Wednesday, July 22, 2015

The Orphan Nuclear Receptor TLX Is an Enhancer of STAT1-Mediated Transcription and Immunity to Toxoplasma gondii

2015 Jul 21;13(7):e1002200. doi: 10.1371/journal.pbio.1002200. eCollection 2015.
 
 
The protozoan parasite, Toxoplasma, like many intracellular pathogens, suppresses interferon gamma (IFN-γ)-induced signal transducer and activator of transcription 1 (STAT1) activity. We exploited this well-defined host-pathogen interaction as the basis for a high-throughput screen, identifying nine transcription factors that enhance STAT1 function in the nucleus, including the orphan nuclear hormone receptor TLX. Expression profiling revealed that upon IFN-γ treatment TLX enhances the output of a subset of IFN-γ target genes, which we found is dependent on TLX binding at those loci. Moreover, infection of TLX deficient mice with the intracellular parasite Toxoplasma results in impaired production of the STAT1-dependent cytokine interleukin-12 by dendritic cells and increased parasite burden in the brain during chronic infection. These results demonstrate a previously unrecognized role for this orphan nuclear hormone receptor in regulating STAT1 signaling and host defense and reveal that STAT1 activity can be modulated in a context-specific manner by such "modifiers."
PMID:
26196739
[PubMed - as supplied by publisher]

Cancer in the parasitic protozoans Trypanosoma brucei and Toxoplasma gondii

2015 Jul 20. pii: 201502599. [Epub ahead of print]
 
 
Cancer is a general name for more than 100 malignant diseases. It is postulated that all cancers start from a single abnormal cell that grows out of control. Untreated cancers can cause serious consequences and deaths. Great progress has been made in cancer research that has significantly improved our knowledge and understanding of the nature and mechanisms of the disease, but the origins of cancer are far from being well understood due to the limitations of suitable model systems and to the complexities of the disease. In view of the fact that cancers are found in various species of vertebrates and other metazoa, here, we suggest that cancer also occurs in parasitic protozoans such as Trypanosoma brucei, a blood parasite, and Toxoplasma gondii, an obligate intracellular pathogen. Without treatment, these protozoan cancers may cause severe disease and death in mammals, including humans. The simpler genomes of these single-cell organisms, in combination with their complex life cycles and fascinating life cycle differentiation processes, may help us to better understand the origins of cancers and, in particular, leukemias.

KEYWORDS:

evolution; malignancy; mammals; single-cell organisms; transmissible
PMID:
26195778
[PubMed - as supplied by publisher]

Alternative splicing mechanisms orchestrating post-transcriptional gene expression: intron retention and the intron-rich genome of apicomplexan parasites

2015 Jul 21. [Epub ahead of print]
 
 
Apicomplexan parasites including Toxoplasma gondii and Plasmodium species have complex life cycles that include multiple hosts and differentiation through several morphologically distinct stages requiring marked changes in gene expression. This review highlights emerging evidence implicating regulation of mRNA splicing as a mechanism to prime these parasites for rapid gene expression upon differentiation. We summarize the most important insights in alternative splicing including its role in regulating gene expression by decreasing mRNA abundance via 'Regulated Unproductive Splicing and Translation'. As a related but less well-understood mechanism, we discuss also our recent work suggesting a role for intron retention for precluding translation of stage specific isoforms of T. gondii glycolytic enzymes. We additionally provide new evidence that intron retention might be a widespread mechanism during parasite differentiation. Supporting this notion, recent genome-wide analysis of Toxoplasma and Plasmodium suggests intron retention is more pervasive than heretofore thought. These findings parallel recent emergence of intron retention being more prevalent in mammals than previously believed, thereby adding to the established roles in plants, fungi and unicellular eukaryotes. Deeper mechanistic studies of intron retention will provide important insight into its role in regulating gene expression in apicomplexan parasites and more general in eukaryotic organisms.
PMID:
26194054
[PubMed - as supplied by publisher]

Parasite manipulation of the invariant chain (Ii/CD74) and the peptide editor H2-DM affects MHC-II antigen presentation during Toxoplasma gondii infection

