Wednesday, February 10, 2016

Basal body structure and composition in the apicomplexans Toxoplasma and Plasmodium

 2016 Feb 4;5:3. doi: 10.1186/s13630-016-0025-5. eCollection 2015.

Abstract

The phylum Apicomplexa encompasses numerous important human and animal disease-causing parasites, including the Plasmodium species, and Toxoplasma gondii, causative agents of malaria and toxoplasmosis, respectively. Apicomplexans proliferate by asexual replication and can also undergo sexual recombination. Most life cycle stages of the parasite lack flagella; these structures only appear on male gametes. Although male gametes (microgametes) assemble a typical 9+2 axoneme, the structure of the templating basal body is poorly defined. Moreover, the relationship between asexual stage centrioles and microgamete basal bodies remains unclear. While asexual stages of Plasmodium lack defined centriole structures, the asexual stages of Toxoplasma and closely related coccidian apicomplexans contain centrioles that consist of nine singlet microtubules and a central tubule. There are relatively few ultra-structural images of Toxoplasma microgametes, which only develop in cat intestinal epithelium. Only a subset of these include sections through the basal body: to date, none have unambiguously captured organization of the basal body structure. Moreover, it is unclear whether this basal body is derived from pre-existing asexual stage centrioles or is synthesized de novo. Basal bodies in Plasmodium microgametes are thought to be synthesized de novo, and their assembly remains ill-defined. Apicomplexan genomes harbor genes encoding δ- and ε-tubulin homologs, potentially enabling these parasites to assemble a typical triplet basal body structure. Moreover, the UNIMOD components (SAS6, SAS4/CPAP, and BLD10/CEP135) are conserved in these organisms. However, other widely conserved basal body and flagellar biogenesis elements are missing from apicomplexan genomes. These differences may indicate variations in flagellar biogenesis pathways and in basal body arrangement within the phylum. As apicomplexan basal bodies are distinct from their metazoan counterparts, it may be possible to selectively target parasite structures in order to inhibit microgamete motility which drives generation of genetic diversity in Toxoplasma and transmission for Plasmodium. 

KEYWORDS: 

Centriole; Coccidia; Flagellum; Malaria; Microgamete; Microtubule organizing center
PMID:
 
26855772
 
[PubMed] 

Tracing amino acid exchange during host-pathogen interaction by combined stable-isotope time-resolved Raman spectral imaging

 2016 Feb 9;6:20811. doi: 10.1038/srep20811.

Abstract

This study investigates the temporal and spatial interchange of the aromatic amino acid phenylalanine (Phe) between human retinal pigment epithelial cell line (ARPE-19) and tachyzoites of the apicomplexan protozoan parasite Toxoplasma gondii (T. gondii). Stable isotope labelling by amino acids in cell culture (SILAC) is combined with Raman micro-spectroscopy to selectively monitor the incorporation of deuterium-labelled Phe into proteins in individual live tachyzoites. Our results show a very rapid uptake of l-Phe(D8) by the intracellular growing parasite. T. gondii tachyzoites are capable of extracting l-Phe(D8) from host cells as soon as it invades the cell. l-Phe(D8) from the host cell completely replaces the l-Phe within T. gondii tachyzoites 7-9 hours after infection. A quantitative model based on Raman spectra allowed an estimation of the exchange rate of Phe as 0.5-1.6 × 10(4) molecules/s. On the other hand, extracellular tachyzoites were not able to consume l-Phe(D8) after 24 hours of infection. These findings further our understanding of the amino acid trafficking between host cells and this strictly intracellular parasite. In particular, this study highlights new aspects of the metabolism of amino acid Phe operative during the interaction between T. gondii and its host cell. 
PMID:
 
26857158
 
[PubMed - in process]

In vitro Activity of Sorghum bicolor Extracts, 3-deoxyanthocyanidins, Against Toxoplasma gondii

