The Toxoplasma Blog: September 2015

Wednesday, September 30, 2015

Ubiquitin systems mark pathogen-containing vacuoles as targets for host defense by guanylate binding proteins

2015 Sep 28. pii: 201515966. [Epub ahead of print]

Haldar AK¹, Foltz C², Finethy R¹, Piro AS¹, Feeley EM¹, Pilla-Moffett DM¹, Komatsu M³, Frickel EM², Coers J⁴.

Many microbes create and maintain pathogen-containing vacuoles (PVs) as an intracellular niche permissive for microbial growth and survival. The destruction of PVs by IFNγ-inducible guanylate binding protein (GBP) and immunity-related GTPase (IRG) host proteins is central to a successful immune response directed against numerous PV-resident pathogens. However, the mechanism by which IRGs and GBPs cooperatively detect and destroy PVs is unclear. We find that host cell priming with IFNγ prompts IRG-dependent association of Toxoplasma- and Chlamydia-containing vacuoles with ubiquitin through regulated translocation of the E3 ubiquitin ligase tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6). This initial ubiquitin labeling elicits p62-mediated escort and deposition of GBPs to PVs, thereby conferring cell-autonomous immunity. Hypervirulent strains of Toxoplasma gondii evade this process via specific rhoptry protein kinases that inhibit IRG function, resulting in blockage of downstream PV ubiquitination and GBP delivery. Our results define a ubiquitin-centered mechanism by which host cells deliver GBPs to PVs and explain how hypervirulent parasites evade GBP-mediated immunity.

KEYWORDS:

Chlamydia; GBPs; Toxoplasma; immunity-related GTPase; interferon

PMID:: 26417105; [PubMed - as supplied by publisher]

Tuesday, September 29, 2015

Functional Analyses of the Toxoplasma gondii DNA Gyrase Holoenzyme: A Janus Topoisomerase with Supercoiling and Decatenation Abilities

2015 Sep 28;5:14491. doi: 10.1038/srep14491.

Lin TY¹, Nagano S¹, Gardiner Heddle J¹.

Abstract

A number of important protozoan parasites including those responsible for toxoplasmosis and malaria belong to the phylum Apicomplexa and are characterised by their possession of a relict plastid, the apicoplast. Being required for survival, apicoplasts are potentially useful drug targets and their attractiveness is increased by the fact that they contain "bacterial" gyrase, a well-established antibacterial drug target. We have cloned and purified the gyrase proteins from the apicoplast of Toxoplasma gondii (the cause of toxoplasmosis), reconstituted the functional enzyme and succeeded in characterising it. We discovered that the enzyme is inhibited by known gyrase inhibitors and that, as well as the expected supercoiling activity, it is also able to decatenate DNA with high efficiency. This unusual dual functionality may be related to the apparent lack of topoisomerase IV in the apicoplast.

PMID:: 26412236; [PubMed - as supplied by publisher]

Monday, September 28, 2015

Clustering of Toxoplasma gondii Infections Within Families of Congenitally Infected Infants

2015 Sep 24. pii: civ721. [Epub ahead of print]

Contopoulos-Ioannidis D¹, Wheeler KM², Ramirez R³, Press C³, Mui E², Zhou Y², Van Tubbergen C², Prasad S², Maldonado Y⁴, Withers S², Boyer KM⁵,Noble AG⁶, Rabiah P⁷, Swisher CN⁶, Heydemann P⁵, Wroblewski K², Karrison T², Grigg ME⁸, Montoya JG⁹, McLeod R².

Author information

Abstract

BACKGROUND:

Family clusters and epidemics of toxoplasmosis in North, Central, and South America led us to determine whether fathers of congenitally infected infants in the National Collaborative Chicago-Based Congenital Toxoplasmosis Study (NCCCTS) have a high incidence of Toxoplasma gondii infection.

METHODS:

We analyzed serum samples collected from NCCCTS families between 1981 and 2013. Paternal serum samples were tested for T. gondii antibodies with immunoglobulin (Ig) G dye test and IgM enzyme-linked immunosorbent assay. Additional testing of paternal serum samples was performed with differential-agglutination and IgG avidity tests when T. gondii IgG and IgM results were positive and serum samples were collected by the 1-year visit of the congenitally infected child. Prevalence of paternal seropositivity and incidence of recent infection were calculated. We analyzed whether certain demographics, maternal parasite serotype, risk factors, or maternal/infant clinical manifestations were associated with paternal T. gondii infection status.

