Nephromyces, a beneficial apicomplexan symbiont in marine animals

Proc Natl Acad Sci U S A. 2010 Aug 24. [Epub ahead of print]

Nephromyces, a beneficial apicomplexan symbiont in marine animals

Saffo MB, McCoy AM, Rieken C, Slamovits CH.

Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138-2902.

Abstract
With malaria parasites (Plasmodium spp.), Toxoplasma, and many other species of medical and veterinary importance its iconic representatives, the protistan phylum Apicomplexa has long been defined as a group composed entirely of parasites and pathogens. We present here a report of a beneficial apicomplexan: the mutualistic marine endosymbiont Nephromyces. For more than a century, the peculiar structural and developmental features of Nephromyces, and its unusual habitat, have thwarted characterization of the phylogenetic affinities of this eukaryotic microbe. Using short-subunit ribosomal DNA (SSU rDNA) sequences as key evidence, with sequence identity confirmed by fluorescence in situ hybridization (FISH), we show that Nephromyces, originally classified as a chytrid fungus, is actually an apicomplexan. Inferences from rDNA data are further supported by the several apicomplexan-like structural features in Nephromyces, including especially the strong resemblance of Nephromyces infective stages to apicomplexan sporozoites. The striking emergence of the mutualistic Nephromyces from a quintessentially parasitic clade accentuates the promise of this organism, and the three-partner symbiosis of which it is a part, as a model for probing the factors underlying the evolution of mutualism, pathogenicity, and infectious disease.

PMID: 20736348 [PubMed - as supplied by publisher]

Toxoplasma Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

J Vis Exp. 2010 Aug 12;(42). pii: 2091. doi: 10.3791/2091.

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Tobin C, Pollard A, Knoll L.

Department of Medical Microbiology and Immunology, University of Wisconsin.

Abstract
Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO(2;) starvation, using medium with high a pH, or the addition of interferon gamma (IFNgamma). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO(2;) and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFNgamma and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii to bradyzoites using a high pH growth medium with low CO(2;) and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFNgamma and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.

PMID: 20736916 [PubMed - in process]

Toxoplasma gondii: Infection natural congenital in cattle and an experimental inoculation of gestating cows with oocysts

Exp Parasitol. 2010 Aug 21. [Epub ahead of print]

Toxoplasma gondii: Infection natural congenital in cattle and an experimental inoculation of gestating cows with oocysts

Costa GH, da Costa AJ, Lopes WD, Bresciani KD, Dos Santos TR, Esper CR, Santana AE.

CPPAR - Animal Health Research Center - Faculdade de Ciências Agrárias e Veterinárias, UNESP, Via de acesso prof. Paulo Donatto Castellani, s/n CEP:14884-900, Jaboticabal, São Paulo-Brasil.

Abstract
Two studies, of a natural infection and an experimental infection, were performed in order to study congenital transmission of Toxoplasma gondii in cattle. In the first study, 50 fetuses were harvested from gestating cows that were eutanasied at a municipal slaughterhouse in Jaboticabal, São Paulo state, Brazil. In the second study, 11 gestating cows were divided into four groups for inoculation with T. gondii: GI consisted of 3 cows inoculated with 1.0 x 10(5) oocysts during their first trimester of gestation; GII consisted of three cows inoculated with 1.0 x 10(5) oocysts during their second trimester of gestation; GIII consisted of three cows inoculated with 1.0 x 10(5) oocysts during their last trimester of gestation; and GIV consisted of two control cows, one during its first and the other during its second trimester of gestation. In both studies, the presence of T. gondii was confirmed both indirectly by immunofluorescence assay (IFAT). In the natural infection experiment, 18% (9/50) of the gestating cows were confirmed to have specific antibodies (IFAT - 1:64) against T. gondii. The bioassay was able to diagnose the presence of T. gondii in the tissue samples from three calves. In the second experiment, the nine cows from groups I, II and III presented with specific antibodies (IFAT) against T. gondii. In contrast, T. gondii could not be detected by IFAT, histopathological examination or the bioassay in any of the nine calves born to cows experimentally infected with T. gondii oocysts. Based on the results from both studies, we conclude that congenital infection of Toxoplasma gondii in cattle, while infrequent, does occur naturally The pathogenicity of the strain of T. gondii may influence the likelihood of this route of transmission.

PMID: 20736009 [PubMed - as supplied by publisher]

Determination of diagnostic value of Toxoplasma gondii recombinant ROP2 and ROP4 antigens in mouse experimental model

Pol J Microbiol. 2010;59(2):137-41.

