Friday, September 30, 2011

Two internal type II NADH dehydrogenases of Toxoplasma gondii are both required for optimal tachyzoite growth

Mol Microbiol. 2011 Oct;82(1):209-21. doi: 10.1111/j.1365-2958.2011.07807.x. Epub 2011 Sep 2.

Two internal type II NADH dehydrogenases of Toxoplasma gondii are both required for optimal tachyzoite growth

Lin SS, Gross U, Bohne W

SourceInstitute of Medical Microbiology, University Medical Center Göttingen, Kreuzbergring 57, Göttingen D-37075, Germany.

Abstract
In many apicomplexan parasites the entry of electrons from NADH into the electron transport chain is governed by type II NADH dehydrogenases (NDH2s) instead of a canonical complex I. Toxoplasma gondii expresses two NDH2 isoforms, TgNDH2-I and TgNDH2-II with no indication for stage-specific regulation. We dissected the orientation of both isoforms by using a split GFP assay and a protease protection assay after selective membrane permeabilization. The two approaches revealed that both TgNDH2 isoforms are internal enzymes facing with their active sites to the mitochondrial matrix. Single knockout mutants displayed a decreased replication rate and a reduced mitochondrial membrane potential, which were both more severe in the Tgndh2-II-deleted than in the Tgndh2-I-deleted mutant. Complementation with a myc-tagged, ectopic copy of the deleted gene restored the growth rate and the mitochondrial membrane potential. However, an overexpression of the remaining intact isoform could not restore the phenotype, suggesting that the two TgNDH2 isoforms are non-redundant and possess functional differences. Together, our studies indicate that although TgNDH2-I and TgNDH2-II are individually non-essential, the expression of both internal isoforms is required to maintain the mitochondrial physiology in T. gondii tachyzoites.

© 2011 Blackwell Publishing Ltd.

PMID:21854467[PubMed - in process]

Population genetics of Toxoplasma gondii: New perspectives from parasite genotypes in wildlife

Vet Parasitol. 2011 Nov 24;182(1):96-111. Epub 2011 Jul 20

Population genetics of Toxoplasma gondii: New perspectives from parasite genotypes in wildlife
Wendte JM, Gibson AK, Grigg ME

SourceMolecular Parasitology Unit, Laboratory of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-0425, USA; Department of Veterinary Pathobiology, Oklahoma State University Center for Veterinary Health Sciences, Stillwater, OK 74074, USA.

Abstract
Toxoplasma gondii, a zoonotic protozoal parasite, is well-known for its global distribution and its ability to infect virtually all warm-blooded vertebrates. Nonetheless, attempts to describe the population structure of T. gondii have been primarily limited to samples isolated from humans and domesticated animals. More recent studies, however, have made efforts to characterize T. gondii isolates from a wider range of host species and geographic locales. These findings have dramatically changed our perception of the extent of genetic diversity in T. gondii and the relative roles of sexual recombination and clonal propagation in the parasite's lifecycle. In particular, identification of novel, disease-causing T. gondii strains in wildlife has raised concerns from both a conservation and public health perspective as to whether distinct domestic and sylvatic parasite gene pools exist. If so, overlap of these cycles may represent regions of high probability of disease emergence. Here, we attempt to answer these key questions by reviewing recent studies of T. gondii infections in wildlife, highlighting those which have advanced our understanding of the genetic diversity and population biology of this important zoonotic pathogen.

Published by Elsevier B.V.

PMID:21824730[PubMed - in process]

A Purification Method for Enrichment of the Toxoplasma gondii Cyst Wall

J Neuroparasitology. 2010 Dec;1. pii: N101001

A Purification Method for Enrichment of the Toxoplasma gondii Cyst Wall

Zhang YW, Halonen SK, Ma YF, Tanowtiz HB, Weiss LM.

SourceDepartment of Pathology, Division of Parasitology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

Abstract
The tissue cyst wall of Toxoplasma gondii is a stage-specific structure that is produced by modification of the bradyzoite-containing parasitophorous vacuole. It is a limiting membrane structure and is critically important for cyst survival and transmission of infection. Studies on the structure and function of the cyst wall should provide new therapeutic strategies for the elimination or prevention of latency during T. gondii infection. The membrane proteins of the T. gondii cyst are an important target for studies of the biochemical and immunological function(s) of the cyst. However, the components of the cyst membrane have been poorly characterized due to the difficulty of purification of these membrane proteins. We developed a lectin DBA (Dolichos biflorus) coated magnetic bead isolation method to isolate T. gondii cyst wall proteins. Our data suggests that this method can isolate cyst wall proteins from both in vitro cell culture or in vivo mouse brain derived tissue cysts. Antibodies to these isolated protein preparations were shown to localize to the cyst wall.

PMID:21687827[PubMed]

Thursday, September 29, 2011

A novel benzodioxole-containing inhibitor of Toxoplasma gondii growth alters the parasite cell cycle

Antimicrob Agents Chemother. 2011 Sep 26. [Epub ahead of print]

A novel benzodioxole-containing inhibitor of Toxoplasma gondii growth alters the parasite cell cycle

Kamau E, Meehan T, Lavine MD, Arrizabalaga G, Mustata G, Boyle J.

SourceDepartment of Biological Sciences, University of Pittsburgh, Pittsburgh, PA. 15260.

Abstract
Toxoplasma gondii is an obligate intracellular parasite that can cause disease in the developing fetus and in immunocompromised humans. Infections can last for the life of the individual, and to date there are no drugs that eliminate chronic cyst stages characteristic of this parasite. In an effort to identify new chemical scaffolds that could form the basis for new therapeutics, we carried out a chemoinformatic screen for compounds that had the potential to interact with members of a superfamily of parasite-secreted kinases, and assayed them for growth inhibition in vitro. Out of 17 candidate compounds we identified one with potent anti-parasitic activity. The compound has an IC50 of ∼2 nM, and structure-function analyses implicate the benzodioxole moiety in its action. The compound does not appear to be cytotoxic to host cells. Using microarray analyses of both parasites and host cells treated with the compound, we found that the levels of very few host cell transcripts are altered by the compound while a large number of parasite transcripts are of different abundance after compound treatment. Gene ontology analyses of parasite transcripts with different abundance revealed an enrichment of cell cycle-related genes, suggesting that the compound alters progression of the parasite through the cell cycle. Assaying nuclear content of treated parasites demonstrated that compound treatment significantly increased the percentage of parasites in the S/M phase of the cell cycle compared to controls. This compound and its analogs represent a novel scaffold with anti-parasitic activity.

PMID:21947387[PubMed - as supplied by publisher]

The first detection of Toxoplasma gondii DNA in environmental fruits and vegetables samples

Eur J Clin Microbiol Infect Dis. 2011 Sep 25. [Epub ahead of print]

The first detection of Toxoplasma gondii DNA in environmental fruits and vegetables samples

Lass A, Pietkiewicz H, Szostakowska B, Myjak P.

SourceDepartment of Tropical Parasitology, Interfaculty Institute of Maritime and Tropical Medicine in Gdynia, Medical University of Gdansk, Gdansk, Poland, anna.ls1@gumed.edu.pl.

Abstract
Toxoplasma gondii infections are prevalent in humans and animals all over the world. The aim of the study was to estimate the occurrence of T. gondii oocysts in fruits and vegetables and determine the genotype of the parasites. A total number of 216 fruits and vegetables samples were taken from shops and home gardens located in the area of northern Poland. Oocysts were recovered with the flocculation method. Then, real-time polymerase chain reaction (PCR) targeting the B1 gene was used for specific T. gondii detection and quantification. Toxoplasma DNA was found in 21 samples. Genotyping at the SAG2 locus showed SAG2 type I and SAG2 type II. This is the first investigation describing T. gondii DNA identification in a large number of fruits and vegetables samples with rapid molecular detection methods. The results showed that fruits and vegetables contaminated with T. gondii may play a role in the prevalence of toxoplasmosis in Poland.

PMID:21948336[PubMed - as supplied by publisher]

Thursday, September 22, 2011

ROP16 Activates STAT3 and STAT6 Resulting in Cytokine Inhibition and Arginase-1-Dependent Growth Control

PLoS Pathog. 2011 Sep;7(9):e1002236. Epub 2011 Sep 8

Toxoplasma gondii Rhoptry Kinase ROP16 Activates STAT3 and STAT6 Resulting in Cytokine Inhibition and Arginase-1-Dependent Growth Control

Butcher BA, Fox BA, Rommereim LM, Kim SG, Maurer KJ, Yarovinsky F, Herbert DR, Bzik DJ, Denkers EY

SourceDepartment of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York, United States of America.

Abstract
The ROP16 kinase of Toxoplasma gondii is injected into the host cell cytosol where it activates signal transducer and activator of transcription (STAT)-3 and STAT6. Here, we generated a ROP16 deletion mutant on a Type I parasite strain background, as well as a control complementation mutant with restored ROP16 expression. We investigated the biological role of the ROP16 molecule during T. gondii infection. Infection of mouse bone marrow-derived macrophages with rop16-deleted (ΔROP16) parasites resulted in increased amounts of IL-12p40 production relative to the ROP16-positive RH parental strain. High level IL-12p40 production in ΔROP16 infection was dependent on the host cell adaptor molecule MyD88, but surprisingly was independent of any previously recognized T. gondii triggered pathway linking to MyD88 (TLR2, TLR4, TLR9, TLR11, IL-1ß and IL-18). In addition, ROP16 was found to mediate the suppressive effects of Toxoplasma on LPS-induced cytokine synthesis in macrophages and on IFN-γ-induced nitric oxide production by astrocytes and microglial cells. Furthermore, ROP16 triggered synthesis of host cell arginase-1 in a STAT6-dependent manner. In fibroblasts and macrophages, failure to induce arginase-1 by ΔROP16 tachyzoites resulted in resistance to starvation conditions of limiting arginine, an essential amino acid for replication and virulence of this parasite. ΔROP16 tachyzoites that failed to induce host cell arginase-1 displayed increased replication and dissemination during in vivo infection. We conclude that encounter between Toxoplasma ROP16 and the host cell STAT signaling cascade has pleiotropic downstream effects that act in multiple and complex ways to direct the course of infection.

PMID:21931552[PubMed - in process]

Determinants of GBP Recruitment to Toxoplasma gondii Vacuoles and the Parasitic Factors That Control It

PLoS One. 2011;6(9):e24434. Epub 2011 Sep 8

Determinants of GBP Recruitment to Toxoplasma gondii Vacuoles and the Parasitic Factors That Control It

Virreira Winter S, Niedelman W, Jensen KD, Rosowski EE, Julien L, Spooner E, Caradonna K, Burleigh BA, Saeij JP, Ploegh HL, Frickel EM

SourceWhitehead Institute for Biomedical Research, Cambridge, Massachusetts, United States of America.

Abstract
IFN-γ is a major cytokine that mediates resistance against the intracellular parasite Toxoplasma gondii. The p65 guanylate-binding proteins (GBPs) are strongly induced by IFN-γ. We studied the behavior of murine GBP1 (mGBP1) upon infection with T. gondii in vitro and confirmed that IFN-γ-dependent re-localization of mGBP1 to the parasitophorous vacuole (PV) correlates with the virulence type of the parasite. We identified three parasitic factors, ROP16, ROP18, and GRA15 that determine strain-specific accumulation of mGBP1 on the PV. These highly polymorphic proteins are held responsible for a large part of the strain-specific differences in virulence. Therefore, our data suggest that virulence of T. gondii in animals may rely in part on recognition by GBPs. However, phagosomes or vacuoles containing Trypanosoma cruzi did not recruit mGBP1. Co-immunoprecipitation revealed mGBP2, mGBP4, and mGBP5 as binding partners of mGBP1. Indeed, mGBP2 and mGBP5 co-localize with mGBP1 in T. gondii-infected cells. T. gondii thus elicits a cell-autonomous immune response in mice with GBPs involved. Three parasitic virulence factors and unknown IFN-γ-dependent host factors regulate this complex process. Depending on the virulence of the strains involved, numerous GBPs are brought to the PV as part of a large, multimeric structure to combat T. gondii.

PMID:21931713[PubMed - in process]

Genetic approaches for understanding virulence in Toxoplasma gondii

Brief Funct Genomics. 2011 Sep 19. [Epub ahead of print]

Genetic approaches for understanding virulence in Toxoplasma gondii

Weilhammer DR, Rasley A

Abstract
Virulence of the protozoan parasite Toxoplasma gondii is highly variable and dependent upon the genotype of the parasite. The application of forward and reverse genetic approaches for understanding the genetic basis of virulence has resulted in the identification of several members of the ROP family as key mediators of virulence. More recently, modern genomic techniques have been used to address strain differences in virulence and have also identified additional members of the ROP family as likely mediators. The development of forward and reverse genetic, as well as modern genomic techniques, and the path to the discovery of the ROP genes as virulence factors is reviewed here.

PMID:21930659[PubMed - as supplied by publisher]

Tuesday, September 20, 2011

A Critical Role for SOCS3 in Innate Resistance to Toxoplasma gondii

Cell Host Microbe. 2011 Sep 15;10(3):224-36

A Critical Role for SOCS3 in Innate Resistance to Toxoplasma gondii

Whitmarsh RJ, Gray CM, Gregg B, Christian DA, May MJ, Murray PJ, Hunter CA

SourceDepartment of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, 380 South University Avenue, Philadelphia, PA 19104, USA.

Abstract
The innate and adaptive immune responses that confer resistance to the intracellular pathogen Toxoplasma gondii critically depend on IL-12 production, which drives interferon-γ (IFN-γ) expression. Certain cytokines can activate STAT3 and limit IL-12 production to prevent infection-associated immune pathology, but T. gondii also directly activates STAT3 to evade host immunity. We show that suppressor of cytokine signaling molecule 3 (SOCS3), a target of STAT3 that limits signaling by the pleiotropic cytokine IL-6, is upregulated in response to infection but is dispensable for the immune-inhibitory effects of T. gondii. Unexpectedly, mice with targeted deletion of SOCS3 in macrophages and neutrophils have reduced IL-12 responses and succumb to toxoplasmosis. Anti-IL-6 administration or IL-12 treatment blocked disease susceptibility, suggesting that in the absence of SOCS3, macrophages are hypersensitive to the anti-inflammatory properties of IL-6. Thus, SOCS3 has a critical role in suppressing IL-6 signals and promoting immune responses to control T. gondii infection.

Copyright © 2011 Elsevier Inc. All rights reserved.

PMID:21925110[PubMed - in process]

Glycosylphosphatidylinositols of Toxoplasma gondii induce matrix metalloproteinase-9 production and degradation of galectin-3

Immunobiology. 2011 Aug 12. [Epub ahead of print]

Glycosylphosphatidylinositols of Toxoplasma gondii induce matrix metalloproteinase-9 production and degradation of galectin-3

Niehus S, Elass E, Coddeville B, Guérardel Y, Schwarz RT, Debierre-Grockiego F

SourceInstitut für Virologie, AG Parasitologie, Philipps-Universität, Marburg, Germany; UPR9022 du CNRS, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, 67084 Strasbourg, France.

