Friday, March 27, 2015

Attenuated Toxoplasma gondii stimulates immunity to pancreatic cancer by manipulation of myeloid cell populations

2015 Mar 24. pii: canimm.0235.2014. [Epub ahead of print]
 
 
Suppressive myeloid cells represent a significant barrier to the generation of productive anti-tumor immune responses to many solid tumors. Eliminating or reprogramming suppressive myeloid cells to abrogate tumor-associated immune suppression is a promising therapeutic approach. We asked whether treatment of established aggressive disseminated pancreatic cancer with the immunotherapeutic attenuated Toxoplasma gondii vaccine strain CPS would trigger tumor-associated myeloid cells to generate therapeutic anti-tumor immune responses. CPS treatment significantly decreased tumor-associated macrophages and markedly increased dendritic cell infiltration of the pancreatic tumor microenvironment. Tumor-resident macrophages and dendritic cells, particularly cells actively invaded by CPS, increased expression of co-stimulatory molecules CD80 and CD86 and concomitantly boosted their production of IL-12. CPS treatment increased CD4+ and CD8+ T cell infiltration into the tumor microenvironment, activated tumor-resident T cells, and increased IFN-γ production by T cell populations. CPS treatment provided a significant therapeutic benefit in pancreatic tumor-bearing mice. This therapeutic benefit depended on IL-12 and IFN-γ production, MyD88 signaling, and CD8+ T cell populations. Although CD4+ T cells exhibited activated effector phenotypes and produced IFN-γ, CD4+ T cells as well as NK cells were not required for the therapeutic benefit. In addition, CD8+ T cells isolated from CPS treated tumor-bearing mice produced IFN-γ after re-exposure to pancreatic tumor antigen, suggesting this immunotherapeutic treatment stimulated tumor cell antigen-specific CD8+ T cell responses. This work highlights the potency and immunotherapeutic efficacy of CPS treatment and demonstrates the significance of targeting tumor-associated myeloid cells as a mechanism to stimulate more effective immunity to pancreatic cancer.
Copyright © 2015, American Association for Cancer Research.
PMID:
25804437
[PubMed - as supplied by publisher]

Wednesday, March 25, 2015

Ectopic Expression of a Neospora caninum Kazal Type Inhibitor Triggers Developmental Defects in Toxoplasma and Plasmodium

2015 Mar 24;10(3):e0121379. doi: 10.1371/journal.pone.0121379.
 
 
Regulated proteolysis is known to control a variety of vital processes in apicomplexan parasites including invasion and egress of host cells. Serine proteases have been proposed as targets for drug development based upon inhibitor studies that show parasite attenuation and transmission blockage. Genetic studies suggest that serine proteases, such as subtilisin and rhomboid proteases, are essential but functional studies have proved challenging as active proteases are difficult to express. Proteinaceous Protease Inhibitors (PPIs) provide an alternative way to address the role of serine proteases in apicomplexan biology. To validate such an approach, a Neospora caninum Kazal inhibitor (NcPI-S) was expressed ectopically in two apicomplexan species, Toxoplasma gondii tachyzoites and Plasmodium berghei ookinetes, with the aim to disrupt proteolytic processes taking place within the secretory pathway. NcPI-S negatively affected proliferation of Toxoplasma tachyzoites, while it had no effect on invasion and egress. Expression of the inhibitor in P. berghei zygotes blocked their development into mature and invasive ookinetes. Moreover, ultra-structural studies indicated that expression of NcPI-S interfered with normal formation of micronemes, which was also confirmed by the lack of expression of the micronemal protein SOAP in these parasites. Our results suggest that NcPI-S could be a useful tool to investigate the function of proteases in processes fundamental for parasite survival, contributing to the effort to identify targets for parasite attenuation and transmission blockage.
PMID:
25803874
[PubMed - as supplied by publisher]

Saturday, March 21, 2015

Yeast Three-Hybrid Screen Identifies TgBRADIN/GRA24 as a Negative Regulator of Toxoplasma gondii Bradyzoite Differentiation

 2015 Mar 19;10(3):e0120331. doi: 10.1371/journal.pone.0120331. eCollection 2015.

Abstract

Differentiation of the protozoan parasite Toxoplasma gondii into its latent bradyzoite stage is a key event in the parasite's life cycle. Compound 2 is an imidazopyridine that was previously shown to inhibit the parasite lytic cycle, in part through inhibition of parasite cGMP-dependent protein kinase. We show here that Compound 2 can also enhance parasite differentiation, and we use yeast three-hybrid analysis to identify TgBRADIN/GRA24 as a parasite protein that interacts directly or indirectly with the compound. Disruption of the TgBRADIN/GRA24 gene leads to enhanced differentiation of the parasite, and the TgBRADIN/GRA24 knockout parasites show decreased susceptibility to the differentiation-enhancing effects of Compound 2. This study represents the first use of yeast three-hybrid analysis to study small-molecule mechanism of action in any pathogenic microorganism, and it identifies a previously unrecognized inhibitor of differentiation in T. gondii. A better understanding of the proteins and mechanisms regulating T. gondii differentiation will enable new approaches to preventing the establishment of chronic infection in this important human pathogen. 
PMID:
 
25789621
 
[PubMed - in process] 

Thursday, March 19, 2015

Proteome-Wide Lysine Acetylation in Cortical Astrocytes and Alterations That Occur during Infection with Brain Parasite Toxoplasma gondii

2015 Mar 18;10(3):e0117966. doi: 10.1371/journal.pone.0117966.
 
 
Lysine acetylation is a reversible post-translational modification (PTM) that has been detected on thousands of proteins in nearly all cellular compartments. The role of this widespread PTM has yet to be fully elucidated, but can impact protein localization, interactions, activity, and stability. Here we present the first proteome-wide survey of lysine acetylation in cortical astrocytes, a subtype of glia that is a component of the blood-brain barrier and a key regulator of neuronal function and plasticity. We identified 529 lysine acetylation sites across 304 proteins found in multiple cellular compartments that largely function in RNA processing/transcription, metabolism, chromatin biology, and translation. Two hundred and seventy-seven of the acetylated lysines we identified on 186 proteins have not been reported previously in any other cell type. We also mapped an acetylome of astrocytes infected with the brain parasite, Toxoplasma gondii. It has been shown that infection with T. gondii modulates host cell gene expression, including several lysine acetyltransferase (KAT) and deacetylase (KDAC) genes, suggesting that the host acetylome may also be altered during infection. In the T. gondii-infected astrocytes, we identified 34 proteins exhibiting a level of acetylation >2-fold and 24 with a level of acetylation more than 2-fold relative to uninfected astrocytes. Our study documents the first acetylome map for cortical astrocytes, uncovers novel lysine acetylation sites, and demonstrates that T. gondii infection produces an altered acetylome.
PMID:
25786129
[PubMed - as supplied by publisher]

Retraction for Xu et al., Reactive Oxygen Species-Triggered Trophoblast Apoptosis Is Initiated by Endoplasmic Reticulum Stress via Activation of Caspase-12, CHOP, and the JNK Pathway in Toxoplasma gondii Infection in Mice

2015 Apr;83(4):1735. doi: 10.1128/IAI.00118-15.
 

PMID:
25784756
[PubMed - in process]

Infection and inflammation in schizophrenia and bipolar disorder: a genome wide study for interactions with genetic variation