Determination of diagnostic value of Toxoplasma gondii recombinant ROP2 and ROP4 antigens in mouse experimental model

Gatkowska J, Dziadek B, Brzostek A, Dziadek J, Dzitko K, Długońska H.

Department of Immunoparasitology, University of Lódź, Poland. gatjus@biol.uni.lodz.pl

Abstract
The aim of this study was to test the potential diagnostic usefulness of recombinant Toxoplasma gondii rhoptry antigens, ROP2 and ROP4, with respect to toxoplasmosis detection and infection phase distinction in laboratory mouce by determining specific serum IgM and IgG antibodies with the use of indirect ELISA technique. The mice antibody response to ROP antigens was significantly higher in the IgM than in the IgG class with the peak on the turn of acute and latent infection, whereas the response to recombinant SAG1 antigen, used as control, revealed preferential synthesis of IgG antibodies with the highest absorbance values measured during latent toxoplasmosis.

PMID: 20734761 [PubMed - in process]

The binding of Toxoplasma gondii glycosylphosphatidylinositols to galectin-3 is required for their recognition by macrophages

J Biol Chem. 2010 Aug 20. [Epub ahead of print]

The binding of Toxoplasma gondii glycosylphosphatidylinositols to galectin-3 is required for their recognition by macrophages

Debierre-Grockiego F, Niehus S, Coddeville B, Elass E, Poirier F, Weingart R, Schmidt RR, Mazurier J, Guerardel Y, Schwarz RT.

UFR Sciences Pharmaceutiques, France;

Abstract
We showed that the production of tumor necrosis factor alpha by macrophages in response to Toxoplasma gondii glycosylphosphatidylinositols (GPIs) requires the expression of both Toll-Like Receptors TLR2 and TLR4, but not of their co-receptor CD14. Galectin-3 is a beta-galactoside-binding protein with immune-regulatory effects, which associates with TLR2. We demonstrate here by using the surface plasmon resonance method that the GPIs of T. gondii bind to human galectin-3 with strong affinity and in a dose-dependent manner. The use of a synthetic glycan and of the lipid moiety cleaved from the GPIs show that both parts are involved in the interaction with galectin-3. GPIs of T. gondii also bind to galectin-1 but with a lower affinity and only through the lipid moiety. At the cellular level, the production of tumor necrosis factor alpha induced by T. gondii GPIs in macrophages depends on the expression of galectin-3 but not of galectin-1. This study is the first identification of a galectin-3 ligand of T. gondii origin, and galectin-3 might be a co-receptor presenting the GPIs to the TLRs on macrophages.

PMID: 20729207 [PubMed - as supplied by publisher]

Modulation of immunity in mice with latent toxoplasmosis-the experimental support for the immunosuppression hypothesis

Parasitol Res. 2010 Aug 19. [Epub ahead of print]

Modulation of immunity in mice with latent toxoplasmosis-the experimental support for the immunosuppression hypothesis of Toxoplasma-induced changes in reproduction of mice and humans

Kaňková S, Holáň V, Zajícová A, Kodym P, Flegr J.

Department of Philosophy and History of Science, Faculty of Science, Charles University in Prague, Vinicná 7, 128 44, Prague 2, Czech Republic.

Abstract
The immunosuppression hypothesis suggests that the increased sex ratio in mice and women with latent toxoplasmosis, retarded embryonic growth in the early phases of pregnancy, prolonged pregnancy of Toxoplasma-infected women, and increased prevalence of toxoplasmosis in mothers of children with Down syndrome can be explained by the presumed immunosuppressive effects of latent toxoplasmosis. Here, we searched for indices of immunosuppression in mice experimentally infected with Toxoplasma gondii. Our results showed that mice in the early phase of latent infection exhibited temporarily increased production of interleukin (IL)-12 and decreased production of IL-10. In accordance with the immunosuppression hypothesis, the mice showed decreased production of IL-2 and nitric oxide and decreased proliferation reaction (synthesis of DNA) in the mixed lymphocyte culture in the early and also in the late phases of latent toxoplasmosis. Since about 30% of the world population are latently infected by T. gondii, the toxoplasmosis-associated immunosuppression might have serious public health consequences.

PMID: 20721578 [PubMed - as supplied by publisher]

Toxoplasma IgG and IgA, but not IgM, antibody titers increase in sera of immunocompetent mice in association with proliferation of tachyzoites

Microbes Infect. 2010 Aug 10. [Epub ahead of print]

Toxoplasma IgG and IgA, but not IgM, antibody titers increase in sera of immunocompetent mice in association with proliferation of tachyzoites in the brain during the chronic stage of infection
Singh J, Graniello C, Ni Y, Payne L, Sa Q, Hester J, Shelton BJ, Suzuki Y.

Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, 1410 Prices Fork Road, Blacksburg, VA 24061.

Abstract
Toxoplasma IgG and IgA, but not IgM, antibody titers were significantly higher in immunocompetent mice with cerebral proliferation of tachyzoites during the chronic stage of infection than those treated with sulfadiazine to inhibit the parasite growth. Their IgG and IgA antibody titers correlated significantly with the amounts of tachyzoite-specific SAG1 mRNA in their brains. In contrast, neither IgG, IgA, nor IgM antibody titers increased following two different doses of challenge infection in chronically infected mice. Increased antibody titers in IgG and IgA but not IgM may be a useful indicator suggesting an occurrence of cerebral tachyzoite growth in immunocompetent individuals chronically infected with T. gondii.

PMID: 20708090 [PubMed - as supplied by publisher]

Activity of the histone deacetylase inhibitor FR235222 on Toxoplasma gondii: inhibition of stage conversion of the parasite cyst form

Antimicrob Agents Chemother. 2010 Aug 16. [Epub ahead of print]

Activity of the histone deacetylase inhibitor FR235222 on Toxoplasma gondii: inhibition of stage conversion of the parasite cyst form and study of new derivative compounds

Maubon D, Bougdour A, Wong YS, Brenier-Pinchart MP, Curt A, Hakimi MA, Pelloux H.

Parasitology-Mycology Laboratory, Département des Agents Infectieux, Centre Hospitalier Universitaire, BP 217, 38043 Grenoble, cedex 09, France; UMR 5163, Laboratoire Adaptation et Pathogénie des Micro-organismes, ATIP+ group, CNRS-Université Joseph Fourier GRENOBLE 1, BP 170, F-38042 Grenoble cedex 9, France; Département de Pharmacochimie Moléculaire, Université de Grenoble, CNRS UMR 5063, CNRS ICMG FR 2607, Bâtiment E, 470 Rue de la Chimie, F-38 041 Grenoble cedex 9, France.

Abstract
Bradyzoite to tachyzoite conversion plays a role in pathogenesis of recrudescence of ocular toxoplasmosis and disease in immune compromised persons. The currently available medicines are ineffective on cysts and fail to prevent reactivation of latent toxoplasmosis. A previous paper showed that the histone deacetylase inhibitor FR235222 has a dramatic effect on tachyzoite growth and induces tachyzoite-to-bradyzoite conversion in vitro. This study shows that FR235222 can target in vitro converted cysts and bradyzoites. Moreover, the compound is active on ex vivo T. gondii cysts. Free bradyzoites isolated after lysis of the cell wall did not proliferate in vitro when the cyst was treated with FR235222. The results imply that this compound is able to cross the T. gondii cystic cell wall. Fluorescent labeling shows that the compound impairs the capacity of the bradyzoites to convert without damaging the cyst wall integrity. In vivo inoculation of formerly treated cysts fails to infect mice when these cysts were treated with FR235222. We used our structural knowledge of FR235222 and its target TgHDAC3 to synthesize new FR235222 derivative compounds. We identified two new molecules that are highly active against tachyzoites. They harbor a better selectivity index that is more suitable for a future in vivo approach in mice with chronic toxoplasmosis. These results identify FR235222 and its derivatives as new lead compounds in the range of therapeutics available in acute and chronic toxoplasmosis.

PMID: 20713670 [PubMed - as supplied by publisher]

Integrative Genomic Approaches Highlight a Family of Parasite-Specific Kinases that Regulate Host Responses

Cell Host Microbe. 2010 Aug 19;8(2):208-218.

Integrative Genomic Approaches Highlight a Family of Parasite-Specific Kinases that Regulate Host Responses

Peixoto L, Chen F, Harb OS, Davis PH, Beiting DP, Brownback CS, Ouloguem D, Roos DS.

Department of Biology and Penn Genome Frontiers Institute, University of Pennsylvania, Philadelphia, PA 19104, USA.

Abstract
Apicomplexan parasites release factors via specialized secretory organelles (rhoptries, micronemes) that are thought to control host cell responses. In order to explore parasite-mediated modulation of host cell signaling pathways, we exploited a phylogenomic approach to characterize the Toxoplasma gondii kinome, defining a 44 member family of coccidian-specific secreted kinases, some of which have been previously implicated in virulence. Comparative genomic analysis suