The Toxoplasma Blog: July 2016

Thursday, July 28, 2016

TGF-β in Toxoplasmosis: Friend or foe?

2016 Jul 20;86:29-35. doi: 10.1016/j.cyto.2016.07.002. [Epub ahead of print]

Zare-Bidaki M1, Assar S1, Hakimi H1, Abdollahi SH2, Nosratabadi R3, Kennedy D4, Arababadi MK5.

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Abstract

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan causing several forms of toxoplasmosis in humans. The main mechanisms that allow the development of the prolonged forms of the disease and its subsequent pathology are yet to be clarified. However, many researchers have hypothesized that immunological and genetic parameters may play crucial roles in the etiology of the disease. Transforming growth factor beta (TGF-β) is a cytokine with a dual role in the regulation of immune responses including those against parasites. However, the relationship between TGF-β and immune responses against T .gondii are not fully understood. The important roles played by TGF-β in the development of Th17 and T regulatory lymphocytes, mucosal immunity and regulation of immune responses have been documented and this provides insights into TGF-β function during parasitic infections such as toxoplasmosis. Therefore, the aim of this review is to collate the current information regarding the status and association of TGF-β with T. gondii infection.

KEYWORDS:

T regulatory lymphocytes; TGF-β; Th17; Toxoplasma gondii

PMID:: 27449809
DOI:: 10.1016/j.cyto.2016.07.002

Secreted Protein Kinases Regulate Cyst Burden During Chronic Toxoplasmosis

2016 Jul 23. doi: 10.1111/cmi.12651. [Epub ahead of print]

Jones NG1, Wang Q1, Sibley LD1.

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Abstract

Toxoplasma gondii is an apicomplexan parasite that secretes a large number of protein kinases and pseudokinases from its rhoptry organelles. Although some rhoptry kinases (ROPKs) act as virulence factors, many remain uncharacterised. In this study, predicted ROPKs were assessed for bradyzoite expression then pritoritised for a reverse genetic analysis in the type II strain Pru that is amenable to targeted disruption. Using CRISPR/Cas9 we engineered C-terminally epitope tagged ROP21 and ROP27 and demonstrated their localization to the PV and cyst matrix. ROP21 and ROP27 were not secreted from microneme, rhoptry, or dense granule organelles, but rather were located in small vesicles consistent with a constitutive pathway. Using CRISPR/Cas9, the genes for ROP21, ROP27, ROP28 and ROP30 were deleted individually and in combination, and the mutant parasites were assessed for growth and their ability to form tissue cysts in mice. All knockouts lines were normal for in vitro growth and bradyzoite differentiation but a combined ∆rop21/∆rop17 knockout led to a 50% reduction in cyst burden in vivo. Our findings question the existing annotation of ROPKs based solely on bioinformatic techniques and yet highlight the importance of secreted kinases in determining the severity of chronic toxoplasmosis.

PMID:: 27450947
DOI:: 10.1111/cmi.12651

Toxoplasma gondii Infection in Mice Impairs Long-Term Fear Memory Consolidation Through Dysfunction of the Cortex and Amygdala

2016 Jul 25. pii: IAI.00217-16. [Epub ahead of print]

Ihara F1, Nishimura M1, Muroi Y2, Mahmoud ME1, Yokoyama N1, Nagamune K3, Nishikawa Y4.

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Abstract

Chronic infection with Toxoplasma gondii becomes established in tissues of the central nervous system, where parasites may directly or indirectly modulate neuronal function. Epidemiological studies reveal that chronic infection in humans is a risk factor for developing mental diseases. However, the mechanisms underlying parasite-induced neuronal dysfunction in the brain remain unclear. Here, we examined memory associated with conditioned fear in mice and found that T. gondii infection impairs consolidation of conditioned fear memory. To examine brain pathology induced by T. gondii infection, we analyzed parasite load and histopathological changes. T. gondii infects all brain areas, yet the cortex exhibits more severe tissue damage than other regions. We measured neurotransmitter levels in the cortex and amygdala because these regions are involved in fear memory expression. Levels of dopamine metabolites, but not dopamine, were increased in the cortex of infected mice compared with those in uninfected mice. By contrast, serotonin levels were decreased in the amygdala and norepinephrine levels were decreased in the cortex and amygdala of infected mice. The levels of cortical dopamine metabolites were associated with the time spent freezing in the fear-conditioning test. These results suggest that T. gondii infection affects fear memory through dysfunction of the cortex and amygdala. Our findings provide insight into the mechanisms underlying neurological changes during T. gondii infection.

PMID:: 27456832
DOI:: 10.1128/IAI.00217-16

Coastal development and precipitation drive pathogen flow from land to sea: evidence from a Toxoplasma gondii and felid host system

2016 Jul 26;6:29252. doi: 10.1038/srep29252.

VanWormer E1, Carpenter TE2, Singh P3,4, Shapiro K1,5, Wallender WW4, Conrad PA1,5, Largier JL6, Maneta MP7, Mazet JA1.

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Abstract

Rapidly developing coastal regions face consequences of land use and climate change including flooding and increased sediment, nutrient, and chemical runoff, but these forces may also enhance pathogen runoff, which threatens human, animal, and ecosystem health. Using the zoonotic parasite Toxoplasma gondii in California, USA as a model for coastal pathogen pollution, we examine the spatial distribution of parasite runoff and the impacts of precipitation and development on projected pathogen delivery to the ocean. Oocysts, the extremely hardy free-living environmental stage of T. gondii shed in faeces of domestic and wild felids, are carried to the ocean by freshwater runoff. Linking spatial pathogen loading and transport models, we show that watersheds with the highest levels of oocyst runoff align closely with regions of increased sentinel marine mammal T. gondii infection. These watersheds are characterized by higher levels of coastal development and larger domestic cat populations. Increases in coastal development and precipitation independently raised oocyst delivery to the ocean (average increases of 44% and 79%, respectively), but dramatically increased parasite runoff when combined (175% average increase). Anthropogenic changes in landscapes and climate can accelerate runoff of diverse pathogens from terrestrial to aquatic environments, influencing transmission to people, domestic animals, and wildlife.

PMID:: 27456911
DOI:: 10.1038/srep29252

Apicoplast fatty acid synthesis is essential for pellicle formation at the end of cytokinesis in Toxoplasma gondii

2016 Jul 25. pii: jcs.185223. [Epub ahead of print]

Martins-Duarte ÉS1, Carias M2, Vommaro R2, Surolia N3, de Souza W1.

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Abstract

The Apicomplexan protozoan Toxoplasma gondii, the causative agent of toxoplasmosis, harbors an apicoplast, a plastid-like organelle with essential metabolic functions. Although the FASII fatty acid biosynthesis pathway located in the apicoplast is essential for parasite survival, the cellular effects of FASII disruption in T. gondii had not been examined in detail. Here, we combined light and electron microscopy techniques - including focused ion-beam scanning electron microscopy (FIB-SEM) - to characterize the effect of FASII disruption in T. gondii, by treatment with the FASII inhibitor triclosan or by inducible knock down of the FASII component acyl carrier protein. Morphological analyses showed that FASII disruption prevented cytokinesis completion in T. gondii tachyzoites, leading to the formation of large masses of "tethered" daughter cells. FIB-SEM showed that tethered daughters had a mature basal complex, but a defect in new membrane addition between daughters resulted in incomplete pellicle formation. Addition of exogenous fatty acids to medium suppressed the formation of tethered daughter cells and supports the notion that FASII is essential to generate lipid substrates required for the final step of parasite division.

PMID:: 27457282
DOI:: 10.1242/jcs.185223

[PubMed - as supplied by publisher]

The Import of Proteins into the Mitochondrion of Toxoplasma gondii

2016 Jul 25. pii: jbc.M116.725069. [Epub ahead of print]

van Dooren GG1, Yeoh LM2, Striepen B3, McFadden GI2.

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Abstract

Outside of well-characterized model eukaryotes, there is relatively little known about the translocons that transport proteins across the two membranes that surround the mitochondrion. Apicomplexans are a phylum of intracellular parasites that cause major diseases in humans and animals, and are evolutionarily distant from model eukaryotes such as yeast. Apicomplexans harbour a mitochondrion that is essential for parasite survival, and is a validated drug target. Here, we demonstrate that the apicomplexan Toxoplasma gondii harbours homologues of proteins from all the major mitochondrial protein translocons present in yeast, suggesting these arose early in eukaryotic evolution. We demonstrate that a T. gondii homologue of Tom22 (TgTom22), a central component of the translocon of the outer mitochondrial membrane (TOM) complex, is essential for parasite survival, mitochondrial protein import, and assembly of the TOM complex. We also identify and characterize a T. gondii homologue of Tom7 (TgTom7) that is important for parasite survival and mitochondrial protein import. Contrary to the role of Tom7 in yeast, TgTom7 is important for TOM complex stability, suggesting the role of this protein has diverged during eukaryotic evolution. Together, our study identifies conserved and modified features of mitochondrial protein import in apicomplexan parasites.

KEYWORDS:

Toxoplasma gondii; intracellular trafficking; mitochondria; parasite; protein import

PMID:: 27458014
DOI:: 10.1074/jbc.M116.725069

Phenotypes Associated with Knockouts of Eight Dense Granule Gene Loci (GRA2-9) in Virulent Toxoplasma gondii

2016 Jul 26;11(7):e0159306. doi: 10.1371/journal.pone.0159306. eCollection 2016.

Rommereim LM1, Bellini V2,3, Fox BA1, Pètre G2,3, Rak C2,3, Touquet B2,3,4, Aldebert D2,3, Dubremetz JF5,6, Cesbron-Delauw MF2,3, Mercier C2,3, Bzik DJ1.

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Abstract

Toxoplasma gondii actively invades host cells and establishes a parasitophorous vacuole (PV) that accumulates many proteins secreted by the dense granules (GRA proteins). To date, at least 23 GRA proteins have been reported, though the function(s) of most of these proteins still remains unknown. We targeted gene knockouts at ten GRA gene loci (GRA1-10) to investigate the cellular roles and essentiality of these classical GRA proteins during acute infection in the virulent type I RH strain. While eight of these genes (GRA2-9) were successfully knocked out, targeted knockouts at the GRA1 and GRA10 loci were not obtained, suggesting these GRA proteins may be essential. As expected, the Δgra2 and Δgra6 knockouts failed to form an intravacuolar network (IVN). Surprisingly, Δgra7 exhibited hyper-formation of the IVN in both normal and lipid-free growth conditions. No morphological alterations were identified in parasite or PV structures in the Δgra3, Δgra4, Δgra5, Δgra8, or Δgra9 knockouts. With the exception of the Δgra3 and Δgra8 knockouts, all of the GRA knockouts exhibited defects in their infection rate in vitro. While the single GRA knockouts did not exhibit reduced replication rates in vitro, replication rate defects were observed in three double GRA knockout strains (Δgra4Δgra6, Δgra3Δgra5 and Δgra3Δgra7). However, the virulence of single or double GRA knockout strains in CD1 mice was not affected. Collectively, our results suggest that while the eight individual GRA proteins investigated in this study (GRA2-9) are not essential, several GRA proteins may provide redundant and potentially important functions during acute infection.

PMID:: 27458822
DOI:: 10.1371/journal.pone.0159306

[PubMed - in process]

Thursday, July 21, 2016

Cryptosporidium and Toxoplasma parasites are inhibited by a benzoxaborole targeting leucyl-tRNA synthetase

2016 Jul 18. pii: AAC.00873-16. [Epub ahead of print]

Palencia A¹, Liu RJ², Lukarska M³, Gut J⁴, Bougdour A⁵, Touquet B⁵, Wang ED², Li X⁶, Alley MR⁶, Freund YR⁶, Rosenthal PJ⁴, Hakimi MA⁵, Cusack S⁷.

