Sunday, May 29, 2011

Toxoplasma gondii aspartic protease 1 is not essential in tachyzoites

Exp Parasitol. 2011 May 15. [Epub ahead of print]

Toxoplasma gondii aspartic protease 1 is not essential in tachyzoites

Polonais V, Shea M, Soldati-Favre D

SourceDepartment of Microbiology and Molecular Medicine, CMU, University of Geneva, 1 Rue Michel-Servet, CH-1211 Geneva 4, Switzerland.

Abstract
Aspartic proteases are important virulence factors for pathogens and are recognized as attractive drug targets. Seven aspartic proteases (ASPs) have been identified in Toxoplasma gondii genome. Bioinformatics and phylogenetic analyses regroup them into five monophyletic groups. Among them, TgASP1, a coccidian specific aspartic protease related to the food vacuole plasmepsins, is associated with the secretory pathway in non-dividing cells and relocalizes in close proximity to the nascent inner membrane complex (IMC) of daughter cells during replication. Despite a potential role for TgASP1 in IMC formation, the generation of a conventional knockout of the TgASP1 gene revealed that this protease is not required for T. gondii tachyzoite survival or for proper IMC biogenesis.

Copyright © 2011. Published by Elsevier Inc.

PMID:21616070[PubMed - as supplied by publisher]

Friday, May 27, 2011

Unusual N-glycan structures are required for trafficking Toxoplasma gondii GAP50 to the inner membrane complex and regulate host cell entry

Mol Cell Proteomics. 2011 May 24. [Epub ahead of print]

Unusual N-glycan structures are required for trafficking Toxoplasma gondii GAP50 to the inner membrane complex and regulate host cell entry through parasite motility

Fauquenoy S, Hovasse A, Sloves PJ, Morelle W, Tchilabalo-Dilezitoko E, Slomianny C, Werkmeister E, Schaeffer C, Van Dorsselaer A, Tomavo S.
SourceCNRS UMR 8204-INSERM U 1019, France;

Abstract
Toxoplasma gondii motility, which is essential for host cell entry, migration through host tissues and invasion, is a unique form of actin-dependent gliding. It is powered by a motor complex mainly composed of myosin heavy chain A (MyoA), myosin light chain 1 (MLC1), gliding associated proteins GAP45 and GAP50, the only integral membrane anchor so far described. In the present study, we have combined glycomic and proteomic approaches to demonstrate that all three potential N-glycosylated sites of GAP50 are occupied by unusual N-glycan structures that are rarely found on mature mammalian glycoproteins. Using site-directed mutagenesis, we show that N-glycosylation is a prerequisite for GAP50 transport from the endoplasmic reticulum to the Golgi apparatus and for its subsequent delivery into the inner complex membrane. Assembly of key partners into the gliding complex, and parasite motility are severely impaired in the unglycosylated GAP50 mutants. Furthermore, comparative affinity purification using N-glycosylated and unglycosylated GAP50 as bait identified three novel hypothetical proteins including the recently described gliding associated protein GAP40, and we demonstrate that N-glycans are required for efficient binding to gliding partners. Collectively, these results provide the first detailed analyses of T. gondii N-glycosylation functions that are vital for parasite motility and host cell entry.

PMID:21610105[PubMed - as supplied by publisher]

Thursday, May 26, 2011

IL-6 Mediates the Susceptibility of Glycoprotein 130 Hypermorphs to Toxoplasma gondii

J Immunol. 2011 May 23. [Epub ahead of print]

IL-6 Mediates the Susceptibility of Glycoprotein 130 Hypermorphs to Toxoplasma gondii

Silver JS, Stumhofer JS, Passos S, Ernst M, Hunter CA

SourceDepartment of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104.

Abstract
IL-6 and IL-27 are closely related cytokines that play critical but distinct roles during infection with Toxoplasma gondii. Thus, IL-6 is required for the development of protective immunity to this pathogen, whereas IL-27 is required to limit infection-induced pathology. Paradoxically, these factors both signal through gp130, but little is known about how the signals downstream of gp130 are integrated to coordinate the immune response to infection. To better understand these events, gp130 Y757F mice that have a mutation in gp130 at the binding site for suppressor of cytokine signaling 3, a critical negative regulator of gp130 signaling, were infected with T. gondii. These mutant mice were acutely susceptible to this challenge, characterized by an early defect in the production of IL-12 and IFN-γ and increased parasite burdens. Consistent with the reduced IL-12 levels, IL-6, but not other gp130 cytokines, was a potent antagonist of IL-12 production by gp130 Y757F macrophages and dendritic cells in vitro. Moreover, in gp130 Y757F mice, blocking IL-6 in vivo, or administration of rIL-12, during infection restored IFN-γ production and protective immunity. Collectively, these studies highlight that a failure to abbreviate IL-6-mediated gp130 signaling results in a profound anti-inflammatory signal that blocks the generation of protective immunity to T. gondii.