Abstract
Toxoplasma gondii glycosylphosphatidylinositols (GPIs) bind to galectin-3 to induce TNF-α production in macrophages via Toll-like receptors 2 and 4. Here we show that T. gondii GPIs stimulate human macrophages to synthesize matrix metalloproteinase-9 in a TNF-α-dependent pathway and degrade extracellular galectin-3.

Copyright © 2011 Elsevier GmbH. All rights reserved.

PMID:21924517[PubMed - as supplied by publisher]

The Treg/Th17 Imbalance in Toxoplasma gondii-Infected Pregnant Mice

Am J Reprod Immunol. 2011 Sep 19. doi: 10.1111/j.1600-0897.2011.01065.x. [Epub ahead of print]

The Treg/Th17 Imbalance in Toxoplasma gondii-Infected Pregnant Mice

Zhang H, Hu X, Liu X, Zhang R, Fu Q, Xu X

SourceDepartment of Immunology, Binzhou Medical College, Yantai, China.

Abstract
Citation Zhang H, Hu X, Liu X, Zhang R, Fu Q, Xu X. The Treg/Th17 Imbalance in Toxoplasma gondii-Infected Pregnant Mice. Am J Reprod Immunol 2011 Aim  To evaluate whether impaired Treg/Th17 balance exists in the pregnant mice infected with Toxoplasma gondii. Method of Study  Regulatory T (Treg) and T-helper type 17 (Th17) cells were measured in both placenta and spleens of the pregnant mice infected with and without T. gondii by flow cytometry. The mRNA and protein expression levels of transforming growth factor-β (TGF-β) and interleukin-17A (IL-17A) were analyzed using real-time PCR and enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of forkhead box P3 (Foxp3), retinoic acid-related orphan receptor γt (RORγt), and IL-6 were also analyzed using real-time PCR. The correlations of the ratio of Treg/Th17 to the mRNA or protein expression level of those factors were analyzed by Spearman's correlation analysis. Data were analyzed by unpaired t-test and paired t-test. Results  The proportion of Tregs or Th17 cells in the placenta and spleens of the T. gondii-infected pregnant mice was significantly lower or higher than in those of non-infected mice, respectively. Upregulation of TGF-β and downregulation of IL-17A were found in the placenta of T. gondii-infected pregnant mice. The ratio of Treg to Th17 was significantly lower in the infected mice than that in the non-infected mice (P < 0.01).The ratio of Treg to Th17 positively or negatively correlated with the protein expression level of TGF-β (r = 0.6204, P < 0.05) or IL-17A (r = -0.6296, P < 0.05), respectively. The ratio also positively correlated with the mRNA expression level of Foxp3 (r = 0.7985, P < 0.01), but negatively correlated with the mRNA expression level of RORγt (r = -0.6153, P < 0.05), and IL-6 (r = -0.7492, P < 0.01). Conclusion  TheTreg/Th17 imbalance exists in the pregnant mice infected with T. gondii, which is associated with the expression of related cytokine and key transcription factors. This result suggests that the embryo loss caused by this parasite may be associated with a reduced ratio of Treg to Th17 cell number.

© 2011 John Wiley & Sons A/S.

PMID:21923716[PubMed - as supplied by publisher]

Effects of Toxoplasma gondii genotype and absence of host MAL/Myd88 on the temporal regulation of gene expression in infected microglial cells

Exp Parasitol. 2011 Sep 6. [Epub ahead of print]

Effects of Toxoplasma gondii genotype and absence of host MAL/Myd88 on the temporal regulation of gene expression in infected microglial cells

Glaser KC, Hagos B, Molestina RE

SourceBEI Resources, American Type Culture Collection, 10801 University Blvd., Manassas, VA 20110, USA; Protistology Department, American Type Culture Collection, 10801 University Blvd., Manassas, VA 20110, USA.

Abstract
The majority of strains of Toxoplasma gondii belong to three distinct clonal lines known as types I, II, and III. The outcome of the immune response to infection is influenced by the parasite strain type. The goal of this study was to examine differences in the kinetics of gene expression in microglial cells infected with types I, II, or III of T. gondii. In addition, a requirement for the integrity of host Toll-like receptor (TLR) signaling in parasite-mediated changes in gene expression was evaluated. Wild type murine microglial cells infected with T. gondii displayed different kinetic patterns of pro-inflammatory cytokine expression that were dependent on the parasite strain type. In general, types II and III elicited higher sustained responses compared to type I which induced fluctuating patterns of cytokine gene expression. Contrary to this, differences in the induction of anti-apoptotic gene expression were minimal among the different type strains throughout infection. Experiments with cells lacking the TLR adaptor molecules MAL and Myd88 showed a dependency on these factors for the pro-inflammatory response but not the anti-apoptotic response. The results show that the outcome of gene expression in T. gondii-infected microglial cells is dependent on the parasite strain type in a time-dependent manner and is selective to particular subsets of genes. The induction of an anti-apoptotic response by T. gondii infection in the absence of TLR signaling reflects a complex level of modulation of host functions by the parasite.

Copyright © 2011 Elsevier Inc. All rights reserved.