 2016 Feb 5. pii: S0014-4894(16)30016-9. doi: 10.1016/j.exppara.2016.02.001. [Epub ahead of print]

Abstract

We investigated dried red leaf extracts of S. bicolor for activity against Toxoplasma gondii tachyzoites. S. bicolor red leaf extracts were obtained by bioassay-guided fractionation using ethanol and ethyl acetate as solvents. Analysis of the crude and fractionated extracts from S. bicolor using electrospray ionization mass spectrometry (ESI-MS) showed that they contained significant amounts of apigeninidin, luteolinidin, 7-methoxyapigeninidin, 5-methoxyapigeninidin, 5-methoxyluteolinidin, 5-7-dimethoxyapigeninidin or 5-7-dimethoxyluteolinidin, based on mass per charge (m/z). When tested in vitro, the IC50s for inhibitory activity against T. gondii tachyzoites' growth of the ethanol and ethyl acetate extracts were 2.3- and 4-fold, respectively, lower than their cytotoxic IC50s in mammalian cells. Ethyl acetate extracts fractionated in chloroform-methanol and chloroform had IC50s against T. gondii that were 56.1- and 3-fold lower than their respective cytotoxic IC50s in mammalian cells. These antiparasitic activities were found to be consistent with those of the respective pure 3-deoxyanthocyanidin compounds identified to be contained in the fractions in significant amounts. Further, we observed that, the position and number of methoxy groups possessed by the 3-deoyanthocyanidins influenced their antiparasitic activity. Together, our findings indicate that S. bicolor red-leaf 3-deoxyanthocyanidins-rich extracts have potent in vitro inhibitory activity against the proliferative stage of T. gondii parasites.
Copyright © 2016. Published by Elsevier Inc.

KEYWORDS: 

3-deoxyanthocyanidins extracts; Sorghum bicolor; Toxoplasma gondii; antiparasitic compounds
PMID:
 
26855040
 
[PubMed - as supplied by publisher]

A Systematic Meta-Analysis of Toxoplasma gondii Prevalence in Food Animals in the United States

 2016 Feb 8. [Epub ahead of print]

Abstract

Toxoplasma gondii is a widely distributed protozoan parasite. The Centers for Disease Control and Prevention reported that T. gondii is one of three pathogens (along with Salmonella and Listeria), that together account for >70% of all deaths due to foodborne illness in the United States. Food animals are reservoirs for T. gondii and act as one of the sources for parasite transmission to humans. Based on limited population-based data, the Food and Agriculture Organization/World Health Organization estimated that approximately 22% of human T. gondii infections are meatborne. The objective of the current study was to conduct a systematic meta-analysis to provide a precise estimation of T. gondii infection prevalence in food animals produced in the United States. Four databases were searched to collect eligible studies. Prevalence was estimated in six animal categories (confinement-raised market pigs, confinement-raised sows, non-confinement-raised pigs, lamb, goats, and non-confinement-raised chickens) by a quality-effects model. A wide variation in prevalence was observed in each animal category. Animals raised outdoors or that have outdoor access had a higher prevalence as compared with animals raised indoors. T. gondii prevalence in non-confinement-raised pigs ranked the highest (31.0%) followed by goats (30.7%), non-confinement-raised chickens (24.1%), lambs (22.0%), confinement-raised sows (16.7%), and confinement-raised market pigs (5.6%). These results indicate that T. gondii-infected animals are a food safety concern. The computed prevalence can be used as an important input in quantitative microbial risk assessment models to further predict public health burden.
PMID:
 
26854596
 
[PubMed - as supplied by publisher]