RESULTS:

Serologic testing revealed a high prevalence (29 of 81; 36%) of T. gondii infection in fathers, relative to the average seropositivity rate of 9.8% for boys and men aged 12-49 years in the United States between 1994 and 2004 (P < .001). Moreover, there was a higher-than-expected incidence of recent infections among fathers with serum samples collected by the 1-year visit of their child (6 of 45; 13%; P < .001). No demographic patterns or clinical manifestations in mothers or infants were associated with paternal infections, except for sandbox exposure.

CONCLUSIONS:

The high prevalence of chronic and incidence of recent T. gondii infections in fathers of congenitally infected children indicates that T. gondii infections cluster within families in North America. When a recently infected person is identified, family clustering and community risk factors should be investigated for appropriate clinical management.

KEYWORDS:

Toxoplasma gondii; clusters; congenital infections; toxoplasmosis

PMID:: 26405150; [PubMed - as supplied by publisher]

Infection by Toxoplasma gondii Induces Amoeboid-Like Migration of Dendritic Cells in a Three-Dimensional Collagen Matrix

2015 Sep 25;10(9):e0139104. doi: 10.1371/journal.pone.0139104.

Kanatani S¹, Uhlén P², Barragan A¹.

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Abstract

Toxoplasma gondii, an obligate intracellular parasite of humans and other warm-blooded vertebrates, invades a variety of cell types in the organism, including immune cells. Notably, dendritic cells (DCs) infected by T. gondii acquire a hypermigratory phenotype that potentiates parasite dissemination by a 'Trojan horse' type of mechanism in mice. Previous studies have demonstrated that, shortly after parasite invasion, infected DCs exhibit hypermotility in 2-dimensional confinements in vitro and enhanced transmigration in transwell systems. However, interstitial migration in vivo involves interactions with the extracellular matrix in a 3-dimensional (3D) space. We have developed a collagen matrix-based assay in a 96-well plate format that allows quantitative locomotion analyses of infected DCs in a 3D confinement over time. We report that active invasion of DCs by T. gondii tachyzoites induces enhanced migration of infected DCs in the collagen matrix. Parasites of genotype II induced superior DC migratory distances than type I parasites. Moreover, Toxoplasma-induced hypermigration of DCs was further potentiated in the presence of the CCR7 chemotactic cue CCL19. Blocking antibodies to integrins (CD11a, CD11b, CD18, CD29, CD49b) insignificantly affected migration of infected DCs in the 3D matrix, contrasting with their inhibitory effects on adhesion in 2D assays. Morphological analyses of infected DCs in the matrix were consistent with the acquisition of an amoeboid-like migratory phenotype. Altogether, the present data show that the Toxoplasma-induced hypermigratory phenotype in a 3D matrix is consistent with integrin-independent amoeboid DC migration with maintained responsiveness to chemotactic and chemokinetic cues. The data support the hypothesis that induction of amoeboid hypermigration and chemotaxis/chemokinesis in infected DCs potentiates the dissemination of T. gondii.

PMID:: 26406763; [PubMed - as supplied by publisher]

Identification of TgAtg8-TgAtg3 interaction in Toxoplasma gondii

2015 Sep 22. pii: S0001-706X(15)30110-8. doi: 10.1016/j.actatropica.2015.09.013. [Epub ahead of print]

Chen D¹, Lin J², Liu Y², Li X³, Chen G⁴, Hua Q⁵, Nie Q⁶, Hu X⁷, Tan F⁸.