The apicomplexan parasites Cryptosporidium and Toxoplasma are serious threats to human health. Cryptosporidiosis is a severe diarrheal disease in malnourished children and immunocompromised individuals, with the only FDA approved drug treatment currently being nitazoxanide. The existing therapies for toxoplasmosis, an important pathology in immunocompromised individuals and pregnant women, also have serious limitations. With the aim of developing alternative therapeutic options to address these health problems, we tested a number of benzoxaboroles, boron-containing compounds shown to be active against various infectious agents, for growth inhibition of Cryptosporidium parasites in mammalian cells. A 3-aminomethyl benzoxaborole, AN6426, with activity in the micromolar range and comparable to nitazoxanide, was identified and further characterised using biophysical measurements of affinity and crystal structures of complexes with the editing domain of Cryptosporidium leucyl-tRNA synthetase (LeuRS). The same compound was shown to be active against Toxoplasma parasites, with the activity being enhanced in the presence of norvaline, an amino acid that can be mischarged by LeuRS. Our observations are consistent with AN6426 inhibiting protein synthesis in both Cryptosporidium and Toxoplasma by forming a covalent adduct with tRNA^Leu in the LeuRS editing active site, which suggest that further exploitation of the benzoxaboroles scaffold is a valid strategy to develop novel, much needed antiparasitic agents.
Copyright © 2016 Palencia et al.

PMID:: 27431220
DOI:: 10.1128/AAC.00873-16

[PubMed - as supplied by publisher]

Global iTRAQ-based proteomic profiling of Toxoplasma gondii oocysts during sporulation

2016 Jul 12;148:12-19. doi: 10.1016/j.jprot.2016.07.010. [Epub ahead of print]

Zhou CX¹, Zhu XQ², Elsheikha HM³, He S⁴, Li Q⁵, Zhou DH⁶, Suo X⁷.

Toxoplasma gondii is a medically and economically important protozoan parasite. However, the molecular mechanisms of its sporulation remain largely unknown. Here, we applied iTRAQ coupled with 2D LC-MS/MS proteomic analysis to investigate the proteomic expression profile of T. gondii oocysts during sporulation. Of the 2095 non-redundant proteins identified, 587 were identified as differentially expressed proteins (DEPs). Based on Gene Ontology enrichment and KEGG pathway analyses the majority of these DEPs were found related to the metabolism of amino acids, carbon and energy. Protein interaction network analysis generated by STRING identified ATP-citrate lyase (ACL), GMP synthase, IMP dehydrogenase (IMPDH), poly (ADP-ribose) glycohydrolase (PARG), and bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) as the top five hubs. We also identified 25 parasite virulence factors that were expressed at relatively high levels in sporulated oocysts compared to non-sporulated oocysts, which might contribute to the infectivity of mature oocysts. Considering the importance of oocysts in the dissemination of toxoplasmosis these findings may help in the search of protein targets with a key role in infectiousness and ecological success of oocysts, creating new opportunities for the development of better means for disease prevention.

BIOLOGICAL SIGNIFICANCE:

The development of new preventative interventions against T. gondii infection relies on an improved understanding of the proteome and chemical pathways of this parasite. To identify proteins required for the development of environmentally resistant and infective T. gondii oocysts, we compared the proteome of non-sporulated (immature) oocysts with the proteome of sporulated (mature, infective) oocysts. iTRAQ 2D-LC-MS/MS analysis revealed proteomic changes that distinguish non-sporulated from sporulated oocysts. Many of the differentially expressed proteins were involved in metabolic pathways and 25 virulence factors were identified upregulated in the sporulated oocysts. This work provides the first quantitative characterization of the proteomic variations that occur in T. gondii oocyst stage during sporulation.
Copyright © 2016. Published by Elsevier B.V.

