Toxoplasma polymorphic effectors determine macrophage polarization and intestinal inflammation

Cell Host Microbe. 2011 Jun 16;9(6):472-83

Toxoplasma polymorphic effectors determine macrophage polarization and intestinal inflammation

Jensen KD, Wang Y, Wojno ED, Shastri AJ, Hu K, Cornel L, Boedec E, Ong YC, Chien YH, Hunter CA, Boothroyd JC, Saeij JP

SourceDepartment of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USA.

Abstract
European and North American strains of the parasite Toxoplasma gondii belong to three distinct clonal lineages, type I, type II, and type III, which differ in virulence. Understanding the basis of Toxoplasma strain differences and how secreted effectors work to achieve chronic infection is a major goal of current research. Here we show that type I and III infected macrophages, a cell type required for host immunity to Toxoplasma, are alternatively activated, while type II infected macrophages are classically activated. The Toxoplasma rhoptry kinase ROP16, which activates STAT6, is responsible for alternative activation. The Toxoplasma dense granule protein GRA15, which activates NF-κB, promotes classical activation by type II parasites. These effectors antagonistically regulate many of the same genes, and mice infected with type II parasites expressing type I ROP16 are protected against Toxoplasma-induced ileitis. Thus, polymorphisms in determinants that modulate macrophage activation influence the ability of Toxoplasma to establish a chronic infection.

Copyright © 2011 Elsevier Inc. All rights reserved.

PMID:21669396[PubMed - in process]

Mol Biochem Parasitol. 2011 Jun 2. [Epub ahead of print]

A transmembrane domain containing pellicle protein of Toxoplasma gondii enhances virulence and invasion after extracellular stress

Payne TM, Lund PJ, Knoll LJ

SourceDepartment of Medical Microbiology and Immunology, University of Wisconsin School of Medicine and Public Health, Microbial Sciences Building Room 3345, 1550 Linden Drive, Madison, WI 53706, United States.

Abstract
To identify Toxoplasma gondii genes important in the establishment of a persistent infection, we previously used signature-tagged mutagenesis to identify mutants with reduced cyst numbers in the brains of mice. One of the mutants, 95C5, has an insertion within a predicted six transmembrane domain protein, which localizes to the parasite pellicle, thus we named it transmembrane pellicle protein 1 (TgTPP1). Although the 95C5 mutant was found be reduced in its ability to form brain cysts, it is defective during acute infection. Addition of TgTPP1 expressed from its endogenous promoter restored the acute lethality of the 95C5 mutant to parental levels. The 95C5 mutant does not have a growth defect in standard tissue culture conditions; however, we found a significant defect in host cell penetration after extracellular stress. Overall, TgTPP1 may function during acute infection by enhancing the parasites ability to invade after extracellular stress.

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

PMID:21669237[PubMed - as supplied by publisher]

The CD8 T-cell road to immunotherapy of toxoplasmosis

Immunotherapy. 2011 Jun;3(6):789-801

The CD8 T-cell road to immunotherapy of toxoplasmosis

Bhadra R, Gigley JP, Khan IA.
SourceDepartment of Microbiology, Immunology & Tropical Medicine, George Washington University, Washington, DC 20037, USA.

Abstract
Toxoplasma gondii infection induces a robust CD8 T-cell immunity that is critical for keeping chronic infection under control. In studies using animal models, it has been demonstrated that the absence of this response can compromise the host ability to keep chronic infection under check. Therapeutic agents that facilitate the induction and maintenance of CD8 T-cell response against the pathogen need to be developed. In the last decade, major strides in understanding the development of effector and memory response, particularly in viral and tumor models, have been made. However, factors involved in the generation of effector or memory response against T. gondii infection have not been extensively investigated. This information will be invaluable in designing immunotherapeutic regimens needed for combating this intracellular pathogen that poses a severe risk for pregnant women and immunocompromised individuals.

PMID:21668315[PubMed - in process]

Cathepsin Proteases in Toxoplasma gondii

Adv Exp Med Biol. 2011;712:49-61.

Cathepsin Proteases in Toxoplasma gondii

Dou Z, Carruthers VB

SourceDepartment of Microbiology and Immunology, University of Michigan School of Medicine, Ann Arbor, Michigan, USA.

Abstract
Cysteine proteases are important for the growth and survival of apicomplexan parasites that infect humans. The apicomplexan Toxoplasma gondii expresses five members of the C1 family of cysteine proteases, including one cathepsin L-like (TgCPL), one cathepsin B-like (TgCPB) and three cathepsin C-like (TgCPC1, 2 and 3) proteases. Recent genetic, biochemical and structural studies reveal that cathepsins function in microneme and rhoptry protein maturation, host cell invasion, replication and nutrient acquisition. here, we review the key features and roles of T. gondii cathepsins and discuss the therapeutic potential for specific inhibitor development.

PMID:21660658[PubMed - in process]

Focus on the ringleader: the role of AMA1 in apicomplexan invasion and replication

Trends Parasitol. 2011 Jun 7. [Epub ahead of print]

Focus on the ringleader: the role of AMA1 in apicomplexan invasion and replication

Tyler JS, Treeck M, Boothroyd JC.

SourceDepartment of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA.

Abstract
Apicomplexan parasites exhibit an unusual mechanism of host cell penetration. A central player in this process is the protein apical membrane antigen 1 (AMA1). Although essential for invasion, the precise functional roles AMA1 plays have been unclear. Several recent studies have provided important functional insight into its role within the multiprotein complex that comprises the moving junction (MJ). Initially formed at the apical tip of the invading parasite, the MJ represents a ring-like region of contact between the surfaces of the invading parasite and the host cell as the invaginated host plasma membrane is forced inward by the penetrating parasite. This review discusses these and other recent insights into AMA1 with particular emphasis on studies conducted in Plasmodium and Toxoplasma.

Copyright © 2011 Elsevier Ltd. All rights reserved.

PMID:21659001[PubMed - as supplied by publisher]

Involvement of a Toxoplasma gondii Chromatin Remodeling Complex Ortholog in Developmental Regulation

PLoS One. 2011;6(5):e19570. Epub 2011 May 31

Involvement of a Toxoplasma gondii Chromatin Remodeling Complex Ortholog in Developmental Regulation

Rooney PJ, Neal LM, Knoll LJ

SourceDepartment of Medical Microbiology and Immunology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, United States of America.

Abstract
The asexual cycle of the parasite Toxoplasma gondii has two developmental stages: a rapidly replicating form called a tachyzoite and a slow growing cyst form called a bradyzoite. While the importance of ATP-independent histone modifications for gene regulation in T. gondii have been demonstrated, ATP-dependent chromatin remodeling pathways have not been examined. In this study we characterized C9, an insertional mutant showing reduced expression of bradyzoite differentiation marker BAG1, in cultured human fibroblasts. This mutant contains an insertion in the gene encoding TgRSC8, which is homologous to the Saccharomyces cerevisiae proteins Rsc8p (remodel the structure of chromatin complex subunit 8) and Swi3p (switch/sucrose non-fermentable [SWI/SNF]) of ATP-dependent chromatin-remodeling complexes. In the C9 mutant, TgRSC8 is the downstream open reading frame on a dicistronic transcript. Though protein was expressed from the downstream gene of the dicistron, TgRSC8 levels were decreased in C9 from those of wild-type parasites, as determined by western immunoblot and flow cytometry. As TgRSC8 localized to the parasite nucleus, we postulated a role in gene regulation. Transcript levels of several markers were assessed by quantitative PCR to test this hypothesis. The C9 mutant displayed reduced steady state transcript levels of bradyzoite-induced genes BAG1, LDH2, SUSA1, and ENO1, all of which were significantly increased with addition of TgRSC8 to the mutant. Transcript levels of some bradyzoite markers were unaltered in C9, or unable to be increased by complementation with TgRSC8, indicating multiple pathways control bradyzoite-upregulated genes. Together, these data suggest a role for TgRSC8 in control of bradyzoite-upregulated gene expression. Thus chromatin remodeling, by both ATP-independent and dependent mechanisms, is an important mode of gene regulation during stage differentiation in parasites.

PMID:21655329[PubMed - in process]

Parasite mitogen-activated protein kinases as drug discovery targets to treat human protozoan pathogens

J Signal Transduct. 2011;2011:971968. Epub 2011 Feb 27

Parasite mitogen-activated protein kinases as drug discovery targets to treat human protozoan pathogens

Brumlik MJ, Pandeswara S, Ludwig SM, Murthy K, Curiel TJ

SourceDepartment of Medicine, School of Medicine, and Program in Immunology and Microbiology, Graduate School of Biomedical Sciences, University of Texas Health Science Center, San Antonio, TX 78229, USA.