2015 Mar 17;10(3):e0116696. doi: 10.1371/journal.pone.0116696. eCollection 2015.
 
 
Inflammation and maternal or fetal infections have been suggested as risk factors for schizophrenia (SZ) and bipolar disorder (BP). It is likely that such environmental effects are contingent on genetic background. Here, in a genome-wide approach, we test the hypothesis that such exposures increase the risk for SZ and BP and that the increase is dependent on genetic variants. We use genome-wide genotype data, plasma IgG antibody measurements against Toxoplasma gondii, Herpes simplex virus type 1, Cytomegalovirus, Human Herpes Virus 6 and the food antigen gliadin as well as measurements of C-reactive protein (CRP), a peripheral marker of inflammation. The subjects are SZ cases, BP cases, parents of cases and screened controls. We look for higher levels of our immunity/infection variables and interactions between them and common genetic variation genome-wide. We find many of the antibody measurements higher in both disorders. While individual tests do not withstand correction for multiple comparisons, the number of nominally significant tests and the comparisons showing the expected direction are in significant excess (permutation p=0.019 and 0.004 respectively). We also find CRP levels highly elevated in SZ, BP and the mothers of BP cases, in agreement with existing literature, but possibly confounded by our inability to correct for smoking or body mass index. In our genome-wide interaction analysis no signal reached genome-wide significance, yet many plausible candidate genes emerged. In a hypothesis driven test, we found multiple interactions among SZ-associated SNPs in the HLA region on chromosome 6 and replicated an interaction between CMV infection and genotypes near the CTNNA3 gene reported by a recent GWAS. Our results support that inflammatory processes and infection may modify the risk for psychosis and suggest that the genotype at SZ-associated HLA loci modifies the effect of these variables on the risk to develop SZ.
PMID:
25781172
[PubMed - in process]

Expression of the glycolytic enzymes enolase and lactate dehydrogenase during the early phase of Toxoplasma differentiation is regulated by an intron retention mechanism

2015 Mar 16. doi: 10.1111/mmi.12999. [Epub ahead of print]
 
 
The intracellular parasite Toxoplasma gondii converts from a rapidly replicating tachyzoite form during acute infection to a quiescent encysted bradyzoite stage that persists inside long lived cells during chronic infection. Bradyzoites adopt reduced metabolism and slow replication while waiting for an opportunity to recrudesce the infection within the host. Inter-conversion between these two developmental stages is characterized by expression of glycolytic isoenzymes that play key roles in parasite metabolism. The parasite genome encodes two isoforms of lactate dehydrogenase (LDH1 and LDH2) and enolase (ENO1 and ENO2) that are expressed in a stage specific manner. Expression of different isoforms of these enzymes allows T. gondii to rapidly adapt to diverse metabolic requirements necessary for either a rapid replication of the tachyzoite stage or a quiescent lifestyle typical of the bradyzoites. Herein we identified unspliced forms of LDH and ENO transcripts produced during transition between these two parasite stages suggestive of an intron retention mechanism to promptly exchange glycolytic isoforms for rapid adaptation to environmental changes. We also identified key regulatory elements in the enolase transcription units, revealing cooperation between the ENO2 5'-untranslated region and the ENO2 intron along with identifying a role for the ENO1 3'-untranslated region in stage specific expression.
This article is protected by copyright. All rights reserved.
PMID:
25777509
[PubMed - as supplied by publisher]

Nonreplicating cyst-defective type II Toxoplasma gondii vaccines stimulate protective immunity to acute and chronic infection

2015 Mar 16. pii: IAI.02756-14. [Epub ahead of print]
 
 
Live attenuated vaccine strains such as type I nonreplicating uracil auxotroph mutants are highly effective in eliciting life-long immunity to virulent acute infection by Toxoplasma gondii. However, it is currently unknown whether vaccine elicited immunity can provide protection against acute infection and also prevent chronic infection. To address this problem we developed nonreverting, nonreplicating, live attenuated uracil auxotroph vaccine strains in the type II Δku80 genetic background by targeting the deletion of the orotidine 5' -monophosphate decarboxylase (OMPDC) and uridine phosphorylase (UP) genes. Deletion of OMPDC induced a severe uracil auxotrophy with loss of replication, loss of virulence in mice, and loss of the ability to develop cysts and chronic infection. Vaccination of mice using type II Δku80Δompdc mutants stimulated a fully protective CD8+ T cell dependent immunity that prevented acute infection by type I and type II strains of T. gondii, and this vaccination also severely reduced or prevented cyst formation after type II challenge infection. Nonreverting, nonreplicating, and non-cyst forming Δompdc mutants provide new tools to examine protective immune responses elicited by vaccination with a live attenuated type II vaccine.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
PMID:
25776745
[PubMed - as supplied by publisher]