Morbid attraction to leopard urine in Toxoplasma-infected chimpanzees

 2016 Feb 8;26(3):R98-R99. doi: 10.1016/j.cub.2015.12.020.

Abstract

Parasites are sometimes capable of inducing phenotypic changes in their hosts to improve transmission [1]. Toxoplasma gondii, a protozoan that infects a broad range of warm-blooded species, is one example that supports the so-called 'parasite manipulation hypothesis': it induces modifications in rodents' olfactory preferences, converting an innate aversion for cat odor into attraction and probably favoring trophic transmission to feline species, its only definitive hosts [2]. In humans, T. gondii induces behavioral modifications such as personality changes, prolonged reaction times and decreased long-term concentration [3]. However, modern humans are not suitable intermediate hosts because they are no longer preyed upon by felines. Consequently, behavioral modifications in infected people are generally assumed to be side effects of toxoplasmosis or residual manipulation traits that evolved in appropriate intermediate hosts. An alternative hypothesis, however, states that these changes result from parasite manipulative abilities that evolved when human ancestors were still under significant feline predation [3,4]. As such, T. gondii also alters olfactory preferences in humans; infected men rate cat urine, but not tiger urine, as pleasant while non-infected men do not [5]. To unravel the origin of Toxoplasma-induced modifications in humans, we performed olfactory tests on a living primate still predated by a feline species. We found in our closest relative, the chimpanzee (Pan troglodytes troglodytes), that Toxoplasma-infected (TI) animals lost their innate aversion towards the urine of leopards (Panthera pardus), their only natural predator. By contrast, we observed no clear difference in the response of TI and Toxoplasma-non-infected (TN) animals towards urine collected from other definitive feline hosts that chimpanzees do not encounter in nature. Although the adaptive value of parasitically induced behavior should be assessed carefully, we suggest that the behavioral modification we report could increase the probability of chimpanzee predation by leopards for the parasite's own benefit. This possible parasite adaptation would hence suggest that Toxoplasma-induced modifications in modern humans are an ancestral legacy of our evolutionary past.
Copyright © 2016 Elsevier Ltd. All rights reserved.
PMID:
 
26859275
 
[PubMed - as supplied by publisher]

The FIKK kinase of Toxoplasma gondii is not essential for the parasite's lytic cycle

 2016 Feb 6. pii: S0020-7519(16)00022-9. doi: 10.1016/j.ijpara.2016.01.001. [Epub ahead of print]

Abstract

FIKK kinases are a novel family of kinases unique to the Apicomplexa. While most apicomplexans encode a single FIKK kinase, Plasmodium falciparum expresses 21 and piroplasms do not encode a FIKK kinase. FIKK kinases share a conserved C-terminal catalytic domain, but the N-terminal region is highly variable and contains no known functional domains. To date, FIKK kinases have been primarily studied in P. falciparum and Plasmodium berghei. Those that have been studied are exported from the parasite and associate with diverse locations in the infected erythrocyte cytosol or membrane. Deletion of individual P. falciparum FIKK kinases indicates that they may play a role in modification of the infected erythrocyte. The current study characterizes the single FIKK gene in Toxoplasma gondii to evaluate the importance of the FIKK kinase in an apicomplexan that has a single FIKK kinase. The TgFIKK gene encoded a protein of approximately 280 kDa. Endogenous tagging of the FIKK protein with Yellow Fluorescent Protein (YFP) showed that the FIKK protein exclusively localized to the posterior end of tachyzoites. A YFP-tagged FIKK and a Ty-tagged FIKK both co-localized with T. gondii membrane occupation and recognition nexus protein (TgMORN1) to the basal complex and were localized apical to inner membrane complex protein-5 (IMC5) and Centrin2. Deletion of TgFIKK, surprisingly, had no detectable effect on the parasite's lytic cycle in vitro in human fibroblast cells or in acute virulence in vivo. Thus, our results clearly show that while the FIKK kinase is expressed in tachyzoites, it is not essential for the lytic cycle of T. gondii.
Copyright © 2016. Published by Elsevier Ltd.

KEYWORDS: 

Basal complex; FIKK kinase; Intracellular pathogen; Parasite; Serine threonine kinases; Toxoplasma
PMID:
 
26859096
 
[PubMed - as supplied by publisher]

Toll-Like Receptor 11 (TLR11) Interacts with Flagellin and Profilin through Disparate Mechanisms

 2016 Feb 9;11(2):e0148987. doi: 10.1371/journal.pone.0148987.

Abstract

Toll-like receptors (TLRs) are innate immune receptors that sense a variety of pathogen-associated molecular patterns (PAMPs) by interacting with them and subsequently initiating signal transduction cascades that elicit immune responses. TLR11 has been shown to interact with two known protein PAMPs: Salmonella and E. coli flagellin FliC and Toxoplasma gondii profilin-like protein. Given the highly divergent biology of these pathogens recognized by TLR11, it is unclear whether common mechanisms are used to recognize these distinct protein PAMPs. Here we show that TLR11 interacts with these two PAMPs using different receptor domains. Furthermore, TLR11 binding to flagellin and profilin exhibits differential dependency on pH and receptor ectodomain cleavage.
PMID:
 
26859749
 
[PubMed - as supplied by publisher]

Friday, February 05, 2016

Toxoplasma gondii Arginine Methyltransferase 1 (PRMT1) Is Necessary for Centrosome Dynamics during Tachyzoite Cell Division

