Ropert C.

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Abstract

During oral infection, mucosal immunity assumes a predominant role. Here, we addressed the role of mast cells (MCs), which are mainly located in mucosa during oral infection with Toxoplasma gondii, using MC-deficient (W/Wv ) mice. We show that in the absence of MCs the resistance of W/Wvmice to oral infection was considerably reduced. W/Wv mice uniformly succumbed within 15 days of infection after administration of cysts of the ME49 strain of T. gondii. The rapid lethality of T. gondii in W/Wv mice correlated with a delayed Th1-cell response, since IFN-γ and IL-12 levels peaked in the later phase of the infection. In vitro, bone marrow-derived MCs (BMMCs) were able to recognize parasite lysate in a MyD88-dependent way, reaffirming the role of this TLR adapter in immune responses to T. gondii. The importance of MCs in vivo was confirmed when W/Wv mice reconstituted with BMMCs derived from control mice retrieved an early strong Th1-cell response and specially a significant IL-12 production. In conclusion, mast cells play an important role for the development of a protective immune response during oral infection with T. gondii. This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

KEYWORDS:

Mast cells; Th1-cell response; Toxoplasma gondii; mucosal immunity; oral infection

PMID:

 

25091816

 

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A RGG motif protein is involved in Toxoplasma gondii stress-mediated response

Mol Biochem Parasitol. 2014 Jul 31. pii: S0166-6851(14)00087-5. doi: 10.1016/j.molbiopara.2014.07.009. [Epub ahead of print]

Boulila Y1, Tomavo S1, Gissot M2.

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Abstract

The molecular mechanisms controlling gene expression are still poorly understood in apicomplexan parasites. Here, we report the characterisation of a homologue of the single strand binding proteins (named TgSsossB) in Toxoplasma gondii. We previously showed that TgSsossB interacts with the TgAlba proteins that are involved in translation regulation. We examined the role of TgSsossB in stress-mediated response, and particularly the role of its Arginine-Glycine-Glycine (RGG) repeats domain. TgSsossB recombinant protein is able to bind to single strand DNA and RNA in a sequence-independent manner, but not to double stranded DNA. We showed that the RGG motif is not involved in this ability to bind to nucleic acid. We produced a mutant tagged strain lacking the RGG motif of TgSsossB using the knock-in strategy. We observed that this strain exhibited a fitness defect compared with the parental parasites. Moreover, the mutant strain produced fewer plaques in stress conditions, a defect that is due to a slow growth phenotype when extracellular parasites are exposed to stress. At the molecular level, we showed that the TgSsossB protein lacking an RGG motif lost its ability to interact with TgAlba2 and an isoform of TgAlba1, indicating that the TgAlba complex is likely non-functional in those parasites. Thus, our findings define the RGG domain of TgSsossB as a protein-protein interaction platform and underline the role of the TgAlba-TgSsossB complex in stress-mediated response.

Copyright © 2014. Published by Elsevier B.V.

KEYWORDS:

Gene expression; RGG motif; Stress; Toxoplasma; Translation

PMID:

 

25089036

 

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Identification and characterization of Toxoplasma SIP, a conserved apicomplexan cytoskeleton protein involved in maintaining the shape, motility and virulence of the parasite

Cell Microbiol. 2014 Aug 3. doi: 10.1111/cmi.12337. [Epub ahead of print]

Lentini G1, Kong-Hap M, El Hajj H, Francia M, Claudet C, Striepen B, Dubremetz JF, Lebrun M.

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Abstract

Apicomplexa possess a complex pellicle that is composed of a plasma membrane and a closely apposed inner membrane complex (IMC) that serves as a support for the actin-myosin motor required for motility and host cell invasion. The IMC consists of longitudinal plates of flattened vesicles, fused together and lined on the cytoplasmic side by a subpellicular network of intermediate filament-like proteins. The spatial organization of the IMC has been well described by electron microscopy, but its composition and molecular organization is largely unknown. Here, we identify a novel protein of the IMC cytoskeletal network in Toxoplasma gondii, called TgSIP, and conserved among apicomplexan parasites. To finely pinpoint the localization of TgSIP, we used structured illumination super resolution microscopy and revealed that it likely decorates the transverse sutures of the plates and the basal end of the IMC. This suggests that TgSIP might contribute to the organization or physical connection among the different components of the IMC. We generated a T. gondii SIP deletion mutant and showed that parasites lacking TgSIP are significantly shorter than wild-type parasites and show defects in gliding motility, invasion and reduced infectivity in mice.

This article is protected by copyright. All rights reserved.

KEYWORDS:

Apicomplexa; Plasmodium; Toxoplasma gondii; inner membrane complex; motility

PMID:

 

25088010

 

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Auranofin Is Highly Efficacious against Toxoplasma gondii In Vitro and in an In Vivo Experimental Model of Acute Toxoplasmosis

PLoS Negl Trop Dis. 2014 Jul 31;8(7):e2973. doi: 10.1371/journal.pntd.0002973. eCollection 2014.

Andrade RM1, Chaparro JD2, Capparelli E2, Reed SL3.

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Abstract

BACKGROUND:

The mainstay of toxoplasmosis treatment targets the folate biosynthetic pathways and has not changed for the last 50 years. The activity of these chemotherapeutic agents is restricted to one lifecycle stage of Toxoplasma gondii, they have significant toxicity, and the impending threat of emerging resistance to these agents makes the discovery of new therapies a priority. We now demonstrate that auranofin, an orally administered gold containing compound that was FDA approved for treatment of rheumatoid arthritis, has activity against Toxoplasma gondii in vitro (IC50 = 0.28 µM) and in vivo (1 mg/kg).

METHODS/PRINCIPAL FINDINGS:

Replication within human foreskin fibroblasts of RH tachyzoites was inhibited by auranofin. At 0.4 µM, auranofin inhibited replication, as measured by percent infected fibroblasts at 24 hrs, (10.94% vs. 24.66% of controls; p = 0.0003) with no effect on parasite invasion (16.95% vs. 12.91% p = 0.4331). After 18 hrs, 62% of extracellular parasites treated with auranofin were non-viable compared to control using an ATP viability assay (p = 0.0003). In vivo, a previously standardized chicken embryo model of acute toxoplasmosis was used. Fourteen day old chicken embryos were injected through the chorioallantoic vein with 1×104 tachyzoites of the virulent RH strain. The treatment group received one dose of auranofin at the time of inoculation (1 mg/kg estimated body weight). On day 5, auranofin-treated chicken embryos were 100% protected against death (p = 0.0002) and had a significantly reduced parasite load as determined by histopathology, immunohistochemistry and by the number of parasites quantified by real-time PCR.

CONCLUSIONS:

These results reveal in vitro and in vivo activity of auranofin against T. gondii, suggesting that it may be an effective alternative treatment for toxoplasmosis.

PMID:

 

25079790

 

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