Tuesday, March 17, 2015

REDHORSE-REcombination and Double crossover detection in Haploid Organisms using next-geneRation SEquencing data

 2015 Feb 26;16(1):133. [Epub ahead of print]

Abstract

BACKGROUND: 

Next-generation sequencing technology provides a means to study genetic exchange at a higher resolution than was possible using earlier technologies. However, this improvement presents challenges as the alignments of next generation sequence data to a reference genome cannot be directly used as input to existing detection algorithms, which instead typically use multiple sequence alignments as input. We therefore designed a software suite called REDHORSE that uses genomic alignments, extracts genetic markers, and generates multiple sequence alignments that can be used as input to existing recombination detection algorithms. In addition, REDHORSE implements a custom recombination detection algorithm that makes use of sequence information and genomic positions to accurately detect crossovers. REDHORSE is a portable and platform independent suite that provides efficient analysis of genetic crosses based on Next-generation sequencing data.

RESULTS: 

We demonstrated the utility of REDHORSE using simulated data and real Next-generation sequencing data. The simulated dataset mimicked recombination between two known haploid parental strains and allowed comparison of detected break points against known true break points to assess performance of recombination detection algorithms. A newly generated NGS dataset from a genetic cross of Toxoplasma gondii allowed us to demonstrate our pipeline. REDHORSE successfully extracted the relevant genetic markers and was able to transform the read alignments from NGS to the genome to generate multiple sequence alignments. Recombination detection algorithm in REDHORSE was able to detect conventional crossovers and double crossovers typically associated with gene conversions whilst filtering out artifacts that might have been introduced during sequencing or alignment. REDHORSE outperformed other commonly used recombination detection algorithms in finding conventional crossovers. In addition, REDHORSE was the only algorithm that was able to detect double crossovers.

CONCLUSION: 

REDHORSE is an efficient analytical pipeline that serves as a bridge between genomic alignments and existing recombination detection algorithms. Moreover, REDHORSE is equipped with a recombination detection algorithm specifically designed for Next-generation sequencing data. REDHORSE is portable, platform independent Java based utility that provides efficient analysis of genetic crosses based on Next-generation sequencing data. REDHORSE is available at http://redhorse.sourceforge.net/.
PMID:
 
25766039
 
[PubMed - as supplied by publisher] 
PMCID:
 
PMC4348101
 

Treatment with anti-toxoplasmic activity (TATA) for toxoplasma positive patients with bipolar disorders or schizophrenia: A cross-sectional study

 2015 Feb 24. pii: S0022-3956(15)00043-6. doi: 10.1016/j.jpsychires.2015.02.011. [Epub ahead of print]

Abstract

OBJECTIVE: 

The association between Toxoplasma gondii seropositivity and respectively Bipolar Disorder (BD) and Schizophrenia/Schizoaffective disorder (SZ) is one of the most studied link between one pathogen and psychiatric disorders. The aim of the present study was thus to retrospectively determine if the administration of an antipsychotic and/or a mood stabilizer having known in vitro Anti-Toxoplasmic Activity (TATA+) was associated with a better clinical outcome in a population of 152 BD or 114 SZ patients and seropositive for T. gondii infection compared to patients receiving a treatment without anti-toxoplasmic activity (TATA-).

METHODS: 

This multicenter study was conducted in an academic public hospital during a 3-years period between 2009 and 2011. All consecutive inpatients and outpatients with SZ or BD diagnosis with a stable treatment for more than 4 weeks were recruited. socio-demographic and clinical characteristics measured with validated scales as well as a serological status for toxoplasmic infection were included. Treatments were classified according to their in vitro antitoxoplasmic activity. A multivariate model was used to determine the clinical characteristics that were significantly different between patients receiving a treatment with no antitoxoplasmic activity compared to others.

RESULTS: 

BD patients with positive serum antibodies against T. gondii presented more lifetime depressive episodes (p = 0.048) after adjustment for age, sex and sociodemographic characteristics when treated by drug having no anti-toxo activity, compared to patients having received drugs with anti-toxo activity. A significant difference was not found in BD toxonegative patients and in SZ toxopositive or toxonegative patients.