2016 Feb 2;7(1). pii: e02094-15. doi: 10.1128/mBio.02094-15.


The arginine methyltransferase family (PRMT) has been implicated in a variety of cellular processes, including signal transduction, epigenetic regulation, and DNA repair pathways. PRMT1 is thought to be responsible for the majority of PRMT activity in Toxoplasma gondii, but its exact function is unknown. To further define the biological function of the PRMT family, we generated T. gondii mutants lacking PRMT1 (Δprmt1) by deletion of the PRMT1 gene. Δprmt1 parasites exhibit morphological defects during cell division and grow slowly, and this phenotype reverses in the Δprmt::PRMT1mRFP complemented strain. Tagged PRMT1 localizes primarily in the cytoplasm with enrichment at the pericentriolar material, and the strain lacking PRMT1 is unable to segregate progeny accurately. Unlike wild-type and complemented parasites, Δprmt1 parasites have abnormal daughter buds, perturbed centrosome stoichiometry, and loss of synchronous replication. Whole-genome expression profiling demonstrated differences in expression of cell-cycle-regulated genes in the Δprmt1 strain relative to the complemented Δprmt1::PRMT1mRFP and parental wild-type strains, but these changes do not correlate with a specific block in cell cycle. Although PRMT1's primary biological function was previously proposed to be methylation of histones, our studies suggest that PRMT1 plays an important role within the centrosome to ensure the proper replication of the parasite.

IMPORTANCE:

Apicomplexan parasites include several important pathogens, including Toxoplasma gondii, a major cause of opportunistic infections and congenital birth defects. These parasites divide using a unique form of cell division called endodyogeny that is different from those of most eukaryotes. PRMT1 is a conserved arginine methyltransferase that was thought to regulate gene expression of T. gondii by modifying histone methylation. Using genetic techniques, we show that disruption of PRMT1 affects the parasite's ability to perform accurate cell division. Our studies reveal an unexpected role for arginine methylation in centrosome biology and regulation of parasite replication.
Copyright © 2016 El Bissati et al.
PMID:
26838719
[PubMed - in process]

A Novel Secreted Protein, MYR1, Is Central to Toxoplasma's Manipulation of Host Cells

2016 Feb 2;7(1). pii: e02231-15. doi: 10.1128/mBio.02231-15.


The intracellular protozoan Toxoplasma gondii dramatically reprograms the transcriptome of host cells it infects, including substantially up-regulating the host oncogene c-myc. By applying a flow cytometry-based selection to infected mouse cells expressing green fluorescent protein fused to c-Myc (c-Myc-GFP), we isolated mutant tachyzoites defective in this host c-Myc up-regulation. Whole-genome sequencing of three such mutants led to the identification of MYR1 (Myc regulation 1; TGGT1_254470) as essential for c-Myc induction. MYR1 is a secreted protein that requires TgASP5 to be cleaved into two stable portions, both of which are ultimately found within the parasitophorous vacuole and at the parasitophorous vacuole membrane. Deletion of MYR1 revealed that in addition to its requirement for c-Myc up-regulation, the MYR1 protein is needed for the ability of Toxoplasma tachyzoites to modulate several other important host pathways, including those mediated by the dense granule effectors GRA16 and GRA24. This result, combined with its location at the parasitophorous vacuole membrane, suggested that MYR1 might be a component of the machinery that translocates Toxoplasma effectors from the parasitophorous vacuole into the host cytosol. Support for this possibility was obtained by showing that transit of GRA24 to the host nucleus is indeed MYR1-dependent. As predicted by this pleiotropic phenotype, parasites deficient in MYR1 were found to be severely attenuated in a mouse model of infection. We conclude, therefore, that MYR1 is a novel protein that plays a critical role in how Toxoplasma delivers effector proteins to the infected host cell and that this is crucial to virulence.

