Tuesday, August 23, 2016

Virus-Like Nanoparticle Vaccine Confers Protection against Toxoplasma gondii

2016 Aug 22;11(8):e0161231. doi: 10.1371/journal.pone.0161231.


The inner membrane complex (IMC) of Toxoplasma gondii as a peripheral membrane system has unique and critical roles in parasite replication, motility and invasion. Disruption of IMC sub-compartment protein produces a severe defect in T. gondii endodyogeny, the form of internal cell budding. In this study, we generated T. gondii virus-like particle particles (VLPs) containing proteins derived from IMC, and investigated their efficacy as a vaccine in mice. VLP vaccination induced Toxoplasma gondii-specific total IgG, IgG1 and IgG2a antibody responses in the sera and IgA antibody responses in the feces. Upon challenge infection with a lethal dose of T. gondii (ME49), all vaccinated mice survived, whereas all naïve control mice died. Vaccinated mice showed significantly reduced cyst load and cyst size in the brain. VLP vaccination also induced IgA and IgG antibody responses in feces and intestines, and antibody-secreting plasma cells, mixed Th1/Th2 cytokines and CD4+/CD8+ T cells from spleen. Taken together, these results indicate that non-replicating VLPs containing inner membrane complex of T. gondii represent a promising strategy for the development of a safe and effective vaccine to control the spread of Toxoplasma gondii infection.
[PubMed - as supplied by publisher]

Monday, August 22, 2016

UAP56 is a conserved crucial component of a divergent mRNA export pathway in Toxoplasma gondii

2016 Aug 20. doi: 10.1111/mmi.13485. [Epub ahead of print]


Nucleo-cytoplasmic RNA export is an essential post-transcriptional step to control gene expression in eukaryotic cells and is poorly understood in apicomplexan parasites. With the exception of UAP56, a component of TREX (Transcription Export) complex, other components of mRNA export machinery are not well conserved in divergent supergroups. Here we use Toxoplasma gondii as a model system to functionally characterize TgUAP56 and its potential interaction factors. We demonstrate that TgUAP56 is crucial for mRNA export and that functional interference leads to significant accumulation of mRNA in the nucleus. It was necessary to employ bioinformatics and phylogenetic analysis to identify orthologs related to mRNA export, which show a remarkable low level of conservation in T. gondii. We adapted a conditional Cas9/CRISPR system to carry out a genetic screen to verify if these factors were involved in mRNA export in T. gondii. Only the disruption of TgRRM_1330 caused accumulation of mRNA in the nucleus as found with TgUAP56. This protein is potentially a divergent partner of TgUAP56, and provides insight into a divergent mRNA export pathway in apicomplexans. This article is protected by copyright. All rights reserved.
© 2016 John Wiley & Sons Ltd.

KEYWORDS:

Key-words: mRNA export pathway; Toxoplasma gondii; conditional Cas9 system
[PubMed - as supplied by publisher]

Saturday, August 13, 2016

TOXOPLASMA GONDII INFECTION POTENTIATES COGNITIVE IMPAIRMENTS OF ALZHEIMER'S DISEASE IN THE BALB/C MICE

 2016 Aug 11. [Epub ahead of print]

Abstract

Here, we hypothesized that in chronic Toxoplasma gondii infection communication among immune cells promotes neuroinflammation through cytokine networks and potentiate cognitive impairments in BALB/c mice with Alzheimer's disease (AD). The animal model of Toxoplasma infection was established by the intraperitoneal inoculation of 20-25 tissue cysts from Tehran strain of T. gondii. We injected amyloid-beta 1-42 peptide (A1-42, 1 and 2 µL) into the hippocampus of BALB/c mice to establish an animal model of AD. The behavioral experiments such as spatial learning and memory were performed using Morris water maze test. The mRNA levels of TNF-, IL-1, IFN-, and inducible nitric oxide synthase (iNOS) were examined by real-time PCR. We found that T. gondii infection caused AD-like symptoms and impaired learning and memory functions of the infected BALB/c mice. We also found that in Toxoplasma infection + A1-42 (1 µL) group, T. gondii infection could potentiate AD in infected mice receiving sub-dose of A1-42 (1 µL) and caused considerable impairment in learning and memory functions similar to AD group. Comparison of the results demonstrated that mRNA levels of IL-1, TNF-, IFN-, and also iNOS significantly (P < 0.001) increased in T. gondii + A1-42 (1 µL) in comparison with the other tested groups. The obtained results showed that chronic T. gondii infection communication among immune cells promotes neuroinflammation through cytokine networks and induce pathological progression of AD in the mice brain, whereas, neuroanatomical Toxoplasma tissue cysts presence in the brain could also affect the behavioral functions in T. gondii infected mice.
PMID:
 