PMID:21924265[PubMed - as supplied by publisher]

Monday, September 19, 2011

Analysis of the glycoproteome of Toxoplasma gondii using lectin affinity chromatography and tandem mass spectrometry

Microbes Infect. 2011 Aug 31. [Epub ahead of print]

Analysis of the glycoproteome of Toxoplasma gondii using lectin affinity chromatography and tandem mass spectrometry

Luo Q, Upadhya R, Zhang H, Madrid-Aliste C, Nieves E, Kim K, Angeletti RH, Weiss LM

SourceDepartment of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA; Laboratory for Macromolecular Analysis and Proteomics, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

Abstract
Glycoproteins are involved in many important molecular recognition processes including invasion, adhesion, differentiation, and development. To identify the glycoproteins of Toxoplasma gondii, a proteomic analysis was undertaken. T. gondii proteins were prepared and fractioned using lectin affinity chromatography. The proteins in each fraction were then separated using SDS-PAGE and identified by tryptic in gel digestion followed by tandem mass spectrometry. Utilizing these methods 132 proteins were identified. Among the identified proteins were 17 surface proteins, 9 microneme proteins, 15 rhoptry proteins, 11 heat shock proteins (HSP), and 32 hypothetical proteins. Several proteins had 1-5 transmembrane domains (TMD) with some being as large as 608.3 kDa. Both lectin-fluorescence labeling and lectin blotting were employed to confirm the presence of carbohydrates on the surface or cytoplasm of T. gondii parasites. PCR demonstrated that selected hypothetical proteins were expressed in T. gondii tachyzoites. This data provides a large-scale analysis of the T. gondii glycoproteome. Studies of the function of glycosylation of these proteins may help elucidate mechanism(s) involved in invasion improving drug therapy as well as identify glycoproteins that may prove to be useful as vaccine candidates.

Copyright © 2011. Published by Elsevier Masson SAS.

PMID:21920448[PubMed - as supplied by publisher]

Friday, September 16, 2011

Toxoplasma gondii induces B7-2 expression through activation of JNK signal transduction

Infect Immun. 2011 Sep 12. [Epub ahead of print]

Toxoplasma gondii induces B7-2 expression through activation of JNK signal transduction

Morgado P, Ong YC, Boothroyd JC, Lodoen MB.

SourceDepartment of Molecular Biology and Biochemistry, 3238 McGaugh Hall, University of California, Irvine, Irvine, CA 92697.

Abstract
Toxoplasma gondii is a globally distributed parasite pathogen that infects virtually all warm-blooded animals. A hallmark of immunity to acute infection is the production of IFN-γ and IL-12, followed by a protective T cell response that is critical for parasite control. Naïve T cell activation requires both TCR stimulation and the engagement of costimulatory receptors. Because of their important function in activating T cells, the expression of co-stimulatory ligands is believed to be under tight control. The molecular mechanisms governing their induction during microbial stimulation, however, are not well understood. We found that all three strains of T. gondii (Types I, II, and III) up-regulated the expression of B7-2, but not B7-1, on the surface of mouse bone marrow-derived macrophages. Additionally, intraperitoneal infection of mice with GFP-expressing parasites resulted in enhanced B7-2 levels specifically on infected, GFP(+)CD11b(+) cells. B7-2 induction occurred at the transcript level, required active parasite invasion, and was not dependent on MyD88 or TRIF. Functional assays demonstrated that T. gondii-infected macrophages stimulated naïve T cell proliferation in a B7-2-dependent manner. Genome-wide transcriptional analysis comparing infected and uninfected macrophages revealed the activation of MAPK signaling in infected cells. Using specific inhibitors against MAPKs, we determined that parasite-induced B7-2 is dependent on JNK, but not ERK or p38 signaling. We also observed that T. gondii-induced B7-2 expression on human peripheral blood monocytes is dependent on JNK signaling, indicating that a common mechanism of B7-2 regulation by T. gondii may exist in both humans and mice.

PMID:21911468[PubMed - as supplied by publisher]

Tuesday, September 13, 2011

A GCN2-like eukaryotic initiation factor-2 kinase increases the viability of extracellular Toxoplasma

Eukaryot Cell. 2011 Sep 9. [Epub ahead of print]

A GCN2-like eukaryotic initiation factor-2 kinase increases the viability of extracellular Toxoplasma gondii parasites

Konrad C, Wek RC, Sullivan WJ Jr

SourceDepartment of Pharmacology & Toxicology.

Abstract
Toxoplasmosis is a significant opportunistic infection caused by the protozoan parasite Toxoplasma gondii, an obligate intracellular pathogen that relies on host cell nutrients for parasite proliferation. Toxoplasma parasites divide until they rupture the host cell, at which point the extracellular parasites must survive until they find a new host cell. Recent studies have indicated that phosphorylation of Toxoplasma eukaryotic translation initiation factor 2-alpha (TgIF2α) plays a key role in promoting parasite viability during times of extracellular stress. Here we report the cloning and characterization of a TgIF2α kinase designated TgIF2K-D that is related to GCN2, an eIF2α kinase known to respond to nutrient starvation in other organisms. TgIF2K-D is present in the cytosol of both intra- and extracellular Toxoplasma and facilitates translational control through TgIF2α phosphorylation in extracellular parasites. We generated a TgIF2K-D knockout parasite and demonstrated that loss of this eIF2α kinase leads to a significant fitness defect that stems from an inability of the parasite to adequately adapt to the environment outside of host cells. This phenotype is consistent with that reported for our non-phosphorylatable TgIF2α mutant (S71A substitution), establishing that TgIF2K-D is the primary eIF2α kinase responsible for promoting extracellular viability of Toxoplasma. These studies suggest that eIF2α phosphorylation and translational control are an important mechanism by which vulnerable extracellular parasites protect themselves while searching for a new host cell. Additionally, TgIF2α is phosphorylated when intracellular parasites are deprived of nutrients, but this can occur independently of TgIF2K-D, indicating that this activity can be mediated by a different TgIF2K.