CONCLUSIONS: 

It seems to be of importance to consider prescribing a drug with a clear anti-toxoplasmic activity (TATA+) for BD patients seropositive to T. gondii, in particular valproate that was found as the mood stabilizer with the highest antitoxoplasmic activity. Prospective randomized controlled trials are warranted to confirm this preliminary data.
Copyright © 2015 Elsevier Ltd. All rights reserved.

KEYWORDS: 

Antipsychotic drugs; Bipolar disorder; Mood stabilizer; Schizophrenia; Toxoplasma gondii; Valproate
PMID:
 
25769398
 
[PubMed - as supplied by publisher]

The apicomplexan glideosome and adhesins - Structures and function

 2015 Mar 9. pii: S1047-8477(15)00054-4. doi: 10.1016/j.jsb.2015.02.008. [Epub ahead of print]

Abstract

The apicomplexan family of pathogens, which includes Plasmodium spp. and Toxoplasma gondii, are primarily obligate intracellular parasites and invade multiple cell types. These parasites express extracellular membrane protein receptors, adhesins, to form specific pathogen-host cell interaction complexes. Various adhesins are used to invade a variety of cell types. The receptors are linked to an actomyosin motor, which is part of a complex comprised of many proteins known as the invasion machinery or glideosome. To date, reviews on invasion have focused primarily on the molecular pathways and signals of invasion, with little or no structural information presented. Over 75 structures of parasite receptors and glideosome proteins have been deposited with the Protein Data Bank. These structures include adhesins, motor proteins, bridging proteins, inner membrane complex and cytoskeletal proteins, as well as co-crystal structures with peptides and antibodies. These structures provide information regarding key interactions necessary for target receptor engagement, machinery complex formation, how force is transmitted, and the basis of inhibitory antibodies. Additionally, these structures can provide starting points for the development of antibodies and inhibitory molecules targeting protein-protein interactions, with the aim to inhibit invasion. This review provides an overview of the parasite adhesin protein families, the glideosome components, glideosome architecture, and discuss recent work regarding alternative models.
Copyright © 2015 Elsevier Inc. All rights reserved.

KEYWORDS: 

Adhesins; Apicomplexa; Cryptosporidium; Glideosome; Invasion machinery; Malaria; Plasmodium; Toxoplasma
PMID:
 
25764948
 
[PubMed - as supplied by publisher]

Thursday, March 12, 2015

New Method for the Orthogonal Labeling and Purification of Toxoplasma gondii Proteins While Inside the Host Cell

2015 Mar 10;6(2). pii: e01628-14. doi: 10.1128/mBio.01628-14.
 
 
Toxoplasma gondii is an obligate intracellular protozoan parasite that is capable of causing severe disease in immunocompromised humans. How T. gondii is able to modulate the host cell to support itself is still poorly understood. Knowledge pertaining to the host-parasite interaction could be bolstered by developing a system to specifically label parasite proteins while the parasite grows inside the host cell. For this purpose, we have created a strain of T. gondii that expresses a mutant Escherichia coli methionyl-tRNA synthetase (MetRS(NLL)) that allows methionine tRNA to be loaded with the azide-containing methionine analog azidonorleucine (Anl). Anl-containing proteins are susceptible to a copper-catalyzed "click" reaction to attach affinity tags for purification or fluorescent tags for visualization. The MetRS(NLL)-Anl system labels nascent T. gondii proteins in an orthogonal fashion, labeling proteins only in MetRS(NLL)-expressing parasites. This system should be useful for nonradioactive pulse-chase studies and purification of nascently translated proteins. Although this approach allows labeling of a diverse array of parasite proteins, secreted parasite proteins appear to be only minimally labeled in MetRS(NLL)-expressing T. gondii. The minimal labeling of secreted proteins is likely a consequence of the selective charging of the initiator tRNA (and not the elongator methionine tRNA) by the heterologously expressed bacterial MetRS.

