Friday, January 30, 2015

Mechanisms of interferon-beta-induced inhibition of Toxoplasma gondii growth in murine macrophages and embryonic fibroblasts

2015 Jan 28. doi: 10.1111/cmi.12423. [Epub ahead of print]
 
 
The apical complex of Toxoplasma gondii enables it to invade virtually all nucleated cells in warm-blooded animals, including humans, making it a parasite of global importance. Anti-T. gondii cellular defense mechanisms depend largely on interferon (IFN)-γ production by immune cells. However, the molecular mechanism of IFN-β-mediated defense remains largely unclear. Here, mouse peritoneal macrophages and murine embryonic fibroblasts (MEFs) primed with recombinant IFN-β and IFN-γ showed different pathways of activation. Treatment of these cells with IFN-β or IFN-γ inhibited T. gondii (Type II PLK strain) growth. Priming macrophages with IFN-β had no effect on inflammatory cytokine expression, inducible nitric oxide synthase or indoleamine 2,3-dioxygenase, nor did it have an effect on their metabolites, nitric oxide and kynurenine, respectively. In contrast, IFN-γ stimulation was characterized by classical macrophage activation and T. gondii elimination. IFN-β activation recruited the immunity-related GTPase M1 (IRGM1) to the parasitophorous vacuole in the macrophages and MEFs. Anti-toxoplasma activities induced by IFN-β were significantly reduced after IRGM1 knockdown in murine macrophages and in IRGM1 deficient MEFs. Thus, this study unravels an alternative pathway of macrophage activation by IFN-β and provides a mechanistic explanation for the contribution of IRGM1 induced by IFN-β to the elimination of T. gondii.
This article is protected by copyright. All rights reserved.
PMID:
25628099
[PubMed - as supplied by publisher]

Mast cells form antibody-dependent degranulatory synapse for dedicated secretion and defence

2015 Jan 28;6:6174. doi: 10.1038/ncomms7174.
 
 
Mast cells are tissue-resident immune cells that play a key role in inflammation and allergy. Here we show that interaction of mast cells with antibody-targeted cells induces the polarized exocytosis of their granules resulting in a sustained exposure of effector enzymes, such as tryptase and chymase, at the cell-cell contact site. This previously unidentified mast cell effector mechanism, which we name the antibody-dependent degranulatory synapse (ADDS), is triggered by both IgE- and IgG-targeted cells. ADDSs take place within an area of cortical actin cytoskeleton clearance in the absence of microtubule organizing centre and Golgi apparatus repositioning towards the stimulating cell. Remarkably, IgG-mediated degranulatory synapses also occur upon contact with opsonized Toxoplasma gondii tachyzoites resulting in tryptase-dependent parasite death. Our results broaden current views of mast cell degranulation by revealing that human mast cells form degranulatory synapses with antibody-targeted cells and pathogens for dedicated secretion and defence.
PMID:
25629393
[PubMed - in process]

Thursday, January 22, 2015

The calcium signaling toolkit of the Apicomplexan parasites Toxoplasma gondii and Plasmodium spp

 2014 Dec 31. pii: S0143-4160(14)00206-1. doi: 10.1016/j.ceca.2014.12.010. [Epub ahead of print]

Abstract

Apicomplexan parasites have complex life cycles, frequently split between different hosts and reliant on rapid responses as the parasites react to changing environmental conditions. Calcium ion (Ca2+) signaling is consequently essential for the cellular and developmental changes that support Apicomplexan parasitism. Apicomplexan genomes reveal a rich repertoire of genes involved in calcium signaling, although many of the genes responsible for observed physiological changes remain unknown. There is evidence, for example, for the presence of a nifedipine-sensitive calcium entry mechanism in Toxoplasma, but the molecular components involved in Ca2+ entry in both Toxoplasma and Plasmodium, have not been identified. The major calcium stores are the endoplasmic reticulum (ER), the acidocalcisomes, and the plant-like vacuole in Toxoplasma, or the food vacuole in Plasmodium spp. Pharmacological evidence suggests that Ca2+ release from intracellular stores may be mediated by inositol 1,4,5-trisphosphate (IP3) or cyclic ADP ribose (cADPR) although there is no molecular evidence for the presence of receptors for these second messengers in the parasites. Several Ca2+-ATPases are present in Apicomplexans and a putative mitochondrial Ca2+/H+ exchanger has been identified. Apicomplexan genomes contain numerous genes encoding Ca2+-binding proteins, with the notable expansion of calcium-dependent protein kinases (CDPKs), whose study has revealed roles in gliding motility, microneme secretion, host cell invasion and egress, and parasite differentiation. Microneme secretion has also been shown to depend on the C2 domain containing protein DOC2 in both Plasmodium spp. and Toxoplasma, providing further evidence for the complex transduction of Ca2+ signals in these organisms. The characterization of these pathways could lead to the discovery of novel drug targets and to a better understanding of the role of Ca2+ in these parasites.
Copyright © 2014 Elsevier Ltd. All rights reserved.