PMID:21908594[PubMed - as supplied by publisher]

The Motility of a Human Parasite, Toxoplasma gondii, Is Regulated by a Novel Lysine Methyltransferase

PLoS Pathog. 2011 Sep;7(9):e1002201. Epub 2011 Sep 1

The Motility of a Human Parasite, Toxoplasma gondii, Is Regulated by a Novel Lysine Methyltransferase

Heaslip AT, Nishi M, Stein B, Hu K

SourceDepartment of Biology, Indiana University, Bloomington, Indiana, United States of America.

Abstract
Protozoa in the phylum Apicomplexa are a large group of obligate intracellular parasites. Toxoplasma gondii and other apicomplexan parasites, such as Plasmodium falciparum, cause diseases by reiterating their lytic cycle, comprising host cell invasion, parasite replication, and parasite egress. The successful completion of the lytic cycle requires that the parasite senses changes in its environment and switches between the non-motile (for intracellular replication) and motile (for invasion and egress) states appropriately. Although the signaling pathway that regulates the motile state switch is critical to the pathogenesis of the diseases caused by these parasites, it is not well understood. Here we report a previously unknown mechanism of regulating the motility activation in Toxoplasma, mediated by a protein lysine methyltransferase, AKMT (for Apical complex lysine (K) methyltransferase). AKMT depletion greatly inhibits activation of motility, compromises parasite invasion and egress, and thus severely impairs the lytic cycle. Interestingly, AKMT redistributes from the apical complex to the parasite body rapidly in the presence of egress-stimulating signals that increase [Ca(2+)] in the parasite cytoplasm, suggesting that AKMT regulation of parasite motility might be accomplished by the precise temporal control of its localization in response to environmental changes.

PMID:21909263[PubMed - in process]

Toxoplasma gondii rhomboid protein 1 (TgROM1) is a potential vaccine candidate against toxoplasmosis

Vet Parasitol. 2011 Aug 16. [Epub ahead of print]

Toxoplasma gondii rhomboid protein 1 (TgROM1) is a potential vaccine candidate against toxoplasmosis

Li J, Han Q, Gong P, Yang T, Ren B, Li S, Zhang X

SourceCollege of Animal Science and Veterinary Medicine, Jilin University, 130062 Changchun, Jilin, China.

Abstract
Infection with the intracellular protozoan parasite Toxoplasma gondii causes serious public health problems and is of great economic importance worldwide. The rhomboid proteins which are responsible for adhesion and invasion of host cells have been suggested as vaccine candidates against toxoplasmosis. A DNA vaccine (pVAX-ROM1) encoding T. gondii rhomboid protein 1 (TgROM1) gene was constructed and the immune response and protective efficacy of this vaccine against lethal challenge in BALB/c mice were evaluated. The results indicated that specific antibody and lymphocyte proliferative responses were elicited in mice receiving pVAX-ROM1. The production levels of IFN-γ, IL-2, IL-4, and IL-10, as well as the percentage of CD4(+) cells in mice vaccinated with pVAX-ROM1 were significantly increased respectively, compared to controls receiving either pVAX1 alone or PBS. After lethal challenge, the mice immunized with pVAX-ROM1 showed an increased survival time compared with the mice in the controls. Our data suggested that a DNA vaccine pVAX-ROM1 encoding T. gondii rhomboid protein 1 triggered strong humoral and cellular responses, and prolonged survival time against T. gondii infection in BALB/c mice.

Copyright © 2011 Elsevier B.V. All rights reserved.

Sunday, September 11, 2011

Toxoplasma gondii: the changing paradigm of congenital toxoplasmosis

Parasitology. 2011 Sep 9:1-3. [Epub ahead of print]
Toxoplasma gondii: the changing paradigm of congenital toxoplasmosis

Lindsay DS, Dubey JP.

SourceDepartment of Biomedical Sciences and Pathobiology, Virginia - Maryland Regional College of Veterinary Medicine, Virginia Tech, 1410 Prices Fork Road, Blacksburg, VA 24061, USA.
Abstract
SUMMARYResearchers have learned much concerning the population biology of Toxoplasma gondii over the past 2 decades. It is now apparent that many atypical genotypes exist besides the typical 3 genotypes (type I, type II and type III) first described from samples from Europe and the United States. These genotypes can differ in pathogenicity and transmissibility from the typical genotypes that have been used in the majority of scientific research over the past 70 years. These differences impact much of what we used to believe as facts about congenital toxoplasmosis (CT) and will be important in developing new recommendations for prevention of CT and the monitoring of women at risk for developing CT. The present review highlights new information on T. gondii genotypes and how this information will change the way we convey information about CT to pregnant women, physicians and students.