2015 Jul 20. pii: IAI.00415-15. [Epub ahead of print]
 
 
Toxoplasma gondii is an obligate intracellular protozoan parasite. This apicomplexan is the causative agent of toxoplasmosis, a leading cause of central nervous system disease in AIDS. It has long been known that T. gondii interferes with major histocompatibility complex (MHC)-II antigen presentation to attenuate CD4+ T cell responses and establish persisting infections. Transcriptional down-regulation of MHC-II genes by T. gondii was previously established, but the precise mechanisms inhibiting MHC-II function are currently unknown. Here, we show that, in addition to transcriptional regulation of MHC-II, the parasite modulates the expression of key components of the MHC-II antigen presentation pathway, namely the MHC-II associated invariant chain (Ii or CD74) and the peptide editor H2-DM in professional antigen presenting cells (pAPCs). Genetic deletion of CD74 restored the ability of infected dendritic cells to present a parasite antigen in the context of MHC-II in vitro. CD74 mRNA and protein levels were surprisingly elevated in infected cells, whereas MHC-II and H2-DM expression was inhibited. CD74 accumulated mainly in the endoplasmic reticulum (ER), and this phenotype required live parasites, but not active replication. Finally, we compared the impact of genetic deletion of CD74 and H2-DM genes on parasite dissemination towards lymphoid organs in mice, as well as activation of CD4+ T cells, and interferon gamma (IFNγ) levels during acute infection. Cyst burdens and survival during the chronic phase of infection were also evaluated in wild-type and knockout mice. These results highlight that the infection is influenced by multiple levels of parasite manipulation of the MHC-II antigen presentation pathway.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
PMID:
26195549
[PubMed - as supplied by publisher]

Research advances in microneme protein 3 of Toxoplasma gondii

2015 Jul 22;8(1):384.
 
 
Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite. It has extensive host populations and is prevalent globally; T. gondii infection can cause a zoonotic parasitic disease. Microneme protein 3 (MIC3) is a secreted protein that is expressed in all stages of the T. gondii life cycle. It has strong immunoreactivity and plays an important role in the recognition, adhesion and invasion of host cells by T. gondii. This article reviews the molecular structure of MIC3, its role in the invasion of host cells by parasites, its relationship with parasite virulence, and its induction of immune protection to lay a solid foundation for an in-depth study of potential diagnostic agents and vaccines for preventing toxoplasmosis.
PMID:
26194005
[PubMed - as supplied by publisher]

Pyrimidinergic Receptor Activation Controls Toxoplasma gondii Infection in Macrophages

2015 Jul 20;10(7):e0133502. doi: 10.1371/journal.pone.0133502.
 
 
Infection by the protozoan parasite Toxoplasma gondii is highly prevalent worldwide and may have serious clinical manifestations in immunocompromised patients. T. gondii is an obligate intracellular parasite that infects almost any cell type in mammalian hosts, including immune cells. The immune cells express purinergic P2 receptors in their membrane - subdivided into P2Y and P2X subfamilies - whose activation is important for infection control. Here, we examined the effect of treatment with UTP and UDP in mouse peritoneal macrophages infected with T. gondii tachyzoites. Treatment with these nucleotides reduced parasitic load by 90%, but did not increase the levels of the inflammatory mediators NO and ROS, nor did it modulate host cell death by apoptosis or necrosis. On the other hand, UTP and UDP treatments induced early egress of tachyzoites from infected macrophages, in a Ca2+-dependent manner, as shown by scanning electron microscopy analysis, and videomicroscopy. In subsequent infections, prematurely egressed parasites had reduced infectivity, and could neither replicate nor inhibit the fusion of lysosomes to the parasitophorous vacuole. The use of selective agonists and antagonists of the receptor subtypes P2Y2 and P2Y4 and P2Y6 showed that premature parasite egress may be mediated by the activation of these receptor subtypes. Our results suggest that the activity of P2Y host cell receptors controls T. gondii infection in macrophages, highlighting the importance of pyrimidinergic signaling for innate immune system response against infection. Finally the P2Y receptors should be considered as new target for the development of drugs against T. gondii infection.
PMID:
26192447
[PubMed - as supplied by publisher]

Monday, July 20, 2015

Elucidating pathways of Toxoplasma gondii invasion in the gastrointestinal tract: Involvement of the tight junction protein occludin