Abstract
Protozoan pathogens are a highly diverse group of unicellular organisms, several of which are significant human pathogens. One group of protozoan pathogens includes obligate intracellular parasites such as agents of malaria, leishmaniasis, babesiosis, and toxoplasmosis. The other group includes extracellular pathogens such as agents of giardiasis and amebiasis. An unfortunate unifying theme for most human protozoan pathogens is that highly effective treatments for them are generally lacking. We will review targeting protozoan mitogen-activated protein kinases (MAPKs) as a novel drug discovery approach towards developing better therapies, focusing on Plasmodia, Leishmania, and Toxoplasma, about which the most is known.

PMID:21637385[PubMed - in process]

Polyparasitism Is Associated with Increased Disease Severity in Toxoplasma gondii-Infected Marine Sentinel Species

PLoS Negl Trop Dis. 2011 May;5(5):e1142. Epub 2011 May 24.

Polyparasitism Is Associated with Increased Disease Severity in Toxoplasma gondii-Infected Marine Sentinel Species

Gibson AK, Raverty S, Lambourn DM, Huggins J, Magargal SL, Grigg ME.
SourceLaboratory of Parasitic Diseases, National Institutes of Health, National Institute of Allergy and Infectious Diseases (NIAID), Bethesda, Maryland, United States of America.

Abstract
In 1995, one of the largest outbreaks of human toxoplasmosis occurred in the Pacific Northwest region of North America. Genetic typing identified a novel Toxoplasma gondii strain linked to the outbreak, in which a wide spectrum of human disease was observed. For this globally-distributed, water-borne zoonosis, strain type is one variable influencing disease, but the inability of strain type to consistently explain variations in disease severity suggests that parasite genotype alone does not determine the outcome of infection. We investigated polyparasitism (infection with multiple parasite species) as a modulator of disease severity by examining the association of concomitant infection of T. gondii and the related parasite Sarcocystis neurona with protozoal disease in wild marine mammals from the Pacific Northwest. These hosts ostensibly serve as sentinels for the detection of terrestrial parasites implicated in water-borne epidemics of humans and wildlife in this endemic region. Marine mammals (151 stranded and 10 healthy individuals) sampled over 6 years were assessed for protozoal infection using multi-locus PCR-DNA sequencing directly from host tissues. Genetic analyses uncovered a high prevalence and diversity of protozoa, with 147/161 (91%) of our sampled population infected. From 2004 to 2009, the relative frequency of S. neurona infections increased dramatically, surpassing that of T. gondii. The majority of T. gondii infections were by genotypes bearing Type I lineage alleles, though strain genotype was not associated with disease severity. Significantly, polyparasitism with S. neurona and T. gondii was common (42%) and was associated with higher mortality and more severe protozoal encephalitis. Our finding of widespread polyparasitism among marine mammals indicates pervasive contamination of waterways by zoonotic agents. Furthermore, the significant association of concomitant infection with mortality and protozoal encephalitis identifies polyparasitism as an important factor contributing to disease severity in marine mammals.

PMID:21629726[PubMed - in process]

Migratory activation of primary cortical microglia upon infection with Toxoplasma gondii

Infect Immun. 2011 May 31. [Epub ahead of print]

Migratory activation of primary cortical microglia upon infection with Toxoplasma gondii

Dellacasa-Lindberg I, Fuks JM, Arrighi RB, Lambert H, Wallin RP, Chambers BJ, Barragan A.
SourceCenter for Infectious Medicine, Dept. of Medicine, Karolinska Institutet, 171 77 Stockholm, Sweden.

Abstract
Disseminated Toxoplasmosis in the central nervous system (CNS) is often accompanied by lethal outcome. Studies in murine models of infection have focused on the role of systemic immunity for control of toxoplasmic encephalitis while knowledge remains limited on the contribution of resident cells with immune functions in the CNS. Here, the role of glia cells was addressed in the setting of recrudescent Toxoplasma infection in mice. Activated astrocytes and microglia were observed in close vicinity of foci with replicating parasites in situ in the brain parenchyma. Toxoplasma gondii tachyzoites were allowed to infect primary microglia and astrocytes in vitro. Microglia were permissive to parasite replication and infected microglia readily transmigrated across transwell membranes and cell monolayers. Thus, infected microglia, but not astrocytes, exhibited a hypermotility phenotype reminiscent of that recently described for infected dendritic cells. In contrast to interferon γ-activated microglia, Toxoplasma-infected microglia did not upregulate MHC class II and the co-stimulatory molecule CD86. Yet, Toxoplasma-infected microglia and astrocytes exhibited increased sensitivity to T cell-mediated killing, leading to rapid parasite transfer to effector T cells in vitro. We hypothesize that glia and T cells, besides their role in triggering anti-parasite immunity, may also act as 'Trojan horses' paradoxically facilitating dissemination of Toxoplasma within the CNS. To our knowledge, this constitutes the first report of migratory activation of a resident CNS cell by an intracellular parasite.

PMID:21628522[PubMed - as supplied by publisher]

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]

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]

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]

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]

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]

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]

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]

Extracellular Toxoplasma gondii tachyzoites do not require carbon source uptake for ATP maintenance, gliding motility and invasion

Int J Parasitol. 2011 Apr 7. [Epub ahead of print]

Extracellular Toxoplasma gondii tachyzoites do not require carbon source uptake for ATP maintenance, gliding motility and invasion in the first hour of their extracellular life

Lin SS, Blume M, von Ahsen N, Gross U, Bohne W.
SourceInstitute of Medical Microbiology, University Medical Center Göttingen, Germany.

Abstract
Apicomplexan parasites undergo metabolic shifts in adaptation to environmental changes. Here, we investigate the metabolic requirements which are responsible for ATP homeostasis in the extracellular stage of Toxoplasma gondii. Surprisingly, we found that freshly released tachyzoites are able to maintain a constant ATP level during the first hour of extracellular incubation without the acquisition of external carbon sources. We further demonstrated that the extent of gliding motility and that of host cell invasion is independent from the availability of external carbon sources during this one hour extracellular period. The ATP level and the invasion efficiency of extracellular parasites were severely decreased by treatment with the glycolysis inhibitor, 2-deoxy-d-glucose, but not by the F(0)F(1)-ATPase inhibitor, oligomycin. This suggests that although the uptake of glucose itself is not required during the 1h incubation period, extracellular parasites depend on the activity of the glycolytic pathway for ATP homeostasis. Furthermore, active glycolysis was evident by the secretion of lactate into the culture medium, even in the absence of external carbon sources. Together, our studies suggest that tachyzoites are independent from external carbon sources within the first hour of their extracellular life, which is the most relevant time span for finding a new host cell, but rely on the glycolytic metabolisation of internal carbon sources for ATP maintenance, gliding motility and host cell invasion.

Copyright © 2011. Published by Elsevier Ltd.

PMID:21515276[PubMed - as supplied by publisher]

Jumbled Genomes: Missing Apicomplexan Synteny

Mol Biol Evol. 2011 Apr 19. [Epub ahead of print]

Jumbled Genomes: Missing Apicomplexan Synteny

Debarry JD, Kissinger JC.
SourceCenter for Tropical and Emerging Global Diseases.

Abstract
Whole-genome comparisons provide insight into genome evolution by informing on gene repertoires, gene gains/losses and genome organization. Most of our knowledge about eukaryotic genome evolution is derived from studies of multi-cellular model organisms. The eukaryotic phylum Apicomplexa contains obligate intracellular protist parasites responsible for a wide range of human and veterinary diseases (e.g. malaria, toxoplasmosis, and theileriosis). We have developed an in silico protein-encoding gene based pipeline to investigate synteny across 12 apicomplexan species from 6 genera. Genome rearrangement between lineages is extensive. Syntenic regions (conserved gene content and order) are rare between lineages and appear to be totally absent across the phylum, with no group of three genes found on the same chromosome and in the same order within 25 kb up- and downstream of any orthologous genes. Conserved synteny between major lineages is limited to small regions in Plasmodium and Theileria/Babesia species and within these conserved regions there are a number of proteins putatively targeted to organelles. The observed overall lack of synteny is surprising considering the divergence times and the apparent absence of transposable elements (TEs) within any of the species examined. TEs are ubiquitous in all other groups of eukaryotes studied to date, and have been shown to be involved in genomic rearrangements. It appears that there are different criteria governing genome evolution within the Apicomplexa relative to other well-studied unicellular and multi-cellular eukaryotes.

PMID:21504890[PubMed - as supplied by publisher]

A Toxoplasma gondii protein with homology to intracellular type Na(+)/H(+) exchangers is important for osmoregulation and invasion

Exp Cell Res. 2011 Apr 9. [Epub ahead of print]

A Toxoplasma gondii protein with homology to intracellular type Na(+)/H(+) exchangers is important for osmoregulation and invasion

Francia ME, Wicher S, Pace DA, Sullivan J, Moreno SN, Arrizabalaga G

SourceDepartment of Biological Sciences, University of Idaho, Life Sciences South Room 142, Moscow, ID 83844, USA.