IMPORTANCE:

Studying how T. gondii modifies the host cell to permit its survival is complicated by the complex protein environment of the host cell. The approach presented in this article provides the first method for specific labeling of T. gondii proteins while the parasite grows inside the host cell. We show that this approach is useful for pulse-chase labeling of parasite proteins during in vitro growth. It should also be applicable during in vivo infections and in other apicomplexan parasites, including Plasmodium spp.
Copyright © 2015 Wier et al.
PMID:
25759504
[PubMed - in process]

The structure of bradyzoite-specific enolase from Toxoplasma gondii reveals insights into its dual cytoplasmic and nuclear functions

2015 Mar 1;71(Pt 3):417-426. Epub 2015 Feb 26.
 
 
In addition to catalyzing a central step in glycolysis, enolase assumes a remarkably diverse set of secondary functions in different organisms, including transcription regulation as documented for the oncogene c-Myc promoter-binding protein 1. The apicomplexan parasite Toxoplasma gondii differentially expresses two nuclear-localized, plant-like enolases: enolase 1 (TgENO1) in the latent bradyzoite cyst stage and enolase 2 (TgENO2) in the rapidly replicative tachyzoite stage. A 2.75 Å resolution crystal structure of bradyzoite enolase 1, the second structure to be reported of a bradyzoite-specific protein in Toxoplasma, captures an open conformational state and reveals that distinctive plant-like insertions are located on surface loops. The enolase 1 structure reveals that a unique residue, Glu164, in catalytic loop 2 may account for the lower activity of this cyst-stage isozyme. Recombinant TgENO1 specifically binds to a TTTTCT DNA motif present in the cyst matrix antigen 1 (TgMAG1) gene promoter as demonstrated by gel retardation. Furthermore, direct physical interactions of both nuclear TgENO1 and TgENO2 with the TgMAG1 gene promoter are demonstrated in vivo using chromatin immunoprecipitation (ChIP) assays. Structural and biochemical studies reveal that T. gondii enolase functions are multifaceted, including the coordination of gene regulation in parasitic stage development. Enolase 1 provides a potential lead in the design of drugs against Toxoplasma brain cysts.

KEYWORDS:

brain parasite; central nervous system; chronic infection; differentiation; moonlighting protein
PMID:
25760592
[PubMed - as supplied by publisher]

Asexual expansion of Toxoplasma gondii merozoites is distinct from tachyzoites

2015 Feb 13;16(1):66. [Epub ahead of print]
 

Abstract

BACKGROUND:

The apicomplexan parasite Toxoplasma gondii is cosmopolitan in nature, largely as a result of its highly flexible life cycle. Felids are its only definitive hosts and a wide range of mammals and birds serve as intermediate hosts. The latent bradyzoite stage is orally infectious in all warm-blooded vertebrates and establishes chronic, transmissible infections. When bradyzoites are ingested by felids, they transform into merozoites in enterocytes and expand asexually as part of their coccidian life cycle. In all other intermediate hosts, however, bradyzoites differentiate exclusively to tachyzoites, and disseminate extraintestinally to many cell types. Both merozoites and tachyzoites undergo rapid asexual population expansion, yet possess different effector fates with respect to the cells and tissues they develop in and the subsequent stages they differentiate into.

RESULTS:

To determine whether merozoites utilize distinct suites of genes to attach, invade, and replicate within feline enterocytes, we performed comparative transcriptional profiling on purified tachyzoites and merozoites. We used high-throughput RNA-Seq to compare the merozoite and tachyzoite transcriptomes. 8323 genes were annotated with sequence reads across the two asexually replicating stages of the parasite life cycle. Metabolism was similar between the two replicating stages. However, significant stage-specific expression differences were measured, with 312 transcripts exclusive to merozoites versus 453 exclusive to tachyzoites. Genes coding for 177 predicted secreted proteins and 64 membrane- associated proteins were annotated as merozoite-specific. The vast majority of known dense-granule (GRA), microneme (MIC), and rhoptry (ROP) genes were not expressed in merozoites. In contrast, a large set of surface proteins (SRS) was expressed exclusively in merozoites.