2015 Jul 13. pii: S1286-4579(15)00142-2. doi: 10.1016/j.micinf.2015.07.001. [Epub ahead of print]
 
 
Toxoplasma gondii is an obligate intracellular parasite infecting one third of the world's population. The small intestine is the parasite's primary route of infection, although the pathway of epithelium transmigration remains unclear. Using an in vitro invasion assay and live imaging we showed that T. gondii (RH) tachyzoites infect and transmigrate between adjacent intestinal epithelial cells in polarized monolayers without altering barrier integrity, despite eliciting the production of specific inflammatory mediators and chemokines. During invasion, T. gondii co-localized with occludin. Reducing the levels of endogenous cellular occludin with specific small interfering RNAs significantly reduced the ability of T. gondii to penetrate between and infect epithelial cells. Furthermore, an in vitroinvasion and binding assays using recombinant occludin fragments established the capacity of the parasite to bind occludin and in particular to the extracellular loops of the protein. These findings provide evidence for occludin playing a role in the invasion of T. gondii in small intestinal epithelial cells.
Copyright © 2015. Published by Elsevier Masson SAS.

KEYWORDS:

Toxoplasma gondii; intestinal epithelial cells; invasion; occludin
PMID:
26183539
[PubMed - as supplied by publisher]

Tuesday, July 14, 2015

Disassembly activities of actin depolymerization factor (ADF) is associated with distinct cellular processes in apicomplexan parasites

2015 Jul 8. pii: mbc.E14-10-1427. [Epub ahead of print]
 
 
Proteins of the actin depolymerizing factor (ADF)/cofilin family have been shown to be crucial for the motility and survival of apicomplexan parasites. However, the mechanisms by which ADF proteins fulfill their function remains poorly understood. In this study we sought to investigate the comparative activities of ADF proteins from Toxoplasma gondii and Plasmodium falciparum, the human malaria parasite, using a conditional T. gondii ADF-knockout line complemented with ADF variants from either species. We show that P. falciparum ADF1 can fully restore native TgADF activity, demonstrating functional conservation between parasites. Strikingly, mutation of a key basic residue (Lys72), previously implicated in disassembly in PfADF1, had no detectable phenotypic effect on parasite growth, motility or development. In contrast, organelle segregation was severely impaired when complementing with a TgADF mutant lacking the corresponding residue (Lys68). Biochemical analyses of each ADF protein confirmed the reduced ability of lysine mutants to mediate actin depolymerization via filament disassembly though not severing, in contrast to previous reports. These data suggest that actin filament disassembly is essential for apicomplexan parasite development but not for motility as well as pointing to genus-specific coevolution between ADF proteins and their native actin.
© 2015 by The American Society for Cell Biology.
PMID:
26157165
[PubMed - as supplied by publisher]

Identification of Toxoplasma gondii protein fractions induce immune response against melanoma in mice

2015 Jul 8. doi: 10.1111/apm.12420. [Epub ahead of print]
 
 
Dendritic cells (DCs) play a crucial role in the initiation of adaptive immune responses against tumor cells. We recently found that protein components of Toxoplasma gondii (T. gondii) could mature DCs efficiently. Therefore, in this study, we aimed to find the most effective protein components of T. gondii which are able to mature DCs and consequently instruct immune responses in tumor-bearing mice. Soluble tachyzoite antigens (STAgs) were fractionated by ammonium sulfate precipitation and subsequently by anion-exchange HPLC. Immature DCs (iDCs) were treated by these protein fractions and were monitored for IL-12p70 and IL-10 production. Moreover, the capacity of mature DCs (mDCs) to induce lymphocyte proliferation was investigated. Ultimately, we analyzed the ability of mDCs in instructing immune responses in tumor-bearing mice. We found that ammonium sulfate fraction one (A1) matured-DCs produced higher IL-12 level and IL-12/IL-10 ratio; therefore, this fraction was selected for further fractionation by anion-exchange HPLC. The results showed that anion-exchange HPLC fraction 14 (C14) matured-DCs secrete higher levels of IL-12p70 and IL-12p70/IL-10 ratio. Survival of the mice matured by A1 fraction increased significantly compared to other groups. Moreover, SDS-PAGE electrophoresis showed that different obtained fractions have distinct proteins based on their size. These results demonstrate that two protein fractions of T. gondii are able to mature DCs more efficient.
© 2015 APMIS. Published by John Wiley & Sons Ltd.