IMPORTANCE:

Toxoplasma gondii is an important human pathogen and a model for the study of intracellular parasitism. Infection of the host cell with Toxoplasma tachyzoites involves the introduction of protein effectors, including many that are initially secreted into the parasitophorous vacuole but must ultimately translocate to the host cell cytosol to function. The work reported here identified a novel protein that is required for this translocation. These results give new insight into a very unusual cell biology process as well as providing a potential handle on a pathway that is necessary for virulence and, therefore, a new potential target for chemotherapy.
Copyright © 2016 Franco et al.
PMID:
26838724
[PubMed - in process]

A MORN1-associated HAD phosphatase in the basal complex is essential for Toxoplasma gondii daughter budding

2016 Feb 3. doi: 10.1111/cmi.12574. [Epub ahead of print]


Apicomplexan parasites replicate by several budding mechanisms with two well-characterized examples being Toxoplasma endodyogeny and Plasmodium schizogony. Completion of budding requires the tapering of the nascent daughter buds toward the basal end, driven by contraction of the basal complex. This contraction is not executed by any of the known cell division associated contractile mechanisms and in order to reveal new components of the unusual basal complex we performed a yeast two-hybrid screen with its major scaffolding protein, TgMORN1. Here we report on a conserved protein with a haloacid dehalogenase (HAD) phosphatase domain, hereafter named HAD2a, identified by yeast two-hybrid. HAD2a has demonstrated enzyme-activity in vitro, localizes to the nascent daughter buds and co-localizes with MORN1 to the basal complex during its contraction. Conditional knockout of HAD2a in Toxoplasma interferes with basal complex assembly, which leads to incomplete cytokinesis and conjoined daughters that ultimately results in disrupted proliferation. In Plasmodium, we further confirmed localization of the HAD2a ortholog to the basal complex toward the end of schizogony. In conclusion, our work highlights an essential role for this HAD phosphatase across apicomplexan budding and suggests a regulatory mechanism of differential phosphorylation on the structure and/or contractile function of the basal complex.
This article is protected by copyright. All rights reserved.
PMID:
26840427
[PubMed - as supplied by publisher]

Extended epigenotype in a Rattus novergicus - Toxoplasma gondii association

2015 Mar 4;8(1):e992743.


Several studies demonstrate that rats (Rattus novergicus) infected with protozoan parasite Toxoplasma gondii exhibit lesser fear to cat odors. This is thought to increase transmission of the parasite to its definitive hosts, i.e. cats. This is an example of extended phenotype where a gene of an organism allegedly creates a phenotype in another organism. We examined a possible proximate mechanism for this phenotype, describing an epigenetic change in arginine vasopressin gene in medial amygdala of male rats. Exogenously mimicking medial amygdala DNA hypomethylation resulted in reduction of fear to cat odors in uninfected animals, thus suggesting sufficiency. Systemic blockade of infection-induced DNA hypomethylation countermanded infection-induced behavioral change, thus suggesting necessity. This leads us to propose an epigenetic basis for this extended phenotype.

KEYWORDS:

DNA methylation; MSRE; behavioral manipulation; fear; nonapeptide; parasite; testosterone
PMID:
26844540
[PubMed - as supplied by publisher]

Gliding Associated Proteins Play Essential Roles during the Formation of the Inner Membrane Complex of Toxoplasma gondii

2016 Feb 4;12(2):e1005403. doi: 10.1371/journal.ppat.1005403. eCollection 2016.


The inner membrane complex (IMC) of apicomplexan parasites is a specialised structure localised beneath the parasite's plasma membrane, and is important for parasite stability and intracellular replication. Furthermore, it serves as an anchor for the myosin A motor complex, termed the glideosome. While the role of this protein complex in parasite motility and host cell invasion has been well described, additional roles during the asexual life cycle are unknown. Here, we demonstrate that core elements of the glideosome, the gliding associated proteins GAP40 and GAP50 as well as members of the GAPM family, have critical roles in the biogenesis of the IMC during intracellular replication. Deletion or disruption of these genes resulted in the rapid collapse of developing parasites after initiation of the cell cycle and led to redistribution of other glideosome components.
PMID:
26845335
[PubMed - as supplied by publisher]