27513205
 
DOI:
 
10.1645/16-28
[PubMed - as supplied by publisher]

Wednesday, August 10, 2016

Toxoplasma gondii TgIST co-opts host chromatin repressors dampening STAT1-dependent gene regulation and IFN-γ-mediated host defenses

2016 Aug 8. pii: jem.20160340. [Epub ahead of print]


An early hallmark of Toxoplasma gondii infection is the rapid control of the parasite population by a potent multifaceted innate immune response that engages resident and homing immune cells along with pro- and counter-inflammatory cytokines. In this context, IFN-γ activates a variety of T. gondii-targeting activities in immune and nonimmune cells but can also contribute to host immune pathology. T. gondii has evolved mechanisms to timely counteract the host IFN-γ defenses by interfering with the transcription of IFN-γ-stimulated genes. We now have identified TgIST (T. gondii inhibitor of STAT1 transcriptional activity) as a critical molecular switch that is secreted by intracellular parasites and traffics to the host cell nucleus where it inhibits STAT1-dependent proinflammatory gene expression. We show that TgIST not only sequesters STAT1 on dedicated loci but also promotes shaping of a nonpermissive chromatin through its capacity to recruit the nucleosome remodeling deacetylase (NuRD) transcriptional repressor. We found that during mice acute infection, TgIST-deficient parasites are rapidly eliminated by the homing Gr1+ inflammatory monocytes, thus highlighting the protective role of TgIST against IFN-γ-mediated killing. By uncovering TgIST functions, this study brings novel evidence on how T. gondii has devised a molecular weapon of choice to take control over a ubiquitous immune gene expression mechanism in metazoans, as a way to promote long-term parasitism.
© 2016 Gay et al.
[PubMed - as supplied by publisher]

Saturday, August 06, 2016

Apicoplast-Localized Lysophosphatidic Acid Precursor Assembly Is Required for Bulk Phospholipid Synthesis in Toxoplasma gondii and Relies on an Algal/Plant-Like Glycerol 3-Phosphate Acyltransferase

 2016 Aug 4;12(8):e1005765. doi: 10.1371/journal.ppat.1005765. eCollection 2016.

Abstract

Most apicomplexan parasites possess a non-photosynthetic plastid (the apicoplast), which harbors enzymes for a number of metabolic pathways, including a prokaryotic type II fatty acid synthesis (FASII) pathway. In Toxoplasma gondii, the causative agent of toxoplasmosis, the FASII pathway is essential for parasite growth and infectivity. However, little is known about the fate of fatty acids synthesized by FASII. In this study, we have investigated the function of a plant-like glycerol 3-phosphate acyltransferase (TgATS1) that localizes to the T. gondii apicoplast. Knock-down of TgATS1 resulted in significantly reduced incorporation of FASII-synthesized fatty acids into phosphatidic acid and downstream phospholipids and a severe defect in intracellular parasite replication and survival. Lipidomic analysis demonstrated that lipid precursors are made in, and exported from, the apicoplast for de novo biosynthesis of bulk phospholipids. This study reveals that the apicoplast-located FASII and ATS1, which are primarily used to generate plastid galactolipids in plants and algae, instead generate bulk phospholipids for membrane biogenesis in T. gondii.

Transcriptomic analysis of mouse liver reveals a potential hepato-enteric pathogenic mechanism in acute Toxoplasma gondii infection

 2016 Aug 3;9(1):427. doi: 10.1186/s13071-016-1716-x.

He JJ1Ma J1,2Elsheikha HM3Song HQ1Huang SY4,5Zhu XQ6,7.

Abstract

BACKGROUND: 

Toxoplasma gondii is a worldwide spread pathogen which can infect all tissues of its host. The transcriptomic responses of infected brain and spleen have been reported. However, our knowledge of the global transcriptomic change in infected liver is limited. Additionally, T. gondii infection represents a highly dynamic process involving complex biological responses of the host at many levels. Herein, we describe such processes at a global level by discovering gene expression changes in mouse livers after acute infection with T. gondii ToxoDB#9 strain.

RESULTS: 

Global transcriptomic analysis identified 2,758 differentially expressed transcripts in infected liver, of which 1,356 were significantly downregulated and 1,402 upregulated. GO and KEGG database analyses showed that host immune responses were upregulated, while the metabolic-related processes/pathways were downregulated, especially xenobiotic metabolism, fatty acid metabolism, energy metabolism, and bile biosynthesis and secretion. The metabolism of more than 800 chemical compounds including anti-Toxoplasma prescribed medicines were predicted to be modulated during acute T. gondii infection due to the downregulation of enzymes involved in xenobiotic metabolism.

CONCLUSIONS: 

To the best of our knowledge, this is the first global transcriptomic analysis of mouse liver infected by T. gondii. The present data indicate that during the early stage of liver infection, T. gondii can induce changes in liver xenobiotic metabolism, upregulating inflammatory response and downregulating hepatocellular PPAR signaling pathway, altering host bile biosynthesis and secretion pathway; these changes could enhance host intestinal dysbacteriosis and thus contribute to the pathological changes of both liver and intestine of infected mice. These findings describe the biological changes in infected liver, providing a potential mechanistic pathway that links hepatic and intestinal pathologies to T. gondii infection.

KEYWORDS: 

Liver function; Mouse liver; RNA-seq; Toxoplasma gondii; Transcriptome

Thursday, August 04, 2016

In Vivo Biotinylation of the Toxoplasma Parasitophorous Vacuole Reveals Novel Dense Granule Proteins Important for Parasite Growth and Pathogenesis

 2016 Aug 2;7(4). pii: e00808-16. doi: 10.1128/mBio.00808-16.

Abstract

Toxoplasma gondii is an obligate intracellular parasite that invades host cells and replicates within a unique parasitophorous vacuole. To maintain this intracellular niche, the parasite secretes an array of dense granule proteins (GRAs) into the nascent parasitophorous vacuole. These GRAs are believed to play key roles in vacuolar remodeling, nutrient uptake, and immune evasion while the parasite is replicating within the host cell. Despite the central role of GRAs in the Toxoplasma life cycle, only a subset of these proteins have been identified, and many of their roles have not been fully elucidated. In this report, we utilize the promiscuous biotin ligase BirA* to biotinylate GRA proteins secreted into the vacuole and then identify those proteins by affinity purification and mass spectrometry. Using GRA-BirA* fusion proteins as bait, we have identified a large number of known and candidate GRAs and verified localization of 13 novel GRA proteins by endogenous gene tagging. We proceeded to functionally characterize three related GRAs from this group (GRA38, GRA39, and GRA40) by gene knockout. While Δgra38 and Δgra40 parasites showed no altered phenotype, disruption of GRA39 results in slow-growing parasites that contain striking lipid deposits in the parasitophorous vacuole, suggesting a role in lipid regulation that is important for parasite growth. In addition, parasites lacking GRA39 showed dramatically reduced virulence and a lower tissue cyst burden in vivo Together, the findings from this work reveal a partial vacuolar proteome of T. gondii and identify a novel GRA that plays a key role in parasite replication and pathogenesis.

IMPORTANCE: 

Most intracellular pathogens reside inside a membrane-bound vacuole within their host cell that is extensively modified by the pathogen to optimize intracellular growth and avoid host defenses. In Toxoplasma, this vacuole is modified by a host of secretory GRA proteins, many of which remain unidentified. Here we demonstrate that in vivo biotinylation of proximal and interacting proteins using the promiscuous biotin ligase BirA* is a powerful approach to rapidly identify vacuolar GRA proteins. We further demonstrate that one factor identified by this approach, GRA39, plays an important role in the ability of the parasite to replicate within its host cell and cause disease.
Copyright © 2016 Nadipuram et al.
PMID:
 
27486190
 
DOI:
 
10.1128/mBio.00808-16
[PubMed - in process]

Blimp-1-mediated CD4 T cell exhaustion causes CD8 T cell dysfunction during chronic toxoplasmosis

 2016 Aug 1. pii: jem.20151995. [Epub ahead of print]

Abstract

CD8, but not CD4, T cells are considered critical for control of chronic toxoplasmosis. Although CD8 exhaustion has been previously reported in Toxoplasma encephalitis (TE)-susceptible model, our current work demonstrates that CD4 not only become exhausted during chronic toxoplasmosis but this dysfunction is more pronounced than CD8 T cells. Exhausted CD4 population expressed elevated levels of multiple inhibitory receptors concomitant with the reduced functionality and up-regulation of Blimp-1, a transcription factor. Our data demonstrates for the first time that Blimp-1 is a critical regulator for CD4 T cell exhaustion especially in the CD4 central memory cell subset. Using a tamoxifen-dependent conditional Blimp-1 knockout mixed bone marrow chimera as well as an adoptive transfer approach, we show that CD4 T cell-intrinsic deletion of Blimp-1 reversed CD8 T cell dysfunction and resulted in improved pathogen control. To the best of our knowledge, this is a novel finding, which demonstrates the role of Blimp-1 as a critical regulator of CD4 dysfunction and links it to the CD8 T cell dysfunctionality observed in infected mice. The critical role of CD4-intrinsic Blimp-1 expression in mediating CD4 and CD8 T cell exhaustion may provide a rational basis for designing novel therapeutic approaches.
© 2016 Hwang et al.
PMID:
 