KEYWORDS:

Toxoplasma gondii ; Dendritic cells; immune response; tumor
PMID:
26152792
[PubMed - as supplied by publisher]

Integration of RNA-seq and proteomics data with genomics for improved genome annotation in Apicomplexan Parasites

2015 Jul 7. doi: 10.1002/pmic.201500253. [Epub ahead of print]
 
 
While high quality genomic sequence data is available for many pathogenic organisms, the corresponding gene annotations are often plagued with inaccuracies that can hinder research that utilizes such genomic data. Experimental validation of gene models is clearly crucial in improving such gene annotations; the field of proteogenomics is an emerging area of research wherein proteomic data is applied to testing and improving genetic models. Krishna et al [Proteomics 2015, 00, 1-11] investigated whether incorporation of RNA-seq data into proteogenomics analyses can contribute significantly to validation studies of genome annotation, in two important parasitic organisms Toxoplasma gondii and Neospora caninum. They applied a systematic approach to combine new and previously published proteomics data from T. gondii and N. caninum with transcriptomics data, leading to substantially improved gene models for these organisms. This study illustrates the importance of incorporating experimental data from both proteomics and RNA-seq studies into routine genome annotation protocols. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.

KEYWORDS:

Apicomplexa; Neospora; Proteogenomics; Proteomics; Protozoa; Toxoplasma
PMID:
26152714
[PubMed - as supplied by publisher]

Friday, July 03, 2015

toxoMine: an integrated omics data warehouse for Toxoplasma gondii systems biology research

 2015 Jun 30;2015. pii: bav066. doi: 10.1093/database/bav066. Print 2015.

Abstract

Toxoplasma gondii (T. gondii) is an obligate intracellular parasite that must monitor for changes in the host environment and respond accordingly; however, it is still not fully known which genetic or epigenetic factors are involved in regulating virulence traits of T. gondii. There are on-going efforts to elucidate the mechanisms regulating the stage transition process via the application of high-throughput epigenomics, genomics and proteomics techniques. Given the range of experimental conditions and the typical yield from such high-throughput techniques, a new challenge arises: how to effectively collect, organize and disseminate the generated data for subsequent data analysis. Here, we describe toxoMine, which provides a powerful interface to support sophisticated integrative exploration of high-throughput experimental data and metadata, providing researchers with a more tractable means toward understanding how genetic and/or epigenetic factors play a coordinated role in determining pathogenicity of T. gondii. As a data warehouse, toxoMine allows integration of high-throughput data sets with public T. gondii data. toxoMine is also able to execute complex queries involving multiple data sets with straightforward user interaction. Furthermore, toxoMine allows users to define their own parameters during the search process that gives users near-limitless search and query capabilities. The interoperability feature also allows users to query and examine data available in other InterMine systems, which would effectively augment the search scope beyond what is available to toxoMine. toxoMine complements the major community database ToxoDB by providing a data warehouse that enables more extensive integrative studies for T. gondii. Given all these factors, we believe it will become an indispensable resource to the greater infectious disease research community.Database URL: http://toxomine.org. 
© The Author(s) 2015. Published by Oxford University Press.
PMID:
 
26130662
 
[PubMed - in process] 

Wednesday, July 01, 2015

Role of CD4+Foxp3+ regulatory T cells in protection induced by a live, attenuated, replicating Type-I vaccine strain of Toxoplasma

2015 Jun 29. pii: IAI.00217-15. [Epub ahead of print]
 
 
Vaccination with the live attenuated Toxoplasma gondii Mic1.3KO strain induced long-lasting immunity against challenge with Toxoplasma gondii Type I and Type II strains. The involvement of T regulatory (Treg) cells in the protection mechanism was investigated. Intraperitoneal injection of Mic1.3KO induced a weak and transient influx of CD4+Foxp3+ T regulatory cells followed by recruitment/expansion of CD4+Foxp3-CD25+ effector cells and control of the parasite at the site of infection. The local and systemic cytokine responses associated with this recruitment of Treg were of TH1/Treg-like type. In contrast, injection of RH, the wild-type strain from which the vaccinal strain is derived, induced a low CD4+Foxp3+ cell influx and uncontrolled multiplication of the parasites at this local site, followed by death of the mice. The associated local and systemic cytokine responses were of TH1/TH17-like type.In addition, in vivo Treg induction in the RH-infected mice with IL2/anti-IL2 complexes induced control of the parasite and a TH1/Treg cytokine response similar to the response after Mic1.3KO vaccination. These results suggest that Treg cells may contribute to the protective response after vaccination with Mic1.3KO.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
PMID:
26123802
[PubMed - as supplied by publisher]