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Abstract

Autophagy is a catabolic process in eukaryotic cells involved in the targeted degradation of cellular organelles and the cytoplasm. Recent works in Toxoplasma gondii suggest that the autophagy processes may serve as an important pathway in modulating parasite survival or death. As an important modulator of Atg8 lipidation and autophagy, Atg8-Atg3 interaction has been attracting increasing attention. However, there is no direct evidence that TgAtg8-TgAtg3 interaction occurs in the parasite. In this study, we firstly found TgAtg8 partially colocalized with TgAtg3 in GFP-TgAtg8 transgenic strains using IFA. Then, lysates from GFP-TgAtg8 tachyzoites were directly subject to large-scale tandem affinity purification with anti-GFP antibody. Western blot and tandem mass spectrometry (MS/MS) analysis determined the interaction between TgAtg8 and TgAtg3. Additionally, we performed real-time interaction analysis with a surface plasmon resonance biosensor using BIAcore system. As expected, the result demonstrated a concentration-dependent increases in resonance signals and indicated the TgAtg8 could bind directly TgAtg3 in vitro. Noteworthily, A KD of 34.9nM obtained from TgAtg8-TgAtg3 interaction indicate a high-affinity between Atg8-Atg3 in Toxoplasma. Furthermore, homology modeling and sequence alignment showed that TgAtg8 has greatest sequence and structural conservation. Within TgAtg3, this protein possesses the core E2 enzymatic activity structure and a truncated handle region which may contain AIM sequence. Taken together, our findings would help elucidate the formation mechanism of autophagosome in Toxoplasma and provide a possibility for looking into parasitic drug targets.

KEYWORDS:

Atg3; Atg8; Protein-Protein interaction; Surface plasmon resonance; Toxoplasma gondii; autophagy

PMID:: 26407821; [PubMed - as supplied by publisher]

Friday, September 25, 2015

Reduction of Toxoplasma gondii development due to inhibition of parasite antioxidant enzymes by a dinuclear iron(III) compound

2015 Sep 21. pii: AAC.00057-15. [Epub ahead of print]

Portes JA¹, Souza TG², Dos Santos TA³, Da Silva LL⁴, Ribeiro TP⁵, Pereira MD⁵, Horn A Jr⁶, Fernandes C⁶, Damatta RA⁴, De Souza W⁷, Seabra SH⁸.

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Abstract

Toxoplasma gondii, which is the agent of toxoplasmosis, is an obligate intracellular protozoan that can infect a wide range of vertebrate cells. Here, we describe the cytotoxic effects of the dinuclear iron compound [Fe(HPClNOL)(SO₄)]₂-μ-oxo, where HPClNOL is the ligand 1-(bis-pyridin-2-ylmethyl-amino)-3-chloropropan-2-ol, on T. gondii infecting LLC-MK2 host cells. This compound was not toxic to LLC-MK2 cells at concentrations up to 200 μM but was very active against the parasite, with an IC₅₀ of 3.6 μM after 48 h of treatment. Cyst formation was observed after treatment as indicated by the appearance of a cyst wall, Dolichos biflorus lectin staining, and scanning and transmission electron microscopy characteristics. Ultraestructural changes were also seen in T. gondii, including membrane blebs and clefts in the cytoplasm with inclusions similar to amylopectin granules, which are typically found in bradyzoites. An analysis of cell death pathways in the parasite revealed that a combination of apoptosis and autophagy was caused by the compound. Fluorescence assays demonstrated that the redox environment in the LLC-MK2 cells becomes oxidant in the presence of the iron compound. Furthermore, a reduction of superoxide dismutase and catalase activities in the treated parasites and the presence of reactive oxygen species within the parasitophorous vacuoles were observed, indicating an impaired protozoan response against these radicals. These findings suggest that this compound disturbs the redox equilibrium of T. gondii, inducing cystogenesis and parasite death.

PMID:: 26392498; [PubMed - as supplied by publisher]

Clinically Available Medicines Demonstrating Anti-Toxoplasma Activity

2015 Sep 21. pii: AAC.02009-15. [Epub ahead of print]

Neville AJ¹, Zach SJ¹, Wang X², Larson JJ¹, Judge AK¹, Davis LA³, Vennerstrom JL², Davis PH¹.

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Abstract

Toxoplasma gondii is an apicomplexan parasite of humans and other mammals, including livestock and companion animals. While chemotherapeutic regimens, including pyrimethamine and sulfadiazine, ameliorate acute or recrudescent disease such as toxoplasmic encephalitis or ocular toxoplasmosis, these drugs are often toxic to the host. Moreover, no approved options are available to treat infected women who are pregnant. Lastly, no drug regimen has shown the ability to eradicate the chronic stage of infection, which is characterized by chemo-resistant intracellular cysts that persist for the life of the host. In an effort to promote additional chemotherapeutic options, we now evaluate clinically available drugs that have shown efficacy in disease models, but which lack clinical case reports. Ideally, less toxic treatments for the acute disease can be identified and developed, with an additional goal of cyst clearance from human and animal hosts.