Abstract
The obligate intracellular parasite Toxoplasma gondii is exposed to a variety of physiological conditions while propagating in an infected organism. The mechanisms by which Toxoplasma overcomes these dramatic changes in its environment are not known. In yeast and plants, ion detoxification and osmotic regulation are controlled by vacuolar compartments. A novel compartment named the plant-like vacuole or vacuolar compartment (PLV/VAC) has recently been described in T.gondii, which could potentially protect extracellular tachyzoites against salt and other ionic stresses. Here, we report the molecular characterization of the vacuolar type Na(+)/H(+) exchanger in T. gondii, TgNHE3, and its co-localization with the PLV/VAC proton-pyrophosphatase (TgVP1). We have created a TgNHE3 knockout strain, which is more sensitive to hyperosmotic shock and toxic levels of sodium, possesses a higher intracellular Ca(2+) concentration [Ca(2+)](i), and exhibits a reduced host invasion efficiency. The defect in invasion correlates with a measurable reduction in the secretion of the adhesin TgMIC2. Overall, our results suggest that the PLV/VAC has functions analogous to those of the vacuolar compartments of plants and yeasts, providing the parasite with a mechanism to resist ionic fluctuations and, potentially, regulate protein trafficking.

Copyright © 2011. Published by Elsevier Inc.

PMID:21501607[PubMed - as supplied by publisher]

Immune response and protective efficacy against homologous challenge in BALB/c mice vaccinated with DNA vaccine

Vet Parasitol. 2011 Mar 12. [Epub ahead of print]

Immune response and protective efficacy against homologous challenge in BALB/c mice vaccinated with DNA vaccine encoding Toxoplasma gondii actin depolymerizing factor gene

Li J, Huang X, Zhang G, Gong P, Zhang X, Wu L.

College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi'an Road, Changchun, Jilin 130062, China.

Abstract
A DNA vaccine (pVAX1-TgADF) encoding Toxoplasma gondii actin depolymerizing factor (ADF) gene was constructed and the immune response and protective efficacy of this vaccine against homologous challenge in BALB/c mice were evaluated. High titers of specific antibody and increases in the percentage of CD4(+) and CD8(+) T lymphocyte cells were observed from BALB/c mice vaccinated with pVAX1-TgADF (P<0.05), when PBS group was used as control. The survival time of BALB/c mice in pVAX1-TgADF group was longer than those in control groups. The numbers of brain cysts in the experimental BALB/c mice immunized with pVAX1-TgADF reduced significantly compared with those in PBS group (P<0.05), and the rate of reduction could reach to around 42.8%. These results suggested that the DNA vaccine pVAX1-TgADF could generate specific humoral and cellular immune responses, prolong survival times, and reduce brain cysts load against T. gondii infection in BALB/c mice.

Copyright © 2011 Elsevier B.V. All rights reserved.
PMID: 21489695 [PubMed - as supplied by publisher]

Toxoplasma in Animals, Food, and Humans: An Old Parasite of New Concern

Foodborne Pathog Dis. 2011 Apr 12. [Epub ahead of print]

Toxoplasma in Animals, Food, and Humans: An Old Parasite of New Concern

Cenci-Goga BT, Rossitto PV, Sechi P, McCrindle CM, Cullor JS.

1 Sezione di Ispezione degli Alimenti di Origine Animale, Facoltà di Medicina Veterinaria, Università degli Studi di Perugia , Perugia, Italy .

Abstract
Abstract All hosts, including humans, can be infected by any one of the three forms of the parasite Toxoplasma gondii that correspond to three morphological stages: tachyzoite, bradyzoite, and sporozoite form. Felids are definitive hosts for T. gondii, which is an intracellular pathogen that infects a wide range of warm-blooded intermediate hosts. Toxoplasmosis is a disease where the interest of the diverse medical and veterinary specialties converge. Awareness needs to be increased that toxoplasmosis can induce clinical disease not only in immunocompromised patients or through congenital infections, but also in healthy patients. This is a review article that aims at illustrating why toxoplasmosis should be regarded a veterinary public health issue and how veterinary practitioners can contribute in controlling the infection.

PMID: 21486145 [PubMed - as supplied by publisher]

Identification of Novel Proteins in Neospora caninum Using an Organelle Purification and Monoclonal Antibody Approach

PLoS One. 2011 Apr 4;6(4):e18383.

Identification of Novel Proteins in Neospora caninum Using an Organelle Purification and Monoclonal Antibody Approach

Sohn CS, Cheng TT, Drummond ML, Peng ED, Vermont SJ, Xia D, Cheng SJ, Wastling JM, Bradley PJ.

Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, Los Angeles, California, United States of America.

Abstract
Neospora caninum is an important veterinary pathogen that causes abortion in cattle and neuromuscular disease in dogs. Neospora has also generated substantial interest because it is an extremely close relative of the human pathogen Toxoplasma gondii, yet does not appear to infect humans. While for Toxoplasma there are a wide array of molecular tools and reagents available for experimental investigation, relatively few reagents exist for Neospora. To investigate the unique biological features of this parasite and exploit the recent sequencing of its genome, we have used an organelle isolation and monoclonal antibody approach to identify novel organellar proteins and develop a wide array of probes for subcellular localization. We raised a panel of forty-six monoclonal antibodies that detect proteins from the rhoptries, micronemes, dense granules, inner membrane complex, apicoplast, mitochondrion and parasite surface. A subset of the proteins was identified by immunoprecipitation and mass spectrometry and reveal that we have identified and localized many of the key proteins involved in invasion and host interaction in Neospora. In addition, we identified novel secretory proteins not previously studied in any apicomplexan parasite. Thus, this organellar monoclonal antibody approach not only greatly enhances the tools available for Neospora cell biology, but also identifies novel components of the unique biological characteristics of this important veterinary pathogen.

PMID: 21483743 [PubMed - in process]

A Novel Toxoplasma gondii Nuclear Factor TgNF3 Is a Dynamic Chromatin-Associated Component, Modulator of Nucleolar Architecture and Parasite Virulence

PLoS Pathog. 2011 Mar;7(3):e1001328. Epub 2011 Mar 31.

A Novel Toxoplasma gondii Nuclear Factor TgNF3 Is a Dynamic Chromatin-Associated Component, Modulator of Nucleolar Architecture and Parasite Virulence

Olguin-Lamas A, Madec E, Hovasse A, Werkmeister E, Callebaut I, Slomianny C, Delhaye S, Mouveaux T, Schaeffer-Reiss C, Van Dorsselaer A, Tomavo S.

Center for Infection and Immunity of Lille, CNRS UMR 8204, INSERM U 1019, Institut Pasteur de Lille, Université Lille Nord de France, Lille, France.

Abstract
In Toxoplasma gondii, cis-acting elements present in promoter sequences of genes that are stage-specifically regulated have been described. However, the nuclear factors that bind to these cis-acting elements and regulate promoter activities have not been identified. In the present study, we performed affinity purification, followed by proteomic analysis, to identify nuclear factors that bind to a stage-specific promoter in T. gondii. This led to the identification of several nuclear factors in T. gondii including a novel factor, designated herein as TgNF3. The N-terminal domain of TgNF3 shares similarities with the N-terminus of yeast nuclear FK506-binding protein (FKBP), known as a histone chaperone regulating gene silencing. Using anti-TgNF3 antibodies, HA-FLAG and YFP-tagged TgNF3, we show that TgNF3 is predominantly a parasite nucleolar, chromatin-associated protein that binds specifically to T. gondii gene promoters in vivo. Genome-wide analysis using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified promoter occupancies by TgNF3. In addition, TgNF3 has a direct role in transcriptional control of genes involved in parasite metabolism, transcription and translation. The ectopic expression of TgNF3 in the tachyzoites revealed dynamic changes in the size of the nucleolus, leading to a severe attenuation of virulence in vivo. We demonstrate that TgNF3 physically interacts with H3, H4 and H2A/H2B assembled into bona fide core and nucleosome-associated histones. Furthermore, TgNF3 interacts specifically to histones in the context of stage-specific gene silencing of a promoter that lacks active epigenetic acetylated histone marks. In contrast to virulent tachyzoites, which express the majority of TgNF3 in the nucleolus, the protein is exclusively located in the cytoplasm of the avirulent bradyzoites. We propose a model where TgNF3 acts essentially to coordinate nucleolus and nuclear functions by modulating nucleosome activities during the intracellular proliferation of the virulent tachyzoites of T. gondii.