PMID:21606248[PubMed - as supplied by publisher]

Human impact on genetic diversity of Toxoplasma gondii: Example of the anthropized environment from French Guiana

Infect Genet Evol. 2011 May 11. [Epub ahead of print]

Human impact on genetic diversity of Toxoplasma gondii: Example of the anthropized environment from French Guiana

Mercier A, Ajzenberg D, Devillard S, Demar MP, de Thoisy B, Bonnabau H, Collinet F, Boukhari R, Blanchet D, Simon S, Carme B, Dardé ML

SourceIFR 145 GEIST, EA 3174 NeuroEpidémiologie Tropicale et Comparée, Faculté de Médecine, 2 rue du Dr Marcland, Limoges, 87025 Limoges Cedex, France.

Abstract
In French Guiana, severe cases of toxoplasmosis in immunocompetent patients are associated with atypical strains of Toxoplasma gondii linked to a wild neotropical rainforest cycle and a higher genetic diversity than usually observed for T. gondii isolates from anthropized environment. This raises the question of the impact of anthropization of the natural environment, on genetic diversity and on the population structure of T. gondii. However, few data are available on strains circulating in the anthropized areas from French Guiana. Seropositive animals originating mainly from anthropized sub-urban areas and punctually from wild environment in French Guiana were analyzed for T. gondii isolation and genotyping. Thirty-three strains were obtained by bioassay in mice and compared with 18 previously reported isolates chiefly originating from the Amazon rainforest. The genotyping analysis performed with 15 microsatellite markers located on 12 different chromosomes revealed a lower genetic diversity in the anthropized environment. Results were analyzed in terms of population structure by clustering methods, Neighbor-joining trees reconstruction based on genetic distances, F(ST,) Mantel's tests and linkage disequilibrium. They clearly showed a genetic differentiation between strains associated to the anthropized environment and those associated to the wild, but with some inbreeding between them. The majority of strains from the anthropized environment were clustered into additional lineages of T. gondii that are common in the Caribbean. In conclusion the two environmental populations "wild" and "anthropized" were genetically well differentiated. The anthropization of the environment seems to be accompanied with a decreased diversity of T. gondii associated with a greater structure of the populations. We detected potential interpenetration and genetic exchanges between these two environmental populations. As a higher pathogenicity in human of "wild" genotypes has been described, the interpenetration of both environments leads to hybridization between strains that may be at risk for human health.

Copyright © 2011. Published by Elsevier B.V.

PMID:21600306[PubMed - as supplied by publisher]

Toxoplasma gondii down modulates cadherin expression in skeletal muscle cells inhibiting myogenesis

BMC Microbiol. 2011 May 18;11(1):110. [Epub ahead of print]

Toxoplasma gondii down modulates cadherin expression in skeletal muscle cells inhibiting myogenesis

Gomes AF, Guimaraes EV, Carvalho L, Correa JR, Mendonca-Lima L, Barbosa HS.
Abstract
ABSTRACT:

BACKGROUND: Toxoplasma gondii belongs to a large and diverse group of obligate intracellular parasitic protozoa. Primary culture of mice skeletal muscle cells (SkMC) was employed as a model for experimental toxoplasmosis studies. The myogenesis of SkMC was reproduced in vitro and the ability of T. gondii tachyzoite forms to infect myoblasts and myotubes and its influence on SkMC myogenesis were analyzed.

RESULTS: In this study we show that, after 24 h of interaction, myoblasts (61%) were more infected with T. gondii than myotubes (38%) and inhibition of myogenesis was about 75%. The role of adhesion molecules such as cadherin in this event was investigated. First, we demonstrate that cadherin localization was restricted to the contact areas between myocytes/myocytes and myocytes/myotubes during the myogenesis process. Immunofluorescence and immunoblotting analysis of parasite-host cell interaction showed a 54% reduction in cadherin expression at 24 h of infection. Concomitantly, a reduction in M-cadherin mRNA levels was observed after 3 and 24 h of T. gondii-host cell interaction.

CONCLUSIONS: These data suggest that T. gondii is able to down regulate M-cadherinexpression, leading to molecular modifications in the host cell surface that interfere with membrane fusion and consequently affect the myogenesis process.