27481131
 
DOI:
 
10.1084/jem.20151995
[PubMed - as supplied by publisher] 

Z-DNA binding protein mediates host control of Toxoplasma gondii infection

 2016 Aug 1. pii: IAI.00511-16. [Epub ahead of print]

Abstract

Intrinsic to Toxoplasma gondii infection is the parasite-induced modulation of the host immune response, which ensures establishment of a chronic life-long infection. This manipulation of the host immune response allows T. gondii to not only dampen the ability of the host to eliminate the parasite, but also to trigger parasite differentiation to the slow growing, encysted bradyzoite form. We previously used RNAseq to profile the transcriptomes of mice and T. gondii during acute and chronic stages of infection. One of the most abundant host transcripts during acute and chronic infection was Z-DNA binding protein 1 (ZBP1). Here, we determined that ZBP1 functions to control T. gondii growth. In activated macrophages isolated from ZBP1 deletion mice (ZBP1-/-), T. gondii has an increased rate of replication and a decreased rate of degradation. We also identified a novel function for ZBP1 as a regulator of nitric oxide (NO) production in activated macrophages, even in the absence of T. gondii infection. Upon stimulation, T. gondii infected ZBP1-/- macrophages display increased pro-inflammatory cytokines compared to wild type macrophages under the same conditions. These in vitro phenotypes recapitulated in vivo with ZBP1-/-mice having increased susceptibility to oral challenge, higher cyst burdens during chronic infection and an elevated inflammatory cytokine response. Taken together, these results highlight a role for ZBP1 in assisting host control of T. gondii infection.
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
PMID:
 
27481249
 
DOI:
 
10.1128/IAI.00511-16
[PubMed - as supplied by publisher]

Pyrimidine pathway dependent and independent functions of the Toxoplasma gondii mitochondrial dihydroorotate dehydrogenase

 2016 Aug 1. pii: IAI.00187-16. [Epub ahead of print]

Abstract

Dihydroorotate dehydrogenase (DHODH) mediates the fourth step of de novo pyrimidine biosynthesis and is a proven drug target for inducing immunosuppression in therapy of human disease as well as a rapidly emerging drug target for treatment of malaria. In Toxoplasma gondii, disruption of the first, fifth, or sixth step of de novo pyrimidine biosynthesis induced uracil auxotrophy. However, previous attempts to generate uracil auxotrophy by genetically deleting the mitochondrial associated DHODH of T. gondii (TgDHODH) failed. To further address the essentiality of TgDHODH, mutant gene alleles deficient in TgDHODH activity were designed to ablate the enzyme activity. Replacement of the endogenous DHODH gene with catalytically deficient DHODH gene alleles induced uracil auxotrophy. Catalytically deficient TgDHODH localized to the mitochondria and parasites retained mitochondrial membrane potential. These results show that TgDHODH is essential for the synthesis of pyrimidines and suggests that TgDHODH is required for a second essential function independent of its role in pyrimidine biosynthesis.
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
PMID:
 
27481247
 
DOI:
 
10.1128/IAI.00187-16
[PubMed - as supplied by publisher]

Wednesday, August 03, 2016

Endothelial cells are a replicative niche for entry of Toxoplasma gondii to the central nervous system

 2016;1. pii: 16001. Epub 2016 Feb 15.

Abstract

An important function of the blood-brain barrier is to exclude pathogens from the central nervous system, but some microorganisms benefit from the ability to enter this site. It has been proposed that Toxoplasma gondii can cross biological barriers as a motile extracellular form that uses transcellular or paracellular migration, or by infecting a host cell that then crosses the blood-brain barrier. Unexpectedly, analysis of acutely infected mice revealed significant numbers of free parasites in the blood and the presence of infected endothelial cells in the brain vasculature. The use of diverse transgenic parasites combined with reporter mice and intravital imaging demonstrated that replication in and lysis of endothelial cells precedes invasion of the central nervous system, and highlight a novel mechanism for parasite entry to the central nervous system.
PMID:
 
27478622