Vacuolar protein sorting mechanisms in apicomplexan parasites

2016 Feb 1. pii: S0166-6851(16)30007-X. doi: 10.1016/j.molbiopara.2016.01.007. [Epub ahead of print]


The phylum Apicomplexa comprises more than 5000 species including pathogens of clinical and economical importance. These obligate intracellular parasites possess a highly complex endomembrane system to build among others three morphologically distinct secretory organelles: rhoptries, micronemes and dense granules. Proteins released by these organelles are essential for invasion and hijacking of the host cell. Due to the complexity of the internal organization of these parasites, a wide panoply of trafficking factors was expected to be required for the correct sorting of proteins towards the various organelles. However, T. gondii and other apicomplexan parasites contain only a core set of these factors and several of the vacuolar protein sorting (VPS) homologs found in most eukaryotes have been lost in this phylum. In this review, we will summarise our current knowledge about the role of trafficking complexes in Toxoplasma gondii, highlighting recent studies focused on complexes formed by VPS proteins. We also present a novel, hypothetical model, suggesting the recycling of parasite membrane and micronemal proteins.
Copyright © 2016. Published by Elsevier B.V.

KEYWORDS:

Multi-subunit tethering complex; Toxoplasma gondii; microneme recycling; vesicular protein sorting
PMID:
26844642
[PubMed - as supplied by publisher]

Thursday, February 04, 2016

The conserved apicomplexan Aurora kinase TgArk3 is involved in endodyogeny, duplication rate and parasite virulence

2016 Feb 2. doi: 10.1111/cmi.12571. [Epub ahead of print]


Aurora kinases are eukaryotic serine/threonine protein kinases that regulate key events associated with chromatin condensation, centrosome and spindle function, and cytokinesis. Elucidating the roles of Aurora kinases in apicomplexan parasites is crucial to understand the cell cycle control during Plasmodium schizogony or Toxoplasma endodyogeny. Here, we report on the localization of two previously uncharacterized Toxoplasma Aurora-related kinases (Ark2 and Ark3) in tachyzoites and of the uncharacterized Ark3 orthologue in Plasmodium falciparum erythrocytic stages. In T. gondii, we show that TgArk2 and TgArk3 concentrate at specific sub-cellular structures linked to parasite division: the mitotic spindle and intranuclear mitotic structures (TgArk2), and the outer core of the centrosome and the budding daughter cells cytoskeleton (TgArk3). By tagging the endogenous PfArk3 gene with the green fluorescent protein (GFP) in live parasites, we show that PfArk3 protein expression peaks late in schizogony and localizes at the periphery of budding schizonts. Disruption of the TgArk2 gene reveals no essential function for tachyzoite propagation in vitro, which is surprising giving that the P. falciparum and P. berghei orthologues are essential for erythrocyte schizogony. In contrast, knock-down of TgArk3 protein results in pronounced defects in parasite division and a major growth deficiency. TgArk3-depleted parasites display several defects, such as reduced parasite growth rate, delayed egress and parasite duplication, defect in rosette formation, reduced parasite size and invasion efficiency and lack of virulence in mice. Our study provides new insights into cell cycle control in Toxoplasma and malaria parasites, and highlights Aurora kinase 3 as potential drug target.

KEYWORDS:

apicomplexa; aurora kinases; centrosome; endodyogeny; plasmodium falciparum; replication; schizogony; spindle pole bodies; tet-inducible system; toxoplasma gondii
PMID:
26833682
[PubMed - as supplied by publisher]

Characterization of an IgG monoclonal antibody targeted to both tissue cyst and sporocyst walls of Toxoplasma gondii

2016 Jan 30. pii: S0014-4894(16)30014-5. doi: 10.1016/j.exppara.2016.01.014. [Epub ahead of print]


Toxoplasma gondii infects animals habiting terrestrial and aquatic environments. Its oocysts and tissue cysts are important for the horizontal transmission of this parasite. The oocyst and tissue cyst walls are crucial for the ability of the parasite to persist in the environment or in animal tissues, respectively. However, the composition of these walls is not well understood. We report the generation of monoclonal antibodies directed against wall components using mice immunized with oocyst antigens of T. gondii. One monoclonal antibody (mAb) G1/19 reacted solely with T. gondii sporozoites. The respective antigen had a relative molecular weight (Mr) of 30 kDa. MAb G1/19 failed to react with sporozoites of any other coccidian parasite species tested (Hammondia hammondi, Hammondia heydorni, Cystoisospora felis, Eimeria bovis, Sarcocystis sp.). Another mAb, designated K8/15-15, recognized antigens in sporocyst walls of the parasite and in the walls of in vivo or in vitro produced tissue cysts, as demonstrated by immunofluorescence and immunoblot assays. Antigens of 80 to a high molecular weight protein of about 350 kDa Mr were recognized by this antibody using antigen extracts from sporocysts, and from in vitro or in vivo generated tissue cysts of the parasite. Tissue cyst and sporocyst walls of H. hammondi and H. heydorni, and tissue cysts of Neospora caninum were also recognized by mAb K8/15-15. Sporocyst walls of C. felis also reacted to this mAb. The cyst walls of Sarcocystis sp. and B. besnoiti were not recognized by mAbK8/15-15. Reactivity by a single mAb against T. gondii antigens in tissue cysts and sporocysts had not been reported previously. MAb K8/15-15 may be a practical tool for the identification of both cysts and sporocysts of the parasite, and may also be potentially employed in proteomic studies on the identification of new components of the cyst and sporocyst walls of T. gondii.
Copyright © 2016. Published by Elsevier Inc.