PMID:: 26392504; [PubMed - as supplied by publisher]

Shared Immune and Repair Markers During Experimental Toxoplasma Chronic Brain Infection and Schizophrenia

2015 Sep 20. pii: sbv134. [Epub ahead of print]

Tomasik J¹, Schultz TL², Kluge W³, Yolken RH⁴, Bahn S¹, Carruthers VB⁵.

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Abstract

Chronic neurologic infection with Toxoplasma gondii is relatively common in humans and is one of the strongest known risk factors for schizophrenia. Nevertheless, the exact neuropathological mechanisms linking T gondii infection and schizophrenia remain unclear. Here we utilize a mouse model of chronic T gondii infection to identify protein biomarkers that are altered in serum and brain samples at 2 time points during chronic infection. Furthermore, we compare the identified biomarkers to those differing between "postmortem" brain samples from 35 schizophrenia patients and 33 healthy controls. Our findings suggest that T gondii infection causes substantial and widespread immune activation indicative of neural damage and reactive tissue repair in the animal model that partly overlaps with changes observed in the brains of schizophrenia patients. The overlapping changes include increases in C-reactive protein (CRP), interleukin-1 beta (IL-1β), interferon gamma (IFNγ), plasminogen activator inhibitor 1 (PAI-1), tissue inhibitor of metalloproteinases 1 (TIMP-1), and vascular cell adhesion molecule 1 (VCAM-1). Potential roles of these factors in the pathogenesis of schizophrenia and toxoplasmosis are discussed. Identifying a defined set of markers shared within the pathophysiological landscape of these diseases could be a key step towards understanding their specific contributions to pathogenesis.

KEYWORDS:

Toxoplasma gondii ; animal model; biomarker

PMID:: 26392628; [PubMed - as supplied by publisher]

A Systematic Review and Meta-Analysis of the Efficacy of Anti-Toxoplasma gondii Medicines in Humans

2015 Sep 22;10(9):e0138204. doi: 10.1371/journal.pone.0138204.

Wei HX¹, Wei SS¹, Lindsay DS², Peng HJ¹.

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Abstract

No effective drug and definitive "gold standard" treatment for Toxoplasma gondii (T. gondii) infection has been available so far, though some medicines have been commonly used in the treatment of T. gondii infection, such as spiramycin, azithromycin, traditional Chinese medicine (TCM), pyrimethamine- sulfadiazine (P-S), trimethoprim-sulfamethoxazole (TMP-SMX), and pyrimethamine-clindamycin (P-C). A systematic review and meta-analysis were performed to compare the efficacies of these conventional medicines in the treatment. Cohort studies for the treatment of acute T. gondii infection were searched from PubMed, Google Scholar, ect. All the cases number for different group extracted from each included literature were input to meta-analysis 3.13 software to calculate the pooled negative conversion rate (NCR), cure rate (CR) or vertical transmission rate based on their sample size and weight. The pooled NCR with 95% confidence intervals (CI) was used to evaluate the overall rate of a diagnosis positive result conversion to a negative result after treatment, which of spiramycin, azithromycin and TCM were 83.4% (95%CI, 72.1%-90.8%), 82.5% (95%CI, 75.9%-87.6%), and 85.5% (95%CI, 71.3%-93.3%) respectively, with no statistical difference between them. The pooled CR with 95% CI was used to evaluate the overall rate of complete disappearance of clinical symptoms for toxoplasmic encephalitis after therapy, which of P-S, TMP-SMX, and P-C were 49.8% (95%CI, 38. 8% -60.8%), 59.9% (95%CI, 48.9%-70.0%), and 47.6% (95%CI, 24.8%-71.4%) respectively, with no statistical difference between them. Primary T. gondii infection in pregnancy was treated mainly with spiramycin alone or combined with other drugs, and the pooled rate of vertical transmission was about 9.9% (95%CI, 5.9%-16.2%) after therapy. Toxoplasmic encephalitis in AIDS patients was usually treated with sulfonamides combined with other drugs and the pooled CR was 49.4% (95%CI, 37.9%-60.9%).

PMID:: 26394212; [PubMed - as supplied by publisher]

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No Evidence of Association between Toxoplasma gondii Infection and Financial Risk Taking in Females

2015 Sep 24;10(9):e0136716. doi: 10.1371/journal.pone.0136716.

Lanchava L¹, Carlson K², Šebánková B³, Flegr J³, Nave G⁴.

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Abstract

BACKGROUND:

Past research linked Toxoplasma gondii (TG) infection in humans with neurological and mental disorders (e.g., schizophrenia, Alzheimer's disease and attention disorders), irregularities of the dopaminergic and testosterone system, and increased likelihood of being involved in traffic accidents.

METHODOLOGY/PRINCIPAL FINDINGS:

We test for an association between TG infection and financial decision-making (DM) using a case-control design in a sample of female Czech students (n = 79). We estimate each subject's risk attitude and loss aversion using an experimental economic task involving real monetary incentives. We find no significant evidence that either measure of decision-making is associated with TG infection.

CONCLUSION:

We were unable to find evidence of an association between TG infection and financial decision-making in females.

PMID:: 26401912; [PubMed - as supplied by publisher]

Tuesday, September 22, 2015

Flt3 Ligand Is Essential for Survival and Protective Immune Responses during Toxoplasmosis

2015 Sep 18. pii: 1500690. [Epub ahead of print]

Dupont CD¹, Harms Pritchard G¹, Hidano S¹, Christian DA¹, Wagage S¹, Muallem G¹, Tait Wojno ED¹, Hunter CA².

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Abstract

Dendritic cells (DCs) are critical for resistance to Toxoplasma gondii, and infection with this pathogen leads to increased numbers of DCs at local sites of parasite replication and in secondary lymphoid organs, but the factors that regulate this expansion are poorly understood. The cytokine Flt3 ligand (Flt3L) is critical for the generation and maintenance of DCs, and Flt3L^-/- mice were found to be highly susceptible to acute toxoplasmosis. This phenotype correlated with decreased production of IL-12 and IFN-γ, as well as impaired NK cell responses. Surprisingly, despite low basal numbers of DCs, Flt3L^-/- mice infected with T. gondii displayed an expansion of CD8α⁺ and CD11b^loCD8α^- DCs. Infection also induced an expansion of parasite-specific CD4⁺ and CD8⁺ T cells in Flt3L^-/- mice; however, these cells were reduced in number and displayed impaired ability to produce IFN-γ relative to wild-type controls. Exogenous IL-12 treatment partially restored NK and T cell responses in Flt3L^-/- mice, as well as acute resistance; however, these mice eventually succumbed to toxoplasmic encephalitis, despite the presence of large numbers of DCs and T cells in the brain. These results highlight the importance of Flt3L for resistance to toxoplasmosis and demonstrate the existence of Flt3L-independent pathways that can mediate infection-induced expansion of DCs and T cell priming.

PMID:: 26385522; [PubMed - as supplied by publisher]

Toxoplasma gondii Toc75 functions in import of stromal but not peripheral apicoplast proteins

2015 Sep 18. doi: 10.1111/tra.12333. [Epub ahead of print]

Sheiner L^1,², Fellows JD¹, Ovciarikova J², Brooks CF¹, Agrawal S¹, Holmes ZC¹, Bietz I³, Flinner N⁴, Heiny S³, Mirus O⁴, Przyborski JM³, Striepen B¹.

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Abstract

Apicomplexa are unicellular parasites causing important human and animal diseases, including malaria and toxoplasmosis. Most of these pathogens possess a relict but essential plastid, the apicoplast. The apicoplast was acquired by secondary endosymbiosis between a red alga and a flagellated eukaryotic protist. As a result the apicoplast is surrounded by four membranes. This complex structure necessitates a system of transport signals and translocons allowing nuclear encoded proteins to find their way to specific apicoplast sub-compartments. Previous studies identified translocons traversing two of the four apicoplast membranes. Here we provide functional support for the role of an apicomplexan Toc75 homolog in apicoplast protein transport. We identify two apicomplexan genes encoding Toc75 and Sam50, both members of the Omp85 protein family. We localize the respective proteins to the apicoplast and the mitochondrion of Toxoplasma and Plasmodium. We show that the Toxoplasma Toc75 is essential for parasite growth and that its depletion results in a rapid defect in the import of apicoplast stromal proteins while the import of proteins of the outer compartments is affected only as the secondary consequence of organelle loss. These observations along with the homology to Toc75 suggest a potential role in transport through the second innermost membrane.

KEYWORDS:

Apicomplexa; Omp85; Plasmodium; Sam50; Toc75; Toxoplasma; apicoplast; complex plastid; protein trafficking

PMID:: 26381927; [PubMed - as supplied by publisher]