KEYWORDS: 

Acidic calcium; Acidocalcisome; Calcium; Calcium entry; Calcium-dependent protein kinases; Microneme; Plasmodium; Toxoplasma gondii
PMID:
 
25605521
 
[PubMed - as supplied by publisher]

Strain hypothesis of Toxoplasma gondii infection on the outcome of human diseases

 2015 Jan 19. doi: 10.1111/apha.12458. [Epub ahead of print]

Abstract

The intracellular protozoan Toxoplasma gondii is an exceptionally successful food- and waterborne parasite that infects approximately 1 billion people worldwide. Genotyping of T. gondii isolates from all continents revealed a complex population structure. Recent research supports the notion that T. gondii genotype may be associated with disease severity. Here, we (1) discuss molecular and serological approaches for designation of T. gondii strain type, (2) overview the literatures on the association of T. gondii strain type and the outcome of human disease, and (3) explore possible mechanisms underlying these strain specific pathology and severity of human toxoplasmosis. Although no final conclusions can be drawn, it is clear that virulent strains (e. g. strains containing type I or atypical alleles) are significantly more often associated with increased frequency and severity of human toxoplasmosis. The significance of highly virulent strains can cause severe diseases in immunocompetent patients and might implicated in brain disorders such as schizophrenia should led to reconsideration of toxoplasmosis. Further studies that combine parasite strain typing and human factor analysis (e.g. immune status and genetic background) are required for better understanding of human susceptibility or resistance to toxoplasmosis. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.

KEYWORDS: 

Toxoplasma gondii ; human factors; mechanisms; outcome of human infection; strain typing; toxoplasmosis; virulence
PMID:
 
25600911
 
[PubMed - as supplied by publisher]

Toxoplasma secretory granules: one population or more?

 2015 Jan 15. pii: S1471-4922(14)00205-0. doi: 10.1016/j.pt.2014.12.002. [Epub ahead of print]

Abstract

In Toxoplasma gondii, dense granules are known as the storage secretory organelles of the so-called GRA proteins (for dense granule proteins), which are destined to the parasitophorous vacuole (PV) and the PV-derived cyst wall. Recently, newly annotated GRA proteins targeted to the host cell nucleus have enlarged this view. Here we provide an update on the latest developments on the Toxoplasma secreted proteins, which to date have been mainly studied at both the tachyzoite and bradyzoite stages, and we point out that recent discoveries could open the issue of a possible, yet uncharacterized, distinct secretory pathway in Toxoplasma.
Copyright © 2014 Elsevier Ltd. All rights reserved.

KEYWORDS: 

GRA proteins; Toxoplasma gondii; dense granules; protozoa
PMID:
 
25599584
 
[PubMed - as supplied by publisher]

Wednesday, January 14, 2015

ATG5 but not ATG7 in Dendritic Cells Enhances IL-2 and IFN-γ Production by TOXOPLASMA GONDII-Reactive CD4+ T Cells

2015 Jan 8. pii: S1286-4579(14)00330-X. doi: 10.1016/j.micinf.2014.12.008. [Epub ahead of print]
 
 
The autophagy proteins (Atg) modulate not only innate but also adaptive immunity against pathogens. We examined the role of dendritic cell Atg5 and Atg7 in the production of IL-2 and IFN-γ by Toxoplasma gondii-reactive CD4+ T cells. T. gondii-reactive mouse CD4+ T cells exhibited unimpaired production of IL-2 and IFN-γ when stimulated with Atg7-deficient mouse dendritic cells that were infected with T. gondii or pulsed with T. gondii lysate antigens. In marked contrast, dendritic cells deficient in Atg5 induced diminished CD4+ T cell production of IL-2 and IFN-γ. This defect was not accompanied by changes in costimulatory ligand expression on dendritic cells or impaired production of IL-12 p70, IL-1β or TNF-α. Knockdown of Irg6a in dendritic cells did not affect CD4+ T cell cytokine production. These results indicate that Atg5 and Atg7 in dendritic cells play differential roles in the modulation of IL-2 and IFN-γ production by T. gondii-reactive CD4+ T cells.
Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