Tuesday, June 30, 2015

Nitric Oxide Production Increases During Toxoplasma gondii Encephalitis in Mice

2015 Jun 23. pii: S0014-4894(15)00169-1. doi: 10.1016/j.exppara.2015.06.009. [Epub ahead of print]
 

Abstract

Toxoplasma gondii is a intracellular parasite with the potential of causing severe encephalitis among immunocompromised human and animals. The aim of this experimental study was to investigate the immunomodulatory and immunopathological role of nitric oxide (NO) in central nervous systems and to identify any correlation between toxoplasmosis neuropathology and investigate the consequences of the cellular responses protect against Toxoplasma gondii. Mice were infected with ME49 strain Toxoplasma gondii and levels of endothelial, neuronal and inducible nitric oxide synthase (eNOS, nNOS, iNOS), glial fibrillary acidic protein (GFAP) and neurofilament (NF) were examined in brain tissues by immunohistochemistry, during the development and establishment of a chronic infection at 10 30 and 60 days post infection.

KEYWORDS:

Toxoplasma gondii; glial fibrillary acidic protein; neurofilament; neuropathology; nitric oxide
PMID:
26115941
[PubMed - as supplied by publisher]

Parasite Calcineurin Regulates Host Cell Recognition and Attachment by Apicomplexans

2015 Jun 24. pii: S1931-3128(15)00251-6. doi: 10.1016/j.chom.2015.06.003. [Epub ahead of print]
 
Apicomplexans invade a variety of metazoan host cells through mechanisms involving host cell receptor engagement and secretion of parasite factors to facilitate cellular attachment. We find that the parasite homolog of calcineurin, a calcium-regulated phosphatase complex central to signal transduction in eukaryotes, also contributes to host cell invasion by the malaria parasite Plasmodium falciparum and related Toxoplasma gondii. Using reverse-genetic and chemical-genetic approaches, we determine that calcineurin critically regulates and stabilizes attachment of extracellular P. falciparum to host erythrocytes before intracellular entry and has similar functions in host cell engagement by T. gondii. Calcineurin-mediated Plasmodium invasion is strongly associated with host receptors required for host cell recognition, and calcineurin function distinguishes this form of receptor-mediated attachment from a second mode of host-parasite adhesion independent of host receptors. This specific role of calcineurin in coordinating physical interactions with host cells highlights an ancestral mechanism for parasitism used by apicomplexans.
Copyright © 2015 Elsevier Inc. All rights reserved.
PMID:
26118996
[PubMed - as supplied by publisher]

Sunday, June 28, 2015

Comparative Analysis of Apicoplast-Targeted Protein Extension Lengths in Apicomplexan Parasites

 2015;2015:452958. doi: 10.1155/2015/452958. Epub 2015 May 31.

Abstract

In general, the mechanism of protein translocation through the apicoplast membrane requires a specific extension of a functionally important region of the apicoplast-targeted proteins. The corresponding signal peptides were detected in many apicomplexans but not in the majority of apicoplast-targeted proteins in Toxoplasma gondii. In T. gondii signal peptides are either much diverged or their extension region is processed, which in either case makes the situation different from other studied apicomplexans. We propose a statistic method to compare extensions of the functionally important regions of apicoplast-targeted proteins. More specifically, we provide a comparison of extension lengths of orthologous apicoplast-targeted proteins in apicomplexan parasites. We focus on results obtained for the model species T. gondii, Neospora caninum, and Plasmodium falciparum. With our method, cross species comparisons demonstrate that, in average, apicoplast-targeted protein extensions in T. gondii are 1.5-fold longer than in N. caninum and 2-fold longer than in P. falciparum. Extensions in P. falciparum less than 87 residues in size are longer than the corresponding extensions in N. caninum and, reversely, are shorter if they exceed 88 residues. 
PMID:
 