PMID: 21483487 [PubMed - in process]

Serum Response Factor Regulates Immediate Early Host Gene Expression in Toxoplasma gondii-Infected Host Cells

PLoS One. 2011 Mar 29;6(3):e18335.

Serum Response Factor Regulates Immediate Early Host Gene Expression in Toxoplasma gondii-Infected Host Cells

Wiley M, Teygong C, Phelps E, Radke J, Blader IJ.

Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America.

Abstract
Toxoplasma gondii is a wide spread pathogen that can cause severe and even fatal disease in fetuses and immune-compromised hosts. As an obligate intracellular parasite, Toxoplasma must alter the environment of its host cell in order to establish its replicative niche. This is accomplished, in part, by secretion of factors into the host cell that act to modulate processes such as transcription. Previous studies demonstrated that genes encoding transcription factors such as c-jun, junB, EGR1, and EGR2 were amongst the host genes that were the most rapidly upregulated following infection. In cells stimulated with growth factors, these genes are regulated by a transcription factor named Serum Response Factor. Serum Response Factor is a ubiquitously expressed DNA binding protein that regulates growth and actin cytoskeleton genes via MAP kinase or actin cytoskeletal signaling, respectively. Here, we report that Toxoplasma infection leads to the rapid activation of Serum Response Factor. Serum Response Factor activation is a Toxoplasma-specific event since the transcription factor is not activated by the closely related protozoan parasite, Neospora caninum. We further demonstrate that Serum Response Factor activation requires a parasite-derived secreted factor that signals via host MAP kinases but independently of the host actin cytoskeleton. Together, these data define Serum Response Factor as a host cell transcription factor that regulates immediate early gene expression in Toxoplasma-infected cells.

PMID: 21479245 [PubMed - as supplied by publisher]

Direct evidence of Toxoplasma-induced changes in serum testosterone in mice

Exp Parasitol. 2011 Mar 30. [Epub ahead of print]

Direct evidence of Toxoplasma-induced changes in serum testosterone in mice

Kaňková S, Kodym P, Flegr J.

Department of Philosophy and History of Science, Faculty of Science, Charles University in Prague, Viničná 7, Prague 2, Czech Republic.

Abstract
Latent toxoplasmosis is known to influence the morphology of infected persons and also increases the probability of the birth of male offspring in both humans and mice. All these traits can be related to the observed differences in the concentration of testosterone between Toxoplasma-infected and Toxoplasma-free subjects. However, it is not possible to decide, using the Toxoplasma-human model, whether toxoplasmosis influences the level of testosterone in the infected host or whether individuals with different levels of testosterone vary in the probability of toxoplasma infection. Here we studied changes in the testosterone levels in the latent phase of toxoplasmosis in laboratory mice artificially infected with cystogenic but relatively virulent strain T38 of T. gondii. We observed decreased testosterone levels in both female and male mice with latent toxoplasmosis in comparison to uninfected controls (P = 0.001). The present results indicate that Toxoplasma infection changes the concentration of serum testosterone in mice and human rather than changed concentration of testosterone influences the probability of the Toxoplasma infection. It is possible that the decrease of testosterone is an adaptive mechanism of infected mice aimed to compensate toxoplasmosis-induced immunosuppression observed during latent Toxoplasma infection.

Copyright © 2011. Published by Elsevier Inc.
PMID: 21458453 [PubMed - as supplied by publisher]

Antibodies to infectious agents and the positive symptom dimension of subclinical psychosis: The TRAILS study

Schizophr Res. 2011 Mar 31. [Epub ahead of print]

Antibodies to infectious agents and the positive symptom dimension of subclinical psychosis: The TRAILS study

Wang H, Yolken RH, Hoekstra PJ, Burger H, Klein HC.

Department of Psychiatry, University Medical Center Groningen, University of Groningen, 9700 RB Groningen, The Netherlands; Department of Pharmacoepidemiology & Pharmacoeconomics, University of Groningen, 9713 AV Groningen, The Netherlands.

Abstract
Infections have been suggested to play a role in the etiology of schizophrenia, but the evidence for this has been inconsistent. Schizophrenia patients have an increased risk of infections as a result of hospitalizations or life style factors. Therefore a study on early subclinical manifestations of psychosis in relation to virus infections is warranted. We examined whether serum antibodies against human Herpes viruses and Toxoplasma gondii were associated with subclinical symptoms of psychosis in adolescents. Data were collected as part of the TRacking Adolescents' Individual Lives Survey (TRAILS) cohort, a large prospective cohort of Dutch adolescents. A total of 1176 participants with an available Community Assessment of Psychic Experiences (CAPE) and an available blood sample were included in this analysis. Solid-enzyme immunoassay methods were used to measure the presence of immunoglobulin G (IgG) antibodies in serum to the Herpes virus family and to T. gondii. There was no significant association between serologic evidence of infection with human Herpes viruses or T. gondii and the risk of subclinical positive experience of psychosis. Subjects with a positive serological reaction to Epstein-Barr Virus (EBV) had higher scores on the positive dimension of psychosis measured by CAPE (b=0.03, P=0.02). This significant association was observed in males, but not in females. The current study suggests that there is no significant association between serological evidence of infection to human Herpes viruses and positive subclinical experience of psychosis, whereas there was an association between EBV infection and subclinical psychotic symptoms in boys.

Copyright © 2010 Elsevier B.V. All rights reserved.
PMID: 21458236 [PubMed - as supplied by publisher]

Identification of the cross-reactive and species-specific antigens between Neospora caninum and Toxoplasma gondii tachyzoites by a proteomics approach

Parasitol Res. 2011 Apr 2. [Epub ahead of print]

Identification of the cross-reactive and species-specific antigens between Neospora caninum and Toxoplasma gondii tachyzoites by a proteomics approach

Zhang H, Lee EG, Yu L, Kawano S, Huang P, Liao M, Kawase O, Zhang G, Zhou J, Fujisaki K, Nishikawa Y, Xuan X.

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan.

Abstract
The characterization of the cross-reactive and species-specific antigens of Neospora caninum and Toxoplasma gondii is important in the exploration to determine the common mechanisms of parasite-host interaction and to improve the serological diagnosis; it is also useful for the selection of the cross-reactive antigens that could be used in the development of vaccines or drugs for controlling the diseases caused by these two parasites. In this study, cross-reactive and species-specific antigens between N. caninum and T. gondii tachyzoites were comprehensively investigated using a proteomics approach with the application of two-dimensional gel electrophoresis, immunoblot analysis, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS), and MALDI-TOF/TOF-MS analysis. Immunoblotting and mass spectrometry analysis revealed that at least 42 individual protein spots of N. caninum were reacted with the anti-N. caninum serum, among which at least 18 protein spots were cross-reacted with the anti-T. gondii serum. Moreover, at least 31 protein spots of T. gondii were reacted with the anti-T. gondii serum, among which at least 19 protein spots were cross-reacted with the anti-N. caninum serum. Furthermore, some new specific proteins were also identified in the N. caninum protein profile by searching Toxoplasma sequences or sequences from other organisms. This study substantiates the usefulness of proteomics in the immunoscreening of the cross-reactive or species-specific antigens of both parasites. In addition, the present study showed that there was significant homology in the antigenic proteome profiles between the two parasites. These observations have implications for the design of multicomponent common vaccines against both parasite infections.

PMID: 21461729 [PubMed - as supplied by publisher]

Apicomplexan apicortins possess a long disordered N-terminal extension

Infect Genet Evol. 2011 Apr 1. [Epub ahead of print]

Apicomplexan apicortins possess a long disordered N-terminal extension

Orosz F.

Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary.

Abstract
A new protein, termed apicortin, has recently been identified, which occurs only in the placozoan animal Trichoplax adhaerens and in the genomes of all currently sequenced apicomplexan parasites, (e.g. Toxoplasma, Plasmodium, etc). Apicortins unite two conserved domains, a DCX motif and a partial p25alpha sequence, which are singly found in diverse other proteins, in doublecortins and TPPPs, respectively. Here I show that although apicortin has a limited phylogenomic distribution, its occurrence is broader than thought previously. It has been identified in another primitive opisthokont, in the chytrid fungus, Spizellomyces punctatus. Apicortins can be divided into two subgroups: apicomplexan and non-apicomplexan ones. The main difference is that the former ones possess a long, N-terminal extension predicted to be disordered, which is missing in the other subgroup. The appearance of the extension in apicomplexan apicortins is an "innovation" of this phylum, and may play a functional role in the protein-protein interactions.

Copyright © 2011. Published by Elsevier B.V.
PMID: 21463710 [PubMed - as supplied by publisher]

Forward targeting of Toxoplasma gondii proproteins to the micronemes involves conserved aliphatic amino acids

Traffic. 2011 Mar 25. doi: 10.1111/j.1600-0854.2011.01192.x. [Epub ahead of print]

Forward targeting of Toxoplasma gondii proproteins to the micronemes involves conserved aliphatic amino acids

Gaji RY, Flammer HP, Carruthers VB.

Department of Microbiology and Immunology, University of Michigan Medical School, 1150 W. Medical Center Dr., Ann Arbor, MI 48109 USA.

Abstract
Like other apicomplexan parasites, Toxoplasma gondii actively invades host cells using a combination of secretory proteins and an acto-myosin motor system. Micronemes are the first set of proteins secreted during invasion that play an essential role in host cell entry. Many microneme proteins (MICs) function in protein complexes, and each complex contains at least one protein that displays a cleavable propeptide. Although MIC propeptides have been implicated in forward targeting to micronemes, the specific amino acids involved have not been identified. It was also not known if the propeptide has a general function in MICs trafficking in T. gondii and other apicomplexans. Here we show that propeptide domains are extensively interchangeable between T. gondii MICs and also with that of Eimeria tenella MIC5 (EtMIC5), suggesting a common mechanism of function. We also performed N-terminal deletion and mutational analysis of M2AP and MIC5 propeptides to show that a valine at position +3 (relative to signal peptidase cleavage) of proM2AP and a leucine at position +1 of proMIC5 are crucial for targeting to micronemes. Valine and leucine are closely related amino acids with similar side chains, implying a similar mode of function, a notion that was confirmed by correct trafficking of TgM2AP-V/L and TgMIC5-L/V substitution mutants. Propeptides of AMA1, MIC3, and EtMIC5 have valine or leucine at or near the N-termini and mutagenesis of these conserved residues validated their role in microneme trafficking. Collectively, our findings suggest that discrete, aliphatic residues at the extreme N-termini of proMICs facilitate trafficking to the micronemes.

© 2011 John Wiley & Sons A/S.
PMID: 21438967 [PubMed - as supplied by publisher]

Polymorphic family of injected pseudokinases is paramount in Toxoplasma virulence

oc Natl Acad Sci U S A. 2011 Mar 21. [Epub ahead of print]

Polymorphic family of injected pseudokinases is paramount in Toxoplasma virulence

Reese ML, Zeiner GM, Saeij JP, Boothroyd JC, Boyle JP.

Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305.

Abstract
Toxoplasma gondii, an obligate intracellular parasite of the phylum Apicomplexa, has the unusual ability to infect virtually any warm-blooded animal. It is an extraordinarily successful parasite, infecting an estimated 30% of humans worldwide. The outcome of Toxoplasma infection is highly dependent on allelic differences in the large number of effectors that the parasite secretes into the host cell. Here, we show that the largest determinant of the virulence difference between two of the most common strains of Toxoplasma is the ROP5 locus. This is an unusual segment of the Toxoplasma genome consisting of a family of 4-10 tandem, highly divergent genes encoding pseudokinases that are injected directly into host cells. Given their hypothesized catalytic inactivity, it is striking that deletion of the ROP5 cluster in a highly virulent strain caused a complete loss of virulence, showing that ROP5 proteins are, in fact, indispensable for Toxoplasma to cause disease in mice. We find that copy number at this locus varies among the three major Toxoplasma lineages and that extensive polymorphism is clustered into hotspots within the ROP5 pseudokinase domain. We propose that the ROP5 locus represents an unusual evolutionary strategy for sampling of sequence space in which the gene encoding an important enzyme has been (i) catalytically inactivated, (ii) expanded in number, and (iii) subject to strong positive selection. Such a strategy likely contributes to Toxoplasma's successful adaptation to a wide host range and has resulted in dramatic differences in virulence.

PMID: 21436047 [PubMed - as supplied by publisher]

T-cell production of matrix metalloproteinases and inhibition of parasite clearance by TIMP-1 during chronic Toxoplasma infection in the brain

ASN Neuro. 2011 Jan 21;3(1). pii: e00049. doi: 10.1042/AN20100027.

T-cell production of matrix metalloproteinases and inhibition of parasite clearance by TIMP-1 during chronic Toxoplasma infection in the brain

Clark RT, Philip Nance J, Noor S, Wilson EH.

Division of Biomedical Sciences, University of California, Riverside, CA 92521, U.S.A.

Abstract
Chronic infection with the intracellular protozoan parasite Toxoplasma gondii leads to tissue remodelling in the brain and a continuous requirement for peripheral leucocyte migration within the CNS (central nervous system). In the present study, we investigate the role of MMPs (matrix metalloproteinases) and their inhibitors in T-cell migration into the infected brain. Increased expression of two key molecules, MMP-8 and MMP-10, along with their inhibitor, TIMP-1 (tissue inhibitor of metalloproteinases-1), was observed in the CNS following infection. Analysis of infiltrating lymphocytes demonstrated MMP-8 and -10 production by CD4+ and CD8+ T-cells. In addition, infiltrating T-cells and CNS resident astrocytes increased their expression of TIMP-1 following infection. TIMP-1-deficient mice had a decrease in perivascular accumulation of lymphocyte populations, yet an increase in the proportion of CD4+ T-cells that had trafficked into the CNS. This was accompanied by a reduction in parasite burden in the brain. Taken together, these findings demonstrate a role for MMPs and TIMP-1 in the trafficking of lymphocytes into the CNS during chronic infection in the brain.

PMID: 21434872 [PubMed - in process]

The Moving Junction Protein RON8 Facilitates Firm Attachment and Host Cell Invasion in Toxoplasma gondii

PLoS Pathog. 2011 Mar;7(3):e1002007. Epub 2011 Mar 10.

The Moving Junction Protein RON8 Facilitates Firm Attachment and Host Cell Invasion in Toxoplasma gondii

Straub KW, Peng ED, Hajagos BE, Tyler JS, Bradley PJ.

Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, California, United States of America.

Abstract
The apicomplexan moving junction (MJ) is a highly conserved structure formed during host cell entry that anchors the invading parasite to the host cell and serves as a molecular sieve of host membrane proteins that protects the parasitophorous vacuole from host lysosomal destruction. While recent work in Toxoplasma and Plasmodium has reinforced the composition of the MJ as an important association of rhoptry neck proteins (RONs) with micronemal AMA1, little is known of the precise role of RONs in the junction or how they are targeted to the neck subcompartment. We report the first functional analysis of a MJ/RON protein by disrupting RON8 in T. gondii. Parasites lacking RON8 are severely impaired in both attachment and invasion, indicating that RON8 enables the parasite to establish a firm clasp on the host cell and commit to invasion. The remaining junction components frequently drag in trails behind invading knockout parasites and illustrate a malformed complex without RON8. Complementation of Δron8 parasites restores invasion and reveals a processing event at the RON8 C-terminus. Replacement of an N-terminal region of RON8 with a mCherry reporter separates regions within RON8 that are necessary for rhoptry targeting and complex formation from those required for function during invasion. Finally, the invasion defects in Δron8 parasites seen in vitro translate to radically impaired virulence in infected mice, promoting a model in which RON8 has a crucial and unprecedented task in committing Toxoplasma to host cell entry.

PMID: 21423671 [PubMed - in process]

Synthesis and biological evaluation of new 2-alkylaminoethyl-1,1-bisphosphonic acids targeting farnesy

Bioorg Med Chem. 2011 Mar 16. [Epub ahead of print]

Synthesis and biological evaluation of new 2-alkylaminoethyl-1,1-bisphosphonic acids against Trypanosoma cruzi and Toxoplasma gondii targeting farnesyl diphosphate synthase

Rosso VS, Szajnman SH, Malayil L, Galizzi M, Moreno SN, Docampo R, Rodriguez JB.

Departamento de Química Orgánica and UMYMFOR (CONICET-FCEyN), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria, C1428EHA Buenos Aires, Argentina; Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, GA 30602, USA.

Abstract
The effect of long-chain 2-alkylaminoethyl-1,1-bisphosphonates against proliferation of the clinically more relevant form of Trypanosoma cruzi, the etiologic agent of American trypanosomiasis (Chagas' disease), and against tachyzoites of Toxoplasma gondii was investigated. Particularly, compound 26 proved to be an extremely potent inhibitor against the intracellular form of T. cruzi, exhibiting IC(50) values at the nanomolar range. This cellular activity was associated with a strong inhibition of the enzymatic activity of T. cruzi farnesyl diphosphate synthase (TcFPPS), which constitutes a valid target for Chagas' disease chemotherapy. Compound 26 was an effective agent against T. cruzi (amastigotes) exhibiting an IC(50) value of 0.67μM, while this compound showed an IC(50) value of 0.81μM against the target enzyme TcFPPS. This drug was less effective against the enzymatic activity of T. cruzi solanesyl diphosphate synthase TcSPPS showing an IC(50) value of 3.2μM. Interestingly, compound 26 was also very effective against T. gondii (tachyzoites) exhibiting IC(50) values of 6.23μM. This cellular activity was also related to the inhibition of the enzymatic activity towards the target enzyme TgFPPS (IC(50)=0.093μM) As bisphosphonate-containing compounds are FDA-approved drugs for the treatment of bone resorption disorders, their potential low toxicity makes them good candidates to control different tropical diseases.

Copyright © 2011 Elsevier Ltd. All rights reserved.
PMID: 21419634 [PubMed - as supplied by publisher]

Toxoplasma gondii Dense Granule Antigen 1 stimulates apoptosis of monocytes through autocrine TGF-β signaling

Apoptosis. 2011 Mar 9. [Epub ahead of print]

Toxoplasma gondii Dense Granule Antigen 1 stimulates apoptosis of monocytes through autocrine TGF-β signaling

D'Angelillo A, De Luna E, Romano S, Bisogni R, Buffolano W, Gargano N, Del Porto P, Del Vecchio L, Petersen E, Romano MF.

Department of Biochemistry and Medical Biotechnology, University of Naples "Federico II", Via S. Pansini 5, 80131, Naples, Italy.

Abstract
Monocyte/macrophages represent the first line of defense against protozoan parasites. Different mechanisms of monocyte suppression by Toxoplasma gondii that sustain parasite invasion and persistence have been described, including apoptosis. In the present study, we investigated the effect of microbial excretory-secretory polypeptides, namely the microneme protein MIC3 and the dense granule antigen GRA1, on apoptosis of monocytes from patients with congenital toxoplasmosis and healthy individuals. We found that GRA1 but not MIC3 could induce apoptosis of monocytes, observing the effect in samples from both Toxoplasma-infected and uninfected individuals, thus ruling out involvement of mechanisms of apoptosis linked to adaptive immunity or a cellular context related to infection. Selective inhibition of TGF-β type I receptors reversed GRA1-induced apoptosis, indicating that this apoptosis involved canonical TGF-β signaling. By using TGF-β-neutralizing antibodies, we showed that monocyte apoptosis required endogenous TGF-β and that GRA1 stimulation activated TGF-β transcription and expression in monocytes but not lymphocytes, suggesting involvement of an autocrine TGF-β-mediated mechanism in GRA1-induced apoptosis.

PMID: 21390541 [PubMed - as supplied by publisher]

Crystal structure of Toxoplasma gondii porphobilinogen synthase: insights on octameric structure and porphobilinogen formation

J Biol Chem. 2011 Mar 7. [Epub ahead of print]

Crystal structure of Toxoplasma gondii porphobilinogen synthase: insights on octameric structure and porphobilinogen formation

Jaffe EK, Shanmugan D, Gardberg A, Dieterich S, Sankaran B, Stewart LJ, Myler PJ, Roos DS.

Fox Chase Cancer Center, United States;

Abstract
Porphobilinogen synthase (PBGS) is essential for heme biosynthesis, but the enzyme of the protozoan parasite Toxoplasma gondii (TgPBGS) differs from that of its human host in several important respects, including subcellular localization, metal ion dependence, and quaternary structural dynamics. We have solved the crystal structure of TgPBGS, which contains an octamer in the crystallographic asymmetric unit. Crystallized in the presence of substrate, each active site contains one molecule of the product porphobilinogen. Unlike prior structures containing a substrate-derived heterocycle directly bound to an active site zinc ion, the product-bound TgPBGS active site contains neither zinc nor magnesium, placing in question the common notion that all PBGS enzymes require an active site metal ion. Unlike human PBGS, the TgPBGS octamer contains magnesium ions at the intersections between pro-octamer dimers, which are presumed to function in allosteric regulation. TgPBGS includes N- and C-terminal regions that differ considerably from previously solved crystal structures. In particular, the C-terminal extension found in all apicomplexan PBGS enzymes forms an intersubunit β-sheet, stabilizing a pro-octamer dimer and preventing formation of hexamers that can form in human PBGS. The TgPBGS structure suggests strategies for the development of parasite-selective PBGS inhibitors.

PMID: 21383008 [PubMed - as supplied by publisher]

Modulation of early {beta}-defensin-2 production as a mechanism developed by Type I Toxoplasma gondii to evade human intestinal immunity

Infect Immun. 2011 Mar 7. [Epub ahead of print]

Modulation of early {beta}-defensin-2 production as a mechanism developed by Type I Toxoplasma gondii to evade human intestinal immunity

Morampudi V, Braun MY, D'Souza S.

Toxoplasmosis Laboratory, Operational Direction Communicable and Infectious Diseases, Scientific Institute of Public Health, 1180 Brussels, Belgium; Institute for Medical Immunology, Faculty of Medicine, Universite Libre de Bruxelles (ULB), 6041 Gosselies, Belgium.

Abstract
We investigated the early innate immune responses induced in human intestinal epithelial cells (IEC) by the three defined Toxoplasma gondii genotype strains. Transcriptome analysis revealed that among differentially expressed genes, β-defensins distinguished the most IEC infected by fast or slow replicating T. gondii genotypes. Although β-defensin 1 and 3 genes were not expressed in host cells early post-infection, the slow replicating Type II and III parasites induced high levels of β-defensin 2 gene expression. Notably, no β-defensin 2 gene expression occurred early after infection with the fast replicating Type I parasite. However, activation of this gene in IEC by Poly I:C treatment prior to infection substantially decreased parasite viability, and pretreatment of parasites with synthetic β-defensin 2 significantly reduced their infectivity of IEC. These findings strongly support the modulation of early β-defensin 2 expression as a mechanism used by Type I T. gondii parasites to mediate immune evasion.

PMID: 21383053 [PubMed - as supplied by publisher]

Toxoplasma gondii protects against H(2) O(2) -induced apoptosis in ARPE-19 cells

Acta Ophthalmol. 2011 Feb 25. doi: 10.1111/j.1755-3768.2011.02113.x. [Epub ahead of print]

Toxoplasma gondii protects against H(2) O(2) -induced apoptosis in ARPE-19 cells through the transcriptional regulation of apoptotic elements and downregulation of the p38 MAPK pathway

Choi SH, Park SJ, Cha GH, Quan JH, Chang NS, Ahn MH, Shin DW, Lee YH.

Department of Ophthalmology, Chungnam National University School of Medicine, Daejeon, Korea Department of Infection Biology and Research Institute for Medical Sciences, Chungnam National University School of Medicine, Daejeon, Korea Department of Environmental Biology and Medical Parasitology, College of Medicine, Hanyang University, Seoul, Korea.

Abstract
Purpose:  Toxoplasmosis, which is caused by the protozoan parasite Toxoplasma gondii, can lead to severe visual impairment. T. gondii inhibits or delays programmed cell death caused by various apoptotic triggers; however, the mechanisms involved in the T. gondii-induced suppression of apoptosis in retinal cells have not been analysed in detail. Methods:  We investigated the role of T. gondii infection in H(2) O(2) -induced apoptosis in human retinal pigment epithelial cells (ARPE-19) by monitoring the activities of apoptosis-regulating molecules and mitogen-activated protein kinases (MAPKs), including p38 MAPK. We also examined the gene downstream from p38 MAPK. Results:  T. gondii infection significantly inhibited the cellular toxicity of H(2) O(2) (500 μm) and increased cell viability in a multiplicity of infection (MOI)-dependent manner by reducing DNA fragmentation and reactive oxygen species (ROS) generation in ARPE-19 cells. Western blot analysis also showed that T. gondii infection prevented the host cell expression of pro-apoptotic factors, such as Bad and Bax, and the activation of caspase-3. Infection with T. gondii increased the expression of the anti-apoptotic factor Bcl-2 in ARPE-19 cells under oxidative stress. In accordance with these findings, Toxoplasma infection was protective enough to suppress the phosphorylation of p38 MAPK following H(2) O(2) treatment. Exposure to H(2) O(2) increased the expression of heme oxygenase-1 (HO-1) in ARPE-19 cells, and its expression was significantly inhibited in H(2) O(2) -treated infected cells. Conclusion:  The protective function of T. gondii infection against ROS-induced apoptosis results from changes in the expression of apoptotic molecules and the downregulation of stress-induced intracellular signalling.

© 2011 The Authors. Acta Ophthalmologica © 2011 Acta Ophthalmologica Scandinavica Foundation.
PMID: 21385331 [PubMed - as supplied by publisher]

Phosphatidylinositol 3-monophosphate is involved in toxoplasma apicoplast biogenesis

PLoS Pathog. 2011 Feb;7(2):e1001286. Epub 2011 Feb 17.

Phosphatidylinositol 3-monophosphate is involved in toxoplasma apicoplast biogenesis

Tawk L, Dubremetz JF, Montcourrier P, Chicanne G, Merezegue F, Richard V, Payrastre B, Meissner M, Vial HJ, Roy C, Wengelnik K, Lebrun M.

UMR 5235 CNRS, Université Montpellier 1 & 2, Montpellier, France.

Abstract
Apicomplexan parasites cause devastating diseases including malaria and toxoplasmosis. They harbour a plastid-like, non-photosynthetic organelle of algal origin, the apicoplast, which fulfils critical functions for parasite survival. Because of its essential and original metabolic pathways, the apicoplast has become a target for the development of new anti-apicomplexan drugs. Here we show that the lipid phosphatidylinositol 3-monophosphate (PI3P) is involved in apicoplast biogenesis in Toxoplasma gondii. In yeast and mammalian cells, PI3P is concentrated on early endosomes and regulates trafficking of endosomal compartments. Imaging of PI3P in T. gondii showed that the lipid was associated with the apicoplast and apicoplast protein-shuttling vesicles. Interference with regular PI3P function by over-expression of a PI3P specific binding module in the parasite led to the accumulation of vesicles containing apicoplast peripheral membrane proteins around the apicoplast and, ultimately, to the loss of the organelle. Accordingly, inhibition of the PI3P-synthesising kinase interfered with apicoplast biogenesis. These findings point to an unexpected implication for this ubiquitous lipid and open new perspectives on how nuclear encoded proteins traffic to the apicoplast. This study also highlights the possibility of developing specific pharmacological inhibitors of the parasite PI3-kinase as novel anti-apicomplexan drugs.

PMID: 21379336 [PubMed - in process]

Rapid discovery of inhibitors of Toxoplasma gondii using hybrid structure-based computational approach

J Comput Aided Mol Des. 2011 Feb 26. [Epub ahead of print]

Rapid discovery of inhibitors of Toxoplasma gondii using hybrid structure-based computational approach

Kortagere S, Mui E, McLeod R, Welsh WJ.

University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ, 08854, USA.

Abstract
Toxoplasma (T.) gondii, the causative agent of toxoplasmosis, is a ubiquitous opportunistic pathogen that infects individuals worldwide, and is a leading cause of severe congenital neurologic and ocular disease in humans. No vaccine to protect humans is available, and hypersensitivity and toxicity limit the use of the few available medicines. Therefore, safer and more effective medicines to treat toxoplasmosis are urgently needed. Using the Hybrid Structure Based (HSB) method, we have previously identified small molecule inhibitors of P. falciparum that seem to target a novel protein-protein interaction between the Myosin tail interacting protein and myosin light chain. This pathway has been hypothesized to be involved in invasion of host erythrocytes by the parasite and is broadly conserved among the apicomplexans. Guided by similar computational drug design approaches, we investigated this series of small molecules as potential inhibitors of T. gondii. Compound C3-21, identified as the most active inhibitor in this series, exhibited an IC(50) value ~500 nM against T. gondii. Among the 16 structural analogs of C3-21 tested thus far, nine additional compounds were identified with IC(50) values <10.0 μM. In vitro assays have revealed that C3-21 markedly limits intracellular growth of T. gondii tachyzoites, but has no effect on host cell human foreskin fibroblasts (HFF) at concentrations more than a log greater than the concentration that inhibits the parasites.

PMID: 21359560 [PubMed - as supplied by publisher]

Base excision repair apurinic/apyrimidinic endonucleases in apicomplexan parasite Toxoplasma gondii

DNA Repair (Amst). 2011 Feb 23. [Epub ahead of print]

Base excision repair apurinic/apyrimidinic endonucleases in apicomplexan parasite Toxoplasma gondii

Onyango DO, Naguleswaran A, Delaplane S, Reed A, Kelley MR, Georgiadis MM, Sullivan WJ Jr.

Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202, United States.

Abstract
DNA repair is essential for cell viability and proliferation. In addition to reactive oxygen produced as a byproduct of their own metabolism, intracellular parasites also have to manage oxidative stress generated as a defense mechanism by the host. The spontaneous loss of DNA bases due to hydrolysis and oxidative DNA damage in intracellular parasites is great, but little is known about the type of DNA repair machineries that exist in these early-branching eukaryotes. However, it is clear, processes similar to DNA base excision repair (BER) must exist to rectify spontaneous and host-mediated damage in Toxoplasma gondii. Here we report that T. gondii, an opportunistic protozoan pathogen, possesses two apurinic/apyrimidinic (AP) endonucleases that function in DNA BER. We characterize the enzymatic activities of Toxoplasma exonuclease III (ExoIII, or Ape1) and endonuclease IV (EndoIV, or Apn1), designated TgAPE and TgAPN, respectively. Over-expression of TgAPN in Toxoplasma conferred protection from DNA damage, and viable knockouts of TgAPN were not obtainable. We generated an inducible TgAPN knockdown mutant using a ligand-controlled destabilization domain to establish that TgAPN is critical for Toxoplasma to recover from DNA damage. The importance of TgAPN and the fact that humans lack any observable APN family activity highlights TgAPN as a promising candidate for drug development to treat toxoplasmosis.

Copyright © 2011 Elsevier B.V. All rights reserved.
PMID: 21353648 [PubMed - as supplied by publisher]

Leishmania infantum and Toxoplasma gondii: Mixed infection of macrophages in vitro and in vivo

Exp Parasitol. 2011 Feb 24. [Epub ahead of print]

Leishmania infantum and Toxoplasma gondii: Mixed infection of macrophages in vitro and in vivo

Christodoulou V, Messaritakis I, Svirinaki E, Tsatsanis C, Antoniou M.

Laboratory of Clinical Bacteriology, Parasitology, Zoonoses, and Geographical Medicine, Faculty of Medicine, University of Crete, Crete, Greece.

Abstract
Although macrophages have a microbicidal role in the immune system they themselves can be infected by pathogens. Often a simultaneous infection by more than one microbe may occur in a single cell. This is the first report of coinfection of macrophages with Toxoplasma gondii and Leishmania infantum, in vitro and in vivo. L. infantum does not cause severe disease in mice but T. gondii, RH strain, is lethal. Cell culture studies using THP-1 macrophages dually infected in vitro revealed that 4.3% harbored both parasites 24hrs after infection. When mice were infected with both parasites on the same day 7.3% of the infected cells carried both parasites 7 days later. Yet, if mice were first infected with L. infantum and then with Toxoplasma (5 days post infection) 18.7% of the macrophages hosted either parasite but concomitant infection could not be found and mice, already harboring L. infantum, survived Toxoplasma's lethal effect.

Copyright © 2011. Published by Elsevier Inc.
PMID: 21354140 [PubMed - as supplied by publisher]

Improved Techniques for Endogenous Epitope Tagging and Gene Deletion in Toxoplasma gondii

J Microbiol Methods. 2011 Feb 22. [Epub ahead of print]

Improved Techniques for Endogenous Epitope Tagging and Gene Deletion in Toxoplasma gondii

Upadhya R, Kim K, Hogue-Angeletti R, Weiss LM.

Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park, Avenue, Bronx, NY 10461.

Abstract
Toxoplasma gondii is an excellent model organism for studies on the biology of the Apicomplexa due to its ease of in vitro cultivation and genetic manipulation. Large-scale reverse genetic studies in T. gondii have, however, been difficult due to the low frequency of homologous recombination. Efforts to ensure homologous recombination have necessitated engineering long flanking regions in the targeting construct. This requirement makes it difficult to engineer chromosomally targeted epitope tags or gene knock out constructs only by restriction enzyme mediated cloning steps. To address this issue we employed multisite Gateway® recombination techniques to generate chromosomal gene manipulation targeting constructs. Incorporation of 1.5 to 2.0 kb flanking homologous sequences in PCR generated targeting constructs resulted in 90% homologous recombination events in wild type T. gondii (RH strain) as determined by epitope tagging and target gene deletion experiments. Furthermore, we report that split marker constructs were equally efficient for targeted gene disruptions using the T. gondii UPRT gene locus as a test case. The methods described in this paper represent an improved strategy for efficient epitope tagging and gene disruptions in T. gondii.

Copyright © 2010 Elsevier B.V. All rights reserved.
PMID: 21352857 [PubMed - as supplied by publisher]

Invasion and intracellular survival by protozoan parasites

Immunol Rev. 2011 Mar;240(1):72-91. doi: 10.1111/j.1600-065X.2010.00990.x.

Invasion and intracellular survival by protozoan parasites

David Sibley L.

Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, USA.

Abstract
Summary:  Intracellular parasitism has arisen only a few times during the long ancestry of protozoan parasites including in diverse groups such as microsporidians, kinetoplastids, and apicomplexans. Strategies used to gain entry differ widely from injection (e.g. microsporidians), active penetration of the host cell (e.g. Toxoplasma), recruitment of lysosomes to a plasma membrane wound (e.g. Trypanosoma cruzi), to host cell-mediated phagocytosis (e.g. Leishmania). The resulting range of intracellular niches is equally diverse ranging from cytosolic (e.g. T. cruzi) to residing within a non-fusigenic vacuole (e.g. Toxoplasma, Encephalitozoon) or a modified phagolysosome (e.g. Leishmania). These lifestyle choices influence access to nutrients, interaction with host cell signaling pathways, and detection by pathogen recognition systems. As such, intracellular life requires a repertoire of adaptations to assure entry-exit from the cell, as well as to thwart innate immune mechanisms and prevent clearance. Elucidating these pathways at the cellular and molecular level may identify key steps that can be targeted to reduce parasite survival or augment immunologic responses and thereby prevent disease.

© 2011 John Wiley & Sons A/S.
PMID: 21349087 [PubMed - in process]

Autophagy in immunity and cell-autonomous defense against intracellular microbes

Immunol Rev. 2011 Mar;240(1):92-104. doi: 10.1111/j.1600-065X.2010.00995.x.

Autophagy in immunity and cell-autonomous defense against intracellular microbes

Deretic V.

Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA.

Abstract
Summary:  Autophagy was viewed until very recently primarily as a metabolic and intracellular biomass and organelle quality and quantity control pathway. It has now been recognized that autophagy represents a bona fide immunologic process with a wide array of roles in immunity. The immunologic functions of autophagy, as we understand them now, span both innate and adaptive immunity. They range from unique and sometimes highly specialized immunologic effectors and regulatory functions (referred to here as type I immunophagy) to generic homeostatic influence on immune cells (type II immunophagy), akin to the effects on survival and homeostasis of other cell types in the body. As a concept-building tool for understanding why and how autophagy is intertwined with immunity, it is useful to consider that the presently complex picture has emerged in increments, starting in part from the realization that autophagy acts as an evolutionarily ancient microbial clearance mechanism defending eukaryotic cells against intracellular pathogens. In this review, we build a stepwise model of how the core axis of autophagy as a cell-autonomous immune defense against microbes evolved into a complex but orderly web of intersections with innate and adaptive immunity processes. The connections between autophagy and conventional immunity systems include Toll-like receptors, Nod-like receptors, RIG-I-like receptors, damage-associated molecular patterns such as HMGB1, other known innate and adaptive immunity receptors and cytokines, sequestasome (p62)-like receptors that act as autophagy adapters, immunity-related GTPase IRGM, innate and adaptive functions of macrophages and dendritic cells, and differential effects on development and homeostasis of T- and B-lymphocyte subsets. The disease contexts covered here include tuberculosis, infections with human immunodeficiency virus and other viruses, Salmonella, Listeria, Shigella, Toxoplasma, and inflammatory disorders such as Crohn's disease and multiple sclerosis.

© 2011 John Wiley & Sons A/S.
PMID: 21349088 [PubMed - in process]

Immunology of Toxoplasma gondii

Immunol Rev. 2011 Mar;240(1):269-85. doi: 10.1111/j.1600-065X.2010.00992.x.

Immunology of Toxoplasma gondii

Munoz M, Liesenfeld O, Heimesaat MM.

Charite Medical School Berlin - Microbiology and Hygiene, Berlin, Germany.

Abstract
Summary:  Toxoplasma gondii is an obligate intracellular parasite. Following oral infection the parasite crosses the intestinal epithelial barrier to disseminate throughout the body and establish latent infection in central nervous tissues. The clinical presentation ranges from asymptomatic to severe neurological disorders in immunocompromised individuals. Since the clinical presentation is diverse and depends, among other factors, on the immune status of the host, in the present review, we introduce parasitological, epidemiological, clinical, and molecular biological aspects of infection with T. gondii to set the stage for an in-depth discussion of host immune responses. Since immune responses in humans have not been investigated in detail the present review is exclusively referring to immune responses in experimental models of infection. Systemic and local immune responses in different models of infection are discussed, and a separate chapter introduces commonly used animal models of infection.

© 2011 John Wiley & Sons A/S.
PMID: 21349099 [PubMed - in process]

Evaluation of Five Antischizophrenic Agents Against Toxoplasma gondii in Human Cell Cultures

J Parasitol. 2011 Feb;97(1):148-51. Epub 2010 Sep 14.

Evaluation of Five Antischizophrenic Agents Against Toxoplasma gondii in Human Cell Cultures

Goodwin DG, Strobl JS, Lindsay DS.

a Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia 24061-0342.

Abstract
Abstract An increasing interest in the association of the presence of antibodies to Toxoplasma gondii and the development of schizophrenia in patients has been generated over the last several years. Some antischizophrenia agents have been shown to have activity against T. gondii in cell culture assays and to ameliorate behavioral changes associated with chronic T. gondii infection in rats. In the present study, we examined the effects of commonly used antipsychotic and mood stabilizing agents (haloperidol, clozapine, fluphenazine, trifluoperazine, and thioridazine) for activity against developing tachyzoites of the RH strain of T. gondii in human fibroblast cell cultures. Neither haloperidol nor clozapine had a measurable effect. Fluphenazine had an IC(50) of 1.7 µM, thioridazine had an IC(50) of 1.2 µM, and trifluoperazine had an IC(50) of 3.8 µM. Our study demonstrates that some agents used to treat schizophrenia have the ability to inhibit T. gondii proliferation in cell culture.

PMID: 21348624 [PubMed - in process]

The C-Terminus of Toxoplasma RON2 Provides the Crucial Link between AMA1 and the Host-Associated Invasion Complex

PLoS Pathog. 2011 Feb 10;7(2):e1001282.

The C-Terminus of Toxoplasma RON2 Provides the Crucial Link between AMA1 and the Host-Associated Invasion Complex

Tyler JS, Boothroyd JC.

Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, United States of America.

Abstract
Host cell invasion by apicomplexan parasites requires formation of the moving junction (MJ), a ring-like apposition between the parasite and host plasma membranes that the parasite migrates through during entry. The Toxoplasma MJ is a secreted complex including TgAMA1, a transmembrane protein on the parasite surface, and a complex of rhoptry neck proteins (TgRON2/4/5/8) described as host cell-associated. How these proteins connect the parasite and host cell has not previously been described. Here we show that TgRON2 localizes to the MJ and that two short segments flanking a hydrophobic stretch near its C-terminus (D3 and D4) independently associate with the ectodomain of TgAMA1. Pre-incubation of parasites with D3 (fused to glutathione S-transferase) dramatically reduces invasion but does not prevent injection of rhoptry bulb proteins. Hence, the entire C-terminal region of TgRON2 forms the crucial bridge between TgAMA1 and the rest of the MJ complex but this association is not required for rhoptry protein injection.

PMID: 21347354 [PubMed - in process]

Genetic mapping identifies novel highly protective antigens for an apicomplexan parasite

PLoS Pathog. 2011 Feb 10;7(2):e1001279.

Genetic mapping identifies novel highly protective antigens for an apicomplexan parasite

Blake DP, Billington KJ, Copestake SL, Oakes RD, Quail MA, Wan KL, Shirley MW, Smith AL.

Institute for Animal Health, Compton, Berkshire, United Kingdom.

Abstract
Apicomplexan parasites are responsible for a myriad of diseases in humans and livestock; yet despite intensive effort, development of effective sub-unit vaccines remains a long-term goal. Antigenic complexity and our inability to identify protective antigens from the pool that induce response are serious challenges in the development of new vaccines. Using a combination of parasite genetics and selective barriers with population-based genetic fingerprinting, we have identified that immunity against the most important apicomplexan parasite of livestock (Eimeria spp.) was targeted against a few discrete regions of the genome. Herein we report the identification of six genomic regions and, within two of those loci, the identification of true protective antigens that confer immunity as sub-unit vaccines. The first of these is an Eimeria maxima homologue of apical membrane antigen-1 (AMA-1) and the second is a previously uncharacterised gene that we have termed 'immune mapped protein-1' (IMP-1). Significantly, homologues of the AMA-1 antigen are protective with a range of apicomplexan parasites including Plasmodium spp., which suggest that there may be some characteristic(s) of protective antigens shared across this diverse group of parasites. Interestingly, homologues of the IMP-1 antigen, which is protective against E. maxima infection, can be identified in Toxoplasma gondii and Neospora caninum. Overall, this study documents the discovery of novel protective antigens using a population-based genetic mapping approach allied with a protection-based screen of candidate genes. The identification of AMA-1 and IMP-1 represents a substantial step towards development of an effective anti-eimerian sub-unit vaccine and raises the possibility of identification of novel antigens for other apicomplexan parasites. Moreover, validation of the parasite genetics approach to identify effective antigens supports its adoption in other parasite systems where legitimate protective antigen identification is difficult.

PMID: 21347348 [PubMed - in process]