PMID:21592384[PubMed - as supplied by publisher]

Virulence differences in Toxoplasma mediated by amplification of a family of polymorphic pseudokinases

Proc Natl Acad Sci U S A. 2011 May 17. [Epub ahead of print]

Virulence differences in Toxoplasma mediated by amplification of a family of polymorphic pseudokinases

Behnke MS, Khan A, Wootton JC, Dubey JP, Tang K, Sibley LD

SourceDepartment of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110.

Abstract
The population structure of Toxoplasma gondii includes three highly prevalent clonal lineages referred to as types I, II, and III, which differ greatly in virulence in the mouse model. Previous studies have implicated a family of serine/threonine protein kinases found in rhoptries (ROPs) as important in mediating virulence differences between strain types. Here, we explored the genetic basis of differences in virulence between the highly virulent type I lineage and moderately virulent type II based on successful genetic cross between these lineages. Genome-wide association revealed that a single quantitative trait locus controls the dramatic difference in lethality between these strain types. Neither ROP16 nor ROP18, previously implicated in virulence of T. gondii, was found to contribute to differences between types I and II. Instead, the major virulence locus contained a tandem cluster of polymorphic alleles of ROP5, which showed similar protein expression between strains. ROP5 contains a conserved serine/threonine protein kinase domain that includes only part of the catalytic triad, and hence, all members are considered to be pseudokinases. Genetic disruption of the entire ROP5 locus in the type I lineage led to complete attenuation of acute virulence, and complementation with ROP5 restored lethality to WT levels. These findings reveal that a locus of polymorphic pseudokinases plays an important role in pathogenesis of toxoplasmosis in the mouse model.

PMID:21586633[PubMed - as supplied by publisher]

Wednesday, May 18, 2011

Control of Toxoplasma reactivation by rescue of dysfunctional CD8+ T-cell response via PD-1-PDL-1 blockade

Proc Natl Acad Sci U S A. 2011 May 16. [Epub ahead of print]

Control of Toxoplasma reactivation by rescue of dysfunctional CD8+ T-cell response via PD-1-PDL-1 blockade

Bhadra R, Gigley JP, Weiss LM, Khan IA

SourceDepartment of Microbiology, Immunology, and Tropical Medicine, George Washington University, Washington, DC 20037.

Abstract
In this study, we document that Toxoplasma gondii differentiation and reactivation are mediated by systemic CD8 T-cell dysfunction during chronic infection. We demonstrate that CD8(+) T-cell exhaustion occurs despite control of parasitemia during early-chronic toxoplasmosis. During later phases, these cells become exhausted, leading to parasite reactivation and mortality. Concomitant with increased CD8(+) T-cell apoptosis and decreased effector response, this dysfunction is characterized by a graded elevation in expression of inhibitory receptor PD-1 on these cells in both lymphoid and nonlymphoid tissue. Blockade of the PD-1-PDL-1 pathway reinvigorates this suboptimal CD8(+) T-cell response, resulting in control of parasite reactivation and prevention of mortality in chronically infected animals. To the best of our knowledge, this report is unique in showing that exposure to a persistent pathogen despite initial control of parasitemia can lead to CD8(+) T-cell dysfunction and parasite reactivation.

PMID:21576466[PubMed - as supplied by publisher]

Modulation of mouse macrophage proteome induced by Toxoplasma gondii tachyzoites in vivo

Parasitol Res. 2011 May 17. [Epub ahead of print]

Modulation of mouse macrophage proteome induced by Toxoplasma gondii tachyzoites in vivo

Zhou DH, Yuan ZG, Zhao FR, Li HL, Zhou Y, Lin RQ, Zou FC, Song HQ, Xu MJ, Zhu XQ

SourceDepartment of Parasitology, College of Veterinary Medicine, South China Agricultural University, Guangzhou, Guangdong Province, 510642, People's Republic of China.