26114107
 
[PubMed - in process]

Kinetic mechanism of Toxoplasma gondii adenosine kinase and the highly efficient utilization of adenosine

 2015 Jun 22. pii: S1096-4959(15)00119-0. doi: 10.1016/j.cbpb.2015.06.006. [Epub ahead of print]

Abstract

Toxoplasma gondii has an extraordinarily ability to utilize adenosine (Ado) as the primary source of all necessary purines in this parasite which lacks de novo purine biosynthesis. The activity of T. gondii adenosine kinase (TgAK, EC 2.7.1.20) is responsible for this efficient salvage of Ado in T. gondii. To fully understand this remarkable efficiency of TgAK in the utilization of Ado, complete kinetic parameters of this enzyme are necessary. Initial velocity and product inhibition studies of TgAK demonstrated that the basic mechanism of this enzyme is a hybrid random bi-uni ping-pong uni-bi. Initial velocity studies showed an intersecting pattern, consistent with substrate-enzyme-co-substrate complex formation and a binding pattern indicating that binding of the substrate interferes with the binding of the co-substrate and vice versa. Estimated kinetic parameters were KAdo = 0.002 ■ 0.0002 mM, KATP = 0.05 ■ 0.008 mM, and Vmax = 920 ■ 35 μmol/min/mg protein. Ado exhibited substrate inhibition suggesting the presence of more than one binding site for Ado on the enzyme. ATP relieved substrate inhibition by Ado. Thus, Ado also binds to the ATP binding site. AMP was competitive with ATP, inferring that AMP binds to the same site as ATP. AMP, ADP and ATP were non-competitive with Ado, therefore, none of these nucleotides binds to the Ado binding site. Combining ATP with ADP was additive. Therefore, the binding of either ATP or ADP does not interfere with the binding of the other. It is concluded that for every ATP consumed, TgAK generates three new AMPs. These findings along with the fact that a wide range of nucleoside 5'-mono, di, and triphosphates could substitute for ATP as phosphate donors in this reaction may explain the efficient and central role played by TgAK in the utilization of Ado as the major source from which all other purines can be synthesized in T. gondii.
Copyright © 2015. Published by Elsevier Inc.

KEYWORDS: 

Adenosine kinase; Enzyme kinetics; Parasite; Purine metabolism; Toxoplasma gondii
PMID:
 
26112826
 
[PubMed - as supplied by publisher]

Could miltefosine be used as a therapy for toxoplasmosis?

 2015 Jun 23. pii: S0014-4894(15)00165-4. doi: 10.1016/j.exppara.2015.06.005. [Epub ahead of print]

Abstract

Toxoplasmosis is a zoonotic protozoal disease affecting more than a billion people worldwide. The shortfalls of the current treatment options necessitate the development of non-toxic and well-tolerated, efficient alternatives especially against the cyst form. The current study was undertaken to investigate, for the first time, the potential potency of miltefosine against Toxoplasma gondii infection in acute and chronic experimental toxoplasmosis. Results showed that there is no evidence of anti-parasitic activity of miltefosine against T. gondii tachyzoites in acute experimental toxoplasmosis. However, anti-parasitic activity of miltefosine against T. gondii cyst stage in chronic experimental toxoplasmosis could not be excluded as demonstrated by significant reduction in brain cyst burden. Moreover, considerable morphological changes in the cysts were revealed by light and electron microscopy study and also by amelioration of pathological changes in the brain. Future studies should focus on enhancement of anti-toxoplasma activity of miltefosine against chronic toxoplasmosis using formulation based nanotechnology. To the best of our knowledge, this is the first study highlighting efficacy of miltefosine against chronic toxoplasmosis, thus, increasing the list of diseases that can be targeted by this drug.
Copyright © 2015. Published by Elsevier Inc.

KEYWORDS: 

Chronic toxoplasmosis; Electron microscopy; Me49 strain; Miltefosine; RH strain; Toxoplasma gondii
PMID:
 
26112396
 
[PubMed - as supplied by publisher]