KEYWORDS:

Oocyst; Proteomics; Sporulation; Toxoplasma gondii; iTRAQ

PMID:: 27422377
DOI:: 10.1016/j.jprot.2016.07.010

Saturday, July 16, 2016

Toxoplasma Effector Recruits the Mi-2/NuRD Complex to Repress STAT1 Transcription and Block IFN-γ-Dependent Gene Expression

2016 Jul 13;20(1):72-82. doi: 10.1016/j.chom.2016.06.006.

Olias P1, Etheridge RD1, Zhang Y2, Holtzman MJ2, Sibley LD3.

Author information

Abstract

Interferon gamma (IFN-γ) is an essential mediator of host defense against intracellular pathogens, including the protozoan parasite Toxoplasma gondii. However, prior T. gondii infection blocks IFN-γ-dependent gene transcription, despite the downstream transcriptional activator STAT1 being activated and bound to cognate nuclear promoters. We identify the parasite effector that blocks STAT1-dependent transcription and show it is associated with recruitment of the Mi-2 nucleosome remodeling and deacetylase (NuRD) complex, a chromatin-modifying repressor. This secreted effector, toxoplasma inhibitor of STAT1-dependent transcription (TgIST), translocates to the host cell nucleus, where it recruits Mi-2/NuRD to STAT1-dependent promoters, resulting in altered chromatin and blocked transcription. TgIST is conserved across strains, underlying their shared ability to block IFN-γ-dependent transcription. TgIST deletion results in increased parasite clearance in IFN-γ-activated cells and reduced mouse virulence, which is restored in IFN-γ-receptor-deficient mice. These findings demonstrate the importance of both IFN-γ responses and the ability of pathogens to counteract these defenses.

KEYWORDS:

acetylation; nuclear transport; phosphorylation; signaling; transcription

PMID:: 27414498
DOI:: 10.1016/j.chom.2016.06.006

[PubMed - in process]

New paradigms for understanding and step changes in treating active and chronic, persistent apicomplexan infections

2016 Jul 14;6:29179. doi: 10.1038/srep29179.

McPhillie M1, Zhou Y2, El Bissati K2, Dubey J3, Lorenzi H4, Capper M5, Lukens AK6,7, Hickman M8, Muench S1, Verma SK3, Weber CR2, Wheeler K2, Gordon J1, Sanders J9, Moulton H9, Wang K10, Kim TK10, He Y10, Santos T11, Woods S12, Lee P8, Donkin D8, Kim E8, Fraczek L2, Lykins J2, Esaa F2, Alibana-Clouser F2, Dovgin S2, Weiss L11, Brasseur G13, Wirth D6,7, Kent M9, Hood L10, Meunieur B14, Roberts CW12, Hasnain SS5, Antonyuk SV5, Fishwick C1, McLeod R2.

Author information

Abstract

Toxoplasma gondii, the most common parasitic infection of human brain and eye, persists across lifetimes, can progressively damage sight, and is currently incurable. New, curative medicines are needed urgently. Herein, we develop novel models to facilitate drug development: EGS strain T. gondii forms cysts in vitro that induce oocysts in cats, the gold standard criterion for cysts. These cysts highly express cytochrome b. Using these models, we envisioned, and then created, novel 4-(1H)-quinolone scaffolds that target the cytochrome bc1 complex Qi site, of which, a substituted 5,6,7,8-tetrahydroquinolin-4-one inhibits active infection (IC50, 30 nM) and cysts (IC50, 4 μM) in vitro, and in vivo (25 mg/kg), and drug resistant Plasmodium falciparum (IC50, <30 abstracttext="" and="" bc1="" billion="" binding="" bradyzoites.="" chronically="" clinically="" co-crystallographic="" complex="" demonstrate="" direct="" encysted="" for="" have="" impact="" improving="" infected="" malaria="" mutant="" nbsp="" nm="" on="" our="" outcomes="" persons="" qi="" relevant="" results="" site.="" studies="" synergy.="" the="" those="" to="" toxoplasmosis="" with="" yeast="">

PMID:: 27412848
DOI:: 10.1038/srep29179

[PubMed - in process]

Saturday, July 09, 2016

An evolutionary conserved zinc finger protein is involved in Toxoplasma gondii mRNA nuclear export

2016 Jul 6. doi: 10.1111/cmi.12644. [Epub ahead of print]

Gissot M1, Hovasse A2, Chaloin L3, Schaeffer-Reiss C2, Van Dorsselaer A2, Tomavo S1.