Abstract
Toxoplasma gondii is an obligate intracellular protozoan parasite, which can invade and multiply within the macrophages of humans and most warm-blooded animals. Macrophages are important effector cells for the control and killing of intracellular T. gondii, and they may also serve as long-term host cells for the replication and survival of the parasite. In the present study, we explored the proteomic profile of macrophages of the specific pathogen-free Kunming mice at 24 h after infection with tachyzoites of the virulent T. gondii RH strain using two-dimensional gel electrophoresis combined with matrix-assisted laser desorption ionization time-of-flight (TOF)/TOF tandem mass spectrometry. Totally, 60 differentially expressed protein spots were identified. Among them, 52 spots corresponded to 38 proteins matching to proteins of the mouse, including actin, enolase, calumenin, vimentin, plastin 2, annexin A1, cathepsin S, arginase-1, arachidonate 12-lipoxygenase, and aminoacylase-1. Functional prediction using Gene Ontology database showed that these proteins were mainly involved in metabolism, structure, protein fate, and immune responses. The findings provided an insight into the interactive relationship between T. gondii and the host macrophages, and will shed new lights on the understanding of molecular mechanisms of T. gondii pathogenesis.

PMID:21584632[PubMed - as supplied by publisher]

Friday, May 13, 2011

Functional genetics in Apicomplexa: Potentials and limits

FEBS Lett. 2011 May 7. [Epub ahead of print]

Functional genetics in Apicomplexa: Potentials and limits

Limenitakis J, Soldati-Favre D

SourceDepartment of Microbiology and Molecular Medicine, CMU, University of Geneva, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland.

Abstract
The Apicomplexans are obligate intracellular protozoan parasites and the causative agents of severe diseases in humans and animals. Although complete genome sequences are available since many years and for several parasites, they are replete with putative genes of unassigned function. Forward and reverse genetic approaches are limited only to a few Apicomplexans that can either be propagated in vitro or in a convenient animal model. This review will compare and contrast the most recent strategies developed for the genetic manipulation of Plasmodium falciparum, Plasmodium berghei and Toxoplasma gondii that have taken advantage of the intrinsic features of their respective genomes. Efforts towards the improvement of the transfection efficiencies in malaria parasites, the development of approaches to study essential genes and the elaboration of high-throughput methods for the identification of gene function will be discussed.

Copyright © 2011. Published by Elsevier B.V.

PMID:21557944[PubMed - as supplied by publisher]

Friday, May 06, 2011

Calcium Uptake and Proton Transport by Acidocalcisomes of Toxoplasma gondii

PLoS One. 2011 Apr 25;6(4):e18390.

Calcium Uptake and Proton Transport by Acidocalcisomes of Toxoplasma gondii

Rohloff P, Miranda K, Rodrigues JC, Fang J, Galizzi M, Plattner H, Hentschel J, Moreno SN

SourceDepartment of Cellular Biology and Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia, United States of America.

Abstract
Acidocalcisomes are acidic calcium stores found in diverse organisms, being conserved from bacteria to humans. They possess an acidic matrix that contains several cations bound to phosphates, which are mainly present in the form of short and long polyphosphate chains. Their matrix is acidified through the action of proton pumps such as a vacuolar proton ATPase and a vacuolar proton pyrophosphatase. Calcium uptake occurs through a Ca(2+)/H(+) countertransporting ATPase located in the membrane of the organelle. Acidocalcisomes have been identified in a variety of microorganisms, including Apicomplexan parasites such as Plasmodium and Eimeria species, and in Toxoplasma gondii. We report the purification and characterization of an acidocalcisome fraction from T. gondii tachyzoites after subcellular fractionation and further discontinuous iodixanol gradient purification. Proton and calcium transport activities in the fraction were characterized by fluorescence microscopy and spectrophotometric methods using acridine orange and arsenazo III, respectively. This work will facilitate the understanding of the function of acidocalcisomes in Apicomplexan parasites, as we can now isolate highly purified fractions that could be used for proteomic analysis to find proteins that may clarify the biogenesis of these organelles.

PMID:21541023[PubMed - in process]

Wednesday, May 04, 2011

Type II Toxoplasma gondii KU80 Knockout Strains Enable Functional Analysis of Genes Required for Cyst Development and Latent Infection

Eukaryot Cell. 2011 Apr 29. [Epub ahead of print]

Type II Toxoplasma gondii KU80 Knockout Strains Enable Functional Analysis of Genes Required for Cyst Development and Latent Infection

Fox BA, Falla A, Rommereim LM, Tomita T, Gigley JP, Mercier C, Cesbron-Delauw MF, Weiss LM, Bzik DJ.
SourceDepartment of Microbiology and Immunology, Dartmouth Medical School, 1 Medical Center Drive, Lebanon, NH 03756 USA.