KEYWORDS:

T cells; autophagy; cytokine; dendritic cells; toxoplasma
PMID:
25578385
 

Persistence of Toxoplasma gondii in the central nervous system: a fine tuned balance between the parasite, the brain and the immune system

2015 Jan 9. doi: 10.1111/pim.12173. [Epub ahead of print]
 
 
Upon infection of humans and animals with Toxoplasma gondii, the parasites persist as intraneuronal cysts that are controlled, but not eliminated by the immune system. In particular, intracerebral T cells are crucial in the control of T. gondii infection and are supported by essential functions from other leukocyte populations. Additionally, brain-resident cells including astrocytes, microglia and neurons contribute to the intracerebral immune response by the production of cytokines, chemokines and expression of immunoregulatory cell surface molecules, such as major histocompatibility (MHC) antigens. However, the in vivo behaviour of these individual cell populations, specifically their interaction during cerebral toxoplasmosis, remains to be elucidated. We discuss here what is known about the function of T cells, recruited myeloid cells and brain-resident cells, with particular emphasis on the potential cross-regulation of these cell populations, in governing cerebral toxoplasmosis. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.

KEYWORDS:

Toxoplasma gondii ; Astrocytes; Microglia; Monocytes; Neurons; T cells
PMID:
25573476
[PubMed - as supplied by publisher]

Sulfamethoxazole-trimethoprim associated with resveratrol for the treatment of toxoplasmosis in mice

2015 Jan 6;79C:17-23. doi: 10.1016/j.micpath.2015.01.001. [Epub ahead of print]
 
 
This study aimed to investigate the influence of sulfamethoxazole-trimethoprim (ST) associated with resveratrol on the enzymatic activities of acetylcholinesterase (AChE), adenylate kinase (AK), pyruvate kinase (PK), and creatine kinase (CK) in the brain of mice experimentally infected by Toxoplasma gondii. For that, 60 mice were divided into ten groups with 6 animals each: groups A to D composed by healthy mice and groups E to J consisting of animals infected by T. gondii (VEG strain). Animals started treatment 20 days post-infection for 10 consecutive days with oral doses of 0.5 mg kg-1 of ST (groups B and F), 100 mg kg-1 of free resveratrol (groups C and G) and inclusion complex of resveratrol (nanoparticles containing resveratrol) (groups D and H), as well as with an association of both drugs (groups I and J). The results showed increased (P < 0.001) AChE activity on infected animals (groups E-J) when compared to not-infected (A) animals, and also uninfected animals treated with ST (group B) had increased AChE activity. AK activity decreased (P < 0.001) in the infected and untreated (group E), differently from the other groups that did not differ. PK activity did not differ between groups (P > 0.05). When comparing control groups (uninfected (A) and infected (E)), we verified a significant (P < 0.001) increase in CK activity in the brain, and it is noteworthy that the animals treated with resveratrol associated with ST (group I and J) had similar CK activity to those animals from the group A. Treatment with the combination of ST and resveratrol was able to reduce (P < 0.05) the number of parasitic cysts in the brain, thus reduced inflammatory infiltrates in the liver, and prevented the occurrence of hepatocytes lesions due to toxoplasmosis in mice. Based on these results, it is possible to conclude that increased AChE and CK activities after T. gondii infection did not change with the treatment of ST-resveratrol association. In addition, decreased AK activity caused by T. gondii infection was normalized by ST-resveratrol treatment. T. gondii infection and treatment does not affect PK activity in brain.
Copyright © 2015 Elsevier Ltd. All rights reserved.

KEYWORDS:

ACh; AChE; ATP; Polyphenol; T. gondii
PMID:
25572158
[PubMed - as supplied by publisher]

Monday, January 12, 2015

Postdoctoral position available

     A post-doctoral fellow position is currently available in Dr. O’Connor’s laboratory at Tufts Medical Center in Boston, MA.  The current focus of our laboratory is natural product drug discovery for the treatment of cryptosporidiosis.  Our collaborative research group (the Philippine Mollusk Symbiont-International Cooperative Biodiversity Group http://www.fic.nih.gov/News/GlobalHealthMatters/september-october-2014/Pages/icbg-grant-awards.aspx) takes an unusual approach to marine natural product drug discovery. We mine the genomes of symbiotic bacteria of marine mollusks to identify those with the potential to express new and unusual bioactive compounds.  Compounds extracted from these bacteria are then screened for activity against Cryptosporidium and Toxoplasma.  Mechanisms of action of the compounds are investigated using Toxoplasma as a model for Cryptosporidium.  There is also the opportunity to conduct research on the symbiotic relationship between bacteria and the mollusks.  We will consider candidates with expertise in any related field such as parasitology/ bacteriology / cell biology/ molecular biology/ genomics/ bioinformatics.  The candidate must possess the ability to work independently and have excellent written and verbal communication skills.  Please note that this position is funded on a NIH post-doctoral training grant. Eligibility requires US citizenship or permanent residency.