Author information

Abstract

Apicomplexan parasites are responsible for some of the most deadly parasitic diseases affecting humans and livestock. There is an urgent need for new medicines that will target apicomplexan-specific pathways. We characterized a Toxoplasma gondii C2H2 zinc finger protein, named TgZNF2, which is conserved among eukaryotes. We constructed an inducible KO strain (iKO-TgZNF2) for this gene where the tgznf2 gene expression is repressed in presence of a tetracycline analog (ATc). We showed that the iKO-TgZNF2 parasites are unable to proliferate after depletion of the TgZNF2 protein. Complementation with a full length copy of the gene restores the phenotype Moreover, the homolog of this protein in the related apicomplexan Plasmodium falciparum was shown to efficiently rescue the phenotype, suggesting that this pathway is likely conserved among apicomplexan parasites. We demonstrated that the iKO-mutant lacking TgZNF2 are arrested during the cell cycle during the G1 phase. We identified potential protein partners of this protein among which are spliceosomal complex and mRNA nuclear export components. We confirmed that TgZNF2 is able to bind in vivo to transcripts but splicing is not perturbed in the ATc treated parasites. Instead, we demonstrated that TgZNF2 depletion leads to the sequestration of polyA+ mRNAs in the nucleus while ribosomal RNAs are not affected. We discovered a conserved protein with specific apicomplexan functional properties that is essential for the survival of T. gondii. TgZNF2 may be crucial to ensure the correct polyA+ mRNA nuclear export, a function that is conserved in P. falciparum.

PMID:: 27385072
DOI:: 10.1111/cmi.12644

[PubMed - as supplied by publisher]

Transgenic Eimeria tenella as a vaccine vehicle: expressing TgSAG1 elicits protective immunity against Toxoplasma gondii infections in chickens and mice

2016 Jul 8;6:29379. doi: 10.1038/srep29379.

Tang X1, Yin G2, Qin M1, Tao G1, Suo J1, Liu X1, Suo X1.

Author information

Abstract

The surface antigen 1 of Toxoplasma gondii (TgSAG1) is a major immunodominant antigen and is widely considered an ideal candidate for the development of an effective recombinant vaccine against toxoplasmosis. Eimeria tenella, an affinis apicomplexan parasite with T. gondii, is a potential vaccine vector carrying exogenous antigens that stimulates specific immune responses. Here, we engineered TgSAG1 into E. tenella and obtained a stably transfected E. tenella line (Et-TgSAG1). We found TgSAG1 localized on the cell surface of Et-TgSAG1, which is similar to its native distribution in T. gondii tachyzoites. We immunized the chickens with Et-TgSAG1 orally and detected TgSAG1-specific immune responses, which partly reduced T. gondii infection. In the mouse model, we immunized the mice with Et-TgSAG1 sporozoites intraperitoneally and challenged them with T. gondii tachyzoites RH strain. We found that the mice immunized with Et-TgSAG1 showed a TgSAG1 specific Th 1-dominant immune response and a prolonged survival time compared with wild-type E. tenella and non-immunized mice. Collectively, our results demonstrated that Et-TgSAG1, utilized as a recombinant vaccine against toxoplasmosis, could be applied in both chickens and mice. Our findings also provide a promising persuasion for the development of transgenic Eimeria as vaccine vectors for use in birds and mammals.

PMID:: 27387302
DOI:: 10.1038/srep29379

[PubMed - as supplied by publisher]