KEYWORDS:

Toxoplasma; immunoblot; monoclonal antibody; oocyst; sporocyst; tissue cyst
PMID:
26836446
[PubMed - as supplied by publisher]

The Apoptotic Role of Metacaspase in Toxoplasma gondii

2016 Jan 19;6:1560. doi: 10.3389/fmicb.2015.01560. eCollection 2015.


Toxoplasma gondii is a major opportunistic pathogen that spreads in a range of animal species and human beings. Quite a few characterizations of apoptosis have been identified in T. gondii treated with apoptosis inducers, but the molecular mechanisms of the pathway are not clearly understood. Metacaspases are caspase-like cysteine proteases that can be found in plants, fungi, and protozoa in which caspases are absent. Metacaspases are multifunctional proteases involved in apoptosis-like cell death, insoluble protein aggregate clearance, and cell proliferation. To investigate whether T. gondii metacaspase (TgMCA) is involved in the apoptosis of the parasites, we generated TgMCA mutant strains. Western blot analysis indicated that the autoproteolytic processing of TgMCA was the same as that for metacaspases of some other species. Indirect immunofluorescence assay (IFA) showed that TgMCA was dispersed throughout the cytoplasm and relocated to the nucleus when the parasites were exposed to the extracellular environment, which indicated the execution of its function in the nucleus. The number of apoptosis parasites was significantly diminished in the TgMCA knockout strain and increased in the TgMCA overexpression strain after treatment with extracellular buffer, as determined by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The lack of TgMCA did not affect the parasite propagation in vitro and virulence in vivo, suggesting that it is probably redundant in parasite propagation. But overexpression of TgMCA reduced the intracellular parasites growth in vitro. The TgMCA knockout strain showed more viability in extracellular buffer compared to the parental and overexpression lines. In this study, we demonstrated that TgMCA contributes to the apoptosis of T. gondii.

KEYWORDS:

T. gondii; apoptosis; knockout; metacaspase; programmed cell death
PMID:
26834715
[PubMed]

Monday, February 01, 2016

Identification of new palmitoylated proteins in Toxoplasma gondii

 2016 Jan 26. pii: S1570-9639(16)30002-4. doi: 10.1016/j.bbapap.2016.01.010. [Epub ahead of print]

Abstract

Protein palmitoylation has been shown to be an important post-translational modification in eukaryotic cells. This modification alters the localization and/or the function of the targeted protein. In recent years, protein palmitoylation has risen in importance in apicomplexan parasites as well. In Toxoplasma gondii, some proteins have been reported to be modified by palmitate. With the development of new techniques that allow the isolation of palmitoylated proteins, this significant post-translational modification has begun to be studied in more detail in T. gondii. Here we describe the palmitoylome of the tachyzoite stage of T. gondii using a combination of the acyl-biotin exchange chemistry method and mass spectrometry analysis. We identified 401 proteins found in multiple cellular compartments, with a wide range of functions that vary from metabolic processes, gliding and host-cell invasion to even regulation of transcription and translation. Besides, we found that more rhoptry proteins than the ones already described for Toxoplasma are palmitoylated, suggesting an important role for this modification in the invasion mechanism of the host-cell. This study documents that protein palmitoylation is a common modification in T. gondii that could have an impact on different cellular processes.
Copyright © 2016. Published by Elsevier B.V.