CONCLUSIONS:

The distinct expression profiles of merozoites and tachyzoites reveal significant additional complexity within the T. gondii life cycle, demonstrating that merozoites are distinct asexual dividing stages which are uniquely adapted to their niche and biological purpose.
PMID:
25757795
[PubMed - as supplied by publisher]

IL-6 driven inflammatory response induces retinal pathology in a model of ocular toxoplasmosis reactivation

2015 Mar 9. pii: IAI.02985-14. [Epub ahead of print]
 
 
Ocular inflammation is one of the consequences of infection with the protozoan parasite Toxoplasma gondii. Even if lesions are self-healing in immunocompetent persons, they pose a life-time risk of reactivation and are a serious threat to vision. As there are virtually no immunological data on reactivating ocular toxoplasmosis, we established a model of direct intravitreal injection of parasites in previously infected mice with a homologous type II strain. Two different mouse strains with variable ability to control retinal infection were studied in order to describe protective and deleterious reaction patterns. In Swiss-Webster mice, which are already relatively resistant to primary infection, no peak of parasite load was observed upon reinfection. In contrast, the susceptible inbred strain C57Bl/6 showed high parasite loads after 7 days, as well as marked deterioration of retinal architecture. Both parameters were back to normal at day 21. C57Bl/6 mice also reacted with a strong local production of inflammatory and Th1 type cytokines like IL-6, IL-17A and IFN-γ, while Swiss-Webster mice only showed moderate expression of the Th2 cytokine IL-31. Interestingly, rapid intraocular production of anti-Toxoplasma antibodies was observed in Swiss-Webster, but not C57Bl/6 mice. We then localized the cellular source of different immune mediators within the retina by immunofluorescence. Finally, neutralization experiments of IFN-γ or IL-6 demonstrated the respective protective and deleterious roles of these cytokines for parasite control and retinal integrity during reinfection. In conclusion, we developed and immunologically characterized a promising mouse model of reactivating ocular toxoplasmosis.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
PMID:
25754200
[PubMed - as supplied by publisher]

Thursday, March 05, 2015

Recombinant expression, purification, and crystallization of the glutaminyl-tRNA synthetase from Toxoplasma gondii

2015 Feb 28. pii: S1046-5928(15)00029-7. doi: 10.1016/j.pep.2015.02.017. [Epub ahead of print]
 
 
Aminoacyl tRNA synthetases play a critical role in protein synthesis by providing precursor transfer-RNA molecules correctly charged with their cognate amino-acids. The essential nature of these enzymes make them attractive targets for designing new drugs against important pathogenic protozoans like Toxoplasma. Because no structural data currently exists for a protozoan glutaminyl-tRNA synthetase (QRS), an understanding of its potential as a drug target and its function in the assembly of the Toxoplasma multi-aminoacyl tRNA (MARS) complex is therefore lacking. Here we describe the optimization of expression and purification conditions that permitted the recovery and crystallization of both domains of the Toxoplasma QRS enzyme from a heterologous E. coli expression system. Expression of full-length QRS was only achieved after the addition of an N-terminal histidine affinity tag and the isolated protein was active on both cellular and in vitro produced Toxoplasma tRNA. Taking advantage of the proteolytic susceptibility of QRS to cleavage into component domains, N-terminal glutathione S-transferase (GST) motif-containing domain fragments were isolated and crystallization conditions discovered. Isolation of the C-terminal catalytic domain was accomplished after subcloning the domain and optimizing expression conditions. Purified catalytic domain survived cryogenic storage and yielded large diffraction-quality crystals over-night after optimization of screening conditions. This work will form the basis of future structural studies into structural-functional relationships of both domains including potential targeted drug-design studies and investigations into the assembly of the Toxoplasma MARS complex.
Copyright © 2015. Published by Elsevier Inc.

KEYWORDS:

Crystallization; Drug discovery; MARS complex; tRNA synthetase
PMID:
25736594
[PubMed - as supplied by publisher]