PMID:21902872[PubMed - as supplied by publisher]

CD4(+) Foxp3(+) Regulatory T cells mediate Toxoplasma gondii-induced T-cell suppression

Eur J Immunol. 2011 Sep 9. doi: 10.1002/eji.201141507. [Epub ahead of print]
CD4(+) Foxp3(+) Regulatory T cells mediate Toxoplasma gondii-induced T-cell suppression through an IL-2-related mechanism but independently of IL-10

Tenorio EP, Fernández J, Castellanos C, Olguín JE, Saavedra R.

SourceDepartamento de Inmunología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico.
Abstract
Acute Toxoplasma gondii infection comprises an immunosuppression stage, characterized by a reduction in T-cell proliferation in vitro. Treg cells maintain the homeostasis of the immune system, but their role in T. gondii-induced suppression has not been addressed. We show herein that immunosuppression, affecting both CD4(+) and CD8(+) T-cell proliferation, concurs with a reduction in Treg-cell number. The residual Treg cells, however, are activated and display an increased suppressive capacity. We show that selective elimination of Treg cells using Foxp3(EGFP) mice leads to a full recovery of CD4(+) and CD8(+) T-cell proliferation. After Treg-cell removal, a reduced production of IL-10 was observed, but IL-2 levels were unchanged. The numbers of IL-10-producing Treg cells also increased during infection, although the in vitro neutralization of this cytokine did not modify T-cell proliferation, suggesting that IL-10 does not mediate the Treg-mediated suppression. However, addition of rIL-2 in vitro fully restored T-cell proliferation from infected animals. Thus, we show that Treg cells mediate the T-cell suppression observed during acute T. gondii infection through an IL-2-dependent mechanism. Our results provide novel insights into the regulation of the immune response against T. gondii.
Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
PMID:21905022[PubMed - as supplied by publisher]

Development of high-throughput methods to quantify cysts of Toxoplasma gondii

Cytometry A. 2011 Sep 8. doi: 10.1002/cyto.a.21138. [Epub ahead of print]
Development of high-throughput methods to quantify cysts of Toxoplasma gondii

Aldebert D, Hypolite M, Cavailles P, Touquet B, Flori P, Loeuillet C, Cesbron-Delauw MF.

SourceLaboratoire Adaptation et Pathogénie des Micro-organismes, UMR 5163 CNRS-UJF Grenoble I, France. delphine.aldebert@ujf-grenoble.fr.
Abstract
Toxplasma is a protozoan parasite, which forms persistent cysts in tissues of chronically infected animals and humans. Cysts can reactivate leading to severe pathologies. They also contribute to the transmission of Toxoplasma infection in humans by ingestion of undercooked meat. Classically, the quantification of cyst burden in tissues uses microscopy methods, which are laborious and time consuming. Here, we have developed automated protocols to quantify cysts, based on flow cytometry or high-throughput microscopy. Brains of rodents infected with cysts of Prugniaud strain were incubated with the FITC-Dolichos biflorus lectin and analyzed by flow cytometry and high-throughput epifluorescence microscopy. The comparison of cyst counts by manual epifluorescence microscopy to flow cytometry or to high-throughput epifluorescence microscopy revealed a good correlation (r = 0.934, r = 0.993, P < 0.001 respectively). High-throughput epifluorescence microscopy was found to be more specific and sensitive than flow cytometry and easier to use for large series of samples. This reliable and easy protocol allow the specific detection of Toxoplasma cysts in brain, even at low concentrations; it could be a new way to detect them in water and in contaminate food. © 2011 International Society for Advancement of Cytometry.
Copyright © 2011 International Society for Advancement of Cytometry.
PMID:21905211[PubMed - as supplied by publisher]

Friday, September 09, 2011

Targeted Disruption of TgPhIL1 in Toxoplasma gondii Results in Altered Parasite Morphology and Fitness

PLoS One. 2011;6(8):e23977. Epub 2011 Aug 25.

Targeted Disruption of TgPhIL1 in Toxoplasma gondii Results in Altered Parasite Morphology and Fitness

Barkhuff WD, Gilk SD, Whitmarsh R, Tilley LD, Hunter C, Ward GE.

SourceDepartment of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont, United States of America.

The inner membrane complex (IMC), a series of flattened vesicles at the periphery of apicomplexan parasites, is thought to be important for parasite shape, motility and replication, but few of the IMC proteins that function in these processes have been identified. TgPhIL1, a Toxoplasma gondii protein that was previously identified through photosensitized labeling with 5-[(125)I] iodonapthaline-1-azide, associates with the IMC and/or underlying cytoskeleton and is concentrated at the apical end of the parasite. Orthologs of TgPhIL1 are found in other apicomplexans, but the function of this conserved protein family is unknown. As a first step towards determining the function of TgPhIL1 and its orthologs, we generated a T. gondii parasite line in which the single copy of TgPhIL1 was disrupted by homologous recombination. The TgPhIL1 knockout parasites have a distinctly different morphology than wild-type parasites, and normal shape is restored in the knockout background after complementation with the wild-type allele. The knockout parasites are outcompeted in culture by parasites expressing functional TgPhIL1, and they generate a reduced parasite load in the spleen and liver of infected mice. These findings demonstrate a role for TgPhIL1 in the morphology, growth and fitness of T. gondii tachyzoites.

PMID:21901148[PubMed - in process]

The organization of the wall filaments and characterization of the matrix structures of Toxoplasma gondii cyst form

Cell Microbiol. 2011 Sep 8. doi: 10.1111/j.1462-5822.2011.01681.x. [Epub ahead of print]
The organization of the wall filaments and characterization of the matrix structures of Toxoplasma gondii cyst form

Lemgruber L, Lupetti P, Martins-Duarte ES, De Souza W, Vommaro RC.

SourceLaboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil. Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Rio de Janeiro, Brazil. Parasitology - Department of Infectious Diseases, University of Heidelberg Medical School, Germany. Laboratory of Cryotechniques for Electron Microscopy, Department of Evolutionary Biology, University of Siena, Italy. Instituto Nacional de Metrologia e Qualidade Industrial - Inmetro, Rio de Janeiro, Brazil.
Abstract

The encystation process is a key step in Toxoplasma gondii life cycle, allowing the parasite to escape from the host immune system and the transmission among the hosts. A detailed characterization of the formation and structure of the cyst stage is essential for a better knowledge of toxoplasmosis. Here we isolated cysts from mice brains and analyzed the cyst wall structure and cyst matrix organization using different electron microscopy techniques. Images obtained showed that the cyst wall presented a filamentous aspect, with circular openings on its surface. The filaments were organized in two layers: a compact one, facing the exterior of the whole cyst and a more loosen one, facing the matrix. Within the cyst wall, we observed tubules and a large number of vesicles. The cyst matrix presented vesicles of different sizes and tubules, which were organized in a network connecting the bradyzoites to each other and to the cyst wall. Large vesicles, with a granular material in their lumen of glycidic nature were observed. Similar vesicles were also found associated with the posterior pole of the bradyzoites and in proximity to the cyst wall.

© 2011 Blackwell Publishing Ltd.

PMID:21899696[PubMed - as supplied by publisher]

Thursday, September 08, 2011

Toxoplasma gondii effectors are master regulators of the inflammatory response

Trends Parasitol. 2011 Sep 3. [Epub ahead of print]

Toxoplasma gondii effectors are master regulators of the inflammatory response

Melo MB, Jensen KD, Saeij JP.
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Toxoplasma is a highly successful parasite that establishes a life-long chronic infection. To do this, it must carefully regulate immune activation and host cell effector mechanisms. Here we review the latest developments in our understanding of how Toxoplasma counteracts the immune response of the host, and in some cases provokes it, through the use of specific parasite effector proteins. An emerging theme from these discoveries is that Toxoplasma effectors are master regulators of the pro-inflammatory response, which elicits many of the toxoplasmacidal mechanisms of the host. We speculate that combinations of these effectors present in certain Toxoplasma strains work to maintain an optimal parasite burden in different hosts to ensure parasite transmission.

Copyright © 2011 Elsevier Ltd. All rights reserved. PMID:21893432[PubMed - as supplied by publisher]

Tuesday, September 06, 2011

Toxoplasma gondii antibody titers and history of suicide attempts in patients with schizophrenia

Schizophr Res. 2011 Sep 2. [Epub ahead of print]

Toxoplasma gondii antibody titers and history of suicide attempts in patients with schizophrenia

Okusaga O, Langenberg P, Sleemi A, Vaswani D, Giegling I, Hartmann AM, Konte B, Friedl M, Groer MW, Yolken RH, Rujescu D, Postolache TT SourceMood and Anxiety Program, University of Maryland School of Medicine, Baltimore, MD, USA; St. Elizabeths Hospital, Psychiatry Residency Training Program, Washington, DC, USA.

Abstract
Toxoplasma gondii (T. gondii) a widespread neurotropic parasite, has been previously associated with schizophrenia and more recently with suicidal behavior. However, no previous study has examined the association of T. gondii with suicidal behavior in schizophrenia patients. 950 individuals diagnosed with schizophrenia by SCID were recruited from the Munich area of Germany. Solid-enzyme immunoassay methods were used to measure IgG plasma antibodies to T. gondii, other neurotropic pathogens and gliadin. Logistic regression models were developed to analyze the association of T. gondii seropositivity or serointensity with history of suicidal behavior. In those younger than the median age of the sample, 38, T. gondii serointensity was associated with history of suicidal behavior (p=0.02), while in the older patients the relationship was not significant (p=0.21). Seropositivity was also associated with history of suicide attempt in younger patients, odds ratio 1.59 (95% CI 1.06 to 2.40), p=0.03. Seropositivity for CMV (p=0.22), HSV-1 (p=0.36) and gliadin (p=0.92) was not related to history of suicide attempt in the entire sample or any age subgroup. T. gondii serology might become, with interaction with vulnerability genes, a candidate biomarker for a subgroup of schizophrenia patients prone to attempting suicide.

Copyright © 2011 Elsevier B.V. All rights reserved. PMID:21890329[PubMed - as supplied by publisher]