KEYWORDS: 

Acyl-biotin exchange; Host-cell invasion; Palmitoylome; Protein identification; Rhoptry; Toxoplasma gondii
PMID:
 
26825284
 
[PubMed - as supplied by publisher]

Toxoplasma gondii peptide ligands open the gate of the HLA class I binding groove

 2016 Jan 29;5. pii: e12556. doi: 10.7554/eLife.12556. [Epub ahead of print]

Abstract

HLA class I presentation of pathogen-derived peptide ligands is essential for CD8+ T-cell recognition of Toxoplasma gondiiinfected cells. Currently, little data exist pertaining to peptides that are presented after T. gondii infection. Herein we purify HLA-A*02:01 complexes from T. gondii infected cells and characterize the peptide ligands using LCMS. We identify 195 T. gondiiencoded ligands originating from both secreted and cytoplasmic proteins. Surprisingly, T. gondii ligands are significantly longer than uninfected host ligands, and these longer pathogen-derived peptides maintain a canonical N-terminal binding core yet exhibit a C-terminal extension of 1-30 amino acids. Structural analysis demonstrates that binding of extended peptides opens the HLA class I F' pocket, allowing the C-terminal extension to protrude through one end of the binding groove. In summary, we demonstrate that unrealized structural flexibility makes MHC class I receptive to parasite-derived ligands that exhibit unique C-terminal peptide extensions.

KEYWORDS: 

human; immunology; infectious disease; microbiology
PMID:
 
26824387
 
[PubMed - as supplied by publisher] 

Wednesday, January 27, 2016

Garcinol inhibits GCN5-mediated lysine acetyltransferase activity and prevents replication of the parasite Toxoplasma gondii

2016 Jan 25. pii: AAC.03059-15. [Epub ahead of print]


Lysine acetylation is a critical post-translational modification that influences protein activity, stability, and binding properties. The acetylation of histone proteins in particular is a well-characterized feature of gene expression regulation. In the protozoan parasite Toxoplasma gondii, a number of lysine acetyltransferases (KATs) contribute to gene expression and are essential for parasite viability. The natural product, garcinol, was recently reported to inhibit enzymatic activities of GCN5 and p300 family KATs in other species. Here we show that garcinol inhibits TgGCN5b, the only nuclear GCN5-family KAT known to be required for Toxoplasma tachyzoite replication. Treatment of tachyzoites with garcinol led to a reduction of global lysine acetylation, particularly on histone H3 and TgGCN5b itself. We also performed RNAseq analysis on intracellular tachyzoites treated with garcinol, which revealed increasing aberrant gene expression coincident with increasing concentrations of garcinol. The majority of the genes that were most significantly affected by garcinol were also associated with TgGCN5b in a previous ChIP-chip analysis. The dysregulated gene expression induced by garcinol significantly inhibits Toxoplasma tachyzoite replication, and the concentrations used exhibit no overt toxicity on human host cells. Garcinol also inhibits Plasmodium falciparum asexual replication with a similar IC50 as Toxoplasma. Together, these data support that pharmacological inhibition of TgGCN5b leads to a catastrophic failure in gene expression control that prevents parasite replication.
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
PMID:
26810649
[PubMed - as supplied by publisher]

Wednesday, January 20, 2016

Infection of male rats with Toxoplasma gondii induces effort-aversion in a T-maze decision-making task

2016 Jan 16. pii: S0889-1591(16)30015-0. doi: 10.1016/j.bbi.2016.01.015. [Epub ahead of print]


Rats chronically infected with protozoan Toxoplasma gondii exhibit greater delay aversion in an inter-temporal task. Moreover Toxoplasma gondii infection also results in dendritic atrophy of basolateral amygdala neurons. Basolateral amygdala is reported to bias decision making towards greater effortful alternatives. In this context, we report that Toxoplasma gondii increases effort aversion in infected male rats. This host-parasite association has been widely studied in the context of loss of innate fear in the infected males. It is suggested that reduced fear towards predators reflects a parasitic behavioral manipulation to enhance trophic transmission of Toxoplasma gondii. Observations reported here extend this paradigm away from a monolithic change in fear and towards a multi-dimensional change in decision making.
Copyright © 2016. Published by Elsevier Inc.

KEYWORDS:

Behavioral manipulation; Decision making; Effort discounting; Operant learning; Parasites
PMID:
26783701
[PubMed - as supplied by publisher]