If interested please contact:


Research Assistant Professor
Division of Geographic Medicine and Infectious Disease
Tufts Medical Center
Box 041,800 Washington St
Boston, MA  02111
tel: 1-617-636-2684
fax: 1-617-636-5292
e-mail:
roconnor@tuftsmedicalcenter.org 

Saturday, January 10, 2015

Diverse Roles for T-bet in the Effector Responses Required for Resistance to Infection

2015 Jan 2. pii: 1401617. [Epub ahead of print]

 
The transcription factor T-bet has been most prominently linked to NK and T cell production of IFN-γ, a cytokine required for the control of a diverse array of intracellular pathogens. Indeed, in mice challenged with the parasite Toxoplasma gondii, NK and T cell responses are characterized by marked increases of T-bet expression. Unexpectedly, T-bet-/- mice infected with T. gondii develop a strong NK cell IFN-γ response that controls parasite replication at the challenge site, but display high parasite burdens at secondary sites colonized by T. gondii and succumb to infection. The loss of T-bet had a modest effect on T cell production of IFN-γ but did not impact on the generation of parasite-specific T cells. However, the absence of T-bet resulted in lower T cell expression of CD11a, Ly6C, KLRG-1, and CXCR3 and fewer parasite-specific T cells at secondary sites of infection, associated with a defect in parasite control at these sites. Together, these data highlight T-bet-independent pathways to IFN-γ production and reveal a novel role for this transcription factor in coordinating the T cell responses necessary to control this infection in peripheral tissues.
Copyright © 2015 by The American Association of Immunologists, Inc.
PMID:
25556247
[PubMed - as supplied by publisher]

Toxoplasma gondii nucleus coding apicoplast protein ACP synthesis and trafficking in delayed death

2015 Jan 7. [Epub ahead of print]
 
 
This study aimed to explore Toxoplasma gondii nucleus coding apicoplast protein acyl carrier protein (ACP) synthesis and trafficking in delayed death. The recombinant T. gondii ACP was expressed by prokaryotic expression method, and anti-ACP polyclonal antibody was obtained from rabbit immune. T. gondii "delayed death" was induced by clindamycin (CLDM), and ACP transcription was determined by real-time PCR assay. The expression of ACP with transit type (t-ACP) and mature type (m-ACP) was determined by Western blotting with anti-ACP polyclonal antibody. The mutant-expressed ACP fused with green fluorescent protein (GFP) tag was constructed by pHX-ACP-GFP. The distribution of ACP in "delayed death" was observed by ACP-GFP fusion protein with a confocal microscope. T. gondii ACP transcription and t-ACP expression had no significant decrease in the early 4 h of "delayed death," but there has been a significant decrease in 6 h. The expression of m-ACP had a significant decrease in 4 h which occurred earlier than the t-ACP expression. The number of brightly dot green fluorescence in ACP-GFP mutant decreased with prolonged time. There was very little brightly dot green fluorescence in ACP-GFP mutant when treated with CLDM for 6 h. CLDM could suppress apicoplast proliferation and induce T. gondii "delayed death"; however, it could not directly suppress nucleus coding ACP transcription and expression. T. gondii lacking of apicoplast had a barrier of transit peptide cleavage and t-ACP could not be transformed into m-ACP. The reason for the decrease in ACP expression could be due to excessive t-ACP synthesis in tachyzoites resulting in a negative feedback for the ACP coding gene transcription.
PMID:
25563610
[PubMed - as supplied by publisher]

Thursday, January 01, 2015

The toxoplasma-host cell junction is anchored to the cell cortex to sustain parasite invasive force