Abstract
Type II Toxoplasma gondii KU80 knockouts (Δku80) deficient in nonhomologous end joining were developed to delete the dominant pathway mediating random integration of targeting episomes. Gene targeting frequency in the type II Δku80Δhxgprt strain measured at the orotate (OPRT) and the uracil (UPRT) phosphoribosyltransferase loci was highly efficient. To assess the potential of the type II Δku80Δhxgprt strain to examine gene function affecting cyst biology and latent stages of infection, we targeted the deletion of four parasite antigen genes (GRA4, GRA6, ROP7, tgd057) that encode characterized CD8(+) T cell epitopes that elicit corresponding antigen-specific CD8(+) T-cell populations associated with control of infection. Cyst development in these type II mutant strains was not found to be strictly dependent on antigen-specific CD8(+) T cell host responses. By contrast, a significant biological role was revealed for the dense granule proteins GRA4 and GRA6 in cyst development as brain tissue cyst burdens were drastically reduced specifically in mutant strains deleted for GRA4 and/or GRA6. Complementation of the Δgra4 and Δgra6 mutant strains using a functional allele of the deleted GRA coding region placed under control of the endogenous UPRT locus was found to significantly restore brain cyst burdens. These results reveal that GRA proteins play a functional role in establishing cyst burdens and latent infection. Collectively, our results suggest that the type II Δku80Δhxgprt genetic background enables a higher throughput functional analysis of the parasite genome to reveal fundamental aspects of parasite biology controlling virulence, pathogenesis, and transmission.

PMID:21531875[PubMed - as supplied by publisher]

Toxoplasma Infection and Later Development of Schizophrenia in Mothers

Am J Psychiatry. 2011 May 2. [Epub ahead of print]

Toxoplasma Infection and Later Development of Schizophrenia in Mothers

Pedersen MG, Stevens H, Pedersen CB, Nørgaard-Pedersen B, Mortensen PB.
SourceNational Center for Register-Based Research, Aarhus University; and Statens Serum Institut, Artillerivej, Copenhagen.

Abstract
Objective: Several studies based on clinical samples have found an association between Toxoplasma gondii infection and schizophrenia, and a case-control study among U.S. military personnel with specimens available from both before and after diagnosis found a positive association between T. gondii immunoglobulin G (IgG) antibody level and schizophrenia. These findings have never been replicated in a prospective cohort study. The purpose of this study was to determine whether mothers infected with T. gondii have an elevated risk of schizophrenia or related disorders and whether the risk depends on IgG antibody level. Method: In a register-based prospective cohort study of 45,609 women born in Denmark, the level of T. gondii-specific IgG antibodies was measured in connection with childbirth between 1992 and 1995. Women were followed up from the date of delivery until 2008. Results: A significant positive association between T. gondii IgG antibody level and schizophrenia spectrum disorders was found. Mothers with the highest IgG level had a relative risk of 1.73 (95% confidence interval [CI]=1.12-2.62) compared with mothers with the lowest IgG level. For schizophrenia, the relative risk was 1.68 (95% CI=0.77-3.46). When the mothers were classified according to IgG level, only those with the highest IgG levels had a significantly higher risk of schizophrenia spectrum disorders. Conclusions: Women with high levels of T. gondii-specific IgG antibodies have a significantly elevated risk of developing schizophrenia spectrum disorders.

PMID:21536690[PubMed - as supplied by publisher]

The moving junction of apicomplexan parasites: a key structure for invasion

Cell Microbiol. 2011 Apr 28. doi: 10.1111/j.1462-5822.2011.01597.x. [Epub ahead of print]

The moving junction of apicomplexan parasites: a key structure for invasion

Besteiro S, Dubremetz JF, Lebrun M.
SourceUMR 5235 CNRS, DIMNP, Université de Montpellier 2, cc 107, 34095 Montpellier cedex 05, France.

Abstract
Most Apicomplexa are obligate intracellular parasites and many are important pathogens of human and domestic animals. For a successful cell invasion, they rely on their own motility and on a firm anchorage to their host cell, depending on the secretion of proteins and the establishment of a structure called the moving junction (MJ). The MJ moves from the apical to the posterior end of the parasite, leading to the internalization of the parasite into a parasitophorous vacuole. Based on recent data obtained in Plasmodium and Toxoplasma, an emerging model emphasizes a cooperative role of secreted parasitic proteins in building the MJ and driving this crucial invasive process. More precisely, the parasite exports the microneme protein AMA1 to its own surface and the rhoptry neck RON2 protein as a receptor inserted into the host cell together with other RON partners. Ongoing and future research will certainly help refining the model by characterizing the molecular organization within the MJ and its interactions with both host and parasite cytoskeleton for anchoring of the complex.

© 2011 Blackwell Publishing Ltd.

PMID:21535344[PubMed - as supplied by publisher]