 2014 Dec 31;12(1):773. [Epub ahead of print]

Abstract

BackgroundThe public health threats imposed by toxoplasmosis worldwide and by malaria in sub-Saharan countries are directly associated with the capacity of their closely related causative agents Toxoplasma and Plasmodium, respectively to colonize and expand inside host cells. Therefore, deciphering how these two Apicomplexan protozoan parasites access their hosting cells has been highlighted as a high priority research with the relevant perspective of designing anti-invasive molecules to prevent diseases. Central to the mechanistic base of invasion for both genera is mechanical force, which is thought to be applied by the parasite at the interface between the two cells following assembly of a unique cell junction but this model lacks direct evidence and has been challenged by recent genetic and cell biology studies. In this work, using parasites expressing the fluorescent core component of this junction, we analyse characteristic features of the kinematics of penetration of more than 1000 invasion events.ResultsThe majority of invasion events occur with a typical forward rotational progression of the parasite through a static junction into a vacuole formed from the invaginating host cell plasma membrane, in which the parasite subsequently replicates. However, if parasites encounter resistance and if the junction is not strongly anchored to the host cell cortex, as when parasites do not secrete the toxofilin protein and therefore are unable to locally remodel the cortical actin cytoskeleton, the junction is capped backwards and travels retrogradely with the host cell membrane along the parasite surface as it is enclosed within a functional vacuole. Kinetic measurements of the invasive trajectories strongly support a similar parasite driven force in both static and capped junctions, both of which lead to successful invasion. However about 20% of toxofilin mutants fail to enter and eventually disengage from the host cell membrane while the secreted RON2 molecules are capped at the posterior pole before being cleaved and released in the medium. By contrast in cells characterized by low cortex tension and high cortical actin dynamics, junction capping and entry failure are drastically reduced.ConclusionThis kinematic analysis of pre-invasive and invasive T. gondii tachyzoite behaviors newly highlights that to invade cells, parasites need to engage their motor with the junction molecular complex where force is efficiently applied only upon proper anchorage to the host cell membrane and cortex.
PMID:
 
25551479
 
[PubMed - as supplied by publisher]

Wednesday, December 31, 2014

Reassessment of the role of aromatic amino acid hydroxylases and the effect of infection by Toxoplasma gondii on host dopamine levels

 2014 Dec 29. pii: IAI.02465-14. [Epub ahead of print]

Abstract

Toxoplasma gondii infection has previously been described to cause infected mice to lose their fear of cat urine. This behavioral manipulation has been proposed to involve alterations of host dopamine pathways due to parasite-encoded aromatic amino acid hydroxylases. Here, we report successful knockout and complementation of the aromatic amino acid hydroxylase AAH2 gene, with no observable phenotype in parasite growth or differentiation in vitro and in vivo. Additionally, expression levels of the two aromatic amino acid hydroxylases were negligible both in tachyzoites and in bradyzoites. Finally, we were unable to confirm previously described effects of parasite infection on host dopamine either in vitro or in vivo, even when AAH2 was over-expressed using the BAG1 promoter. Together, these data indicate that AAH enzymes in the parasite do not cause global or regional alterations of dopamine in the host brain, although they may locally affect this pathway. Additionally, our findings suggest alternative roles for the AHH enzymes in T. gondii since AAH1 is essential for growth in non-dopaminergic cells.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
PMID:
 
25547791
 
[PubMed - as supplied by publisher]

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

 2014 Dec 9;(94). doi: 10.3791/52237.

Abstract

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma establishes a lifelong persistent infection in the brain. While this brain infection is asymptomatic in most immunocompetent people, in the developing fetus or immunocompromised individuals such as acquired immune deficiency syndrome (AIDS) patients, this predilection for and persistence in the brain can lead to devastating neurologic disease. Thus, it is clear that the brain-Toxoplasma interaction is critical to the symptomatic disease produced by Toxoplasma, yet we have little understanding of the cellular or molecular interaction between cells of the central nervous system (CNS) and the parasite. In the mouse model of CNS toxoplasmosis it has been known for over 30 years that neurons are the cells in which the parasite persists, but little information is available about which part of the neuron is generally infected (soma, dendrite, axon) and if this cellular relationship changes between strains. In part, this lack is secondary to the difficulty of imaging and visualizing whole infected neurons from an animal. Such images would typically require serial sectioning and stitching of tissue imaged by electron microscopy or confocal microscopy after immunostaining. By combining several techniques, the method described here enables the use of thick sections (160 µm) to identify and image whole cells that contain cysts, allowing three-dimensional visualization and analysis of individual, chronically infected neurons without the need for immunostaining, electron microscopy, or serial sectioning and stitching. Using this technique, we can begin to understand the cellular relationship between the parasite and the infected neuron.
PMID:
 
25549001
 
[PubMed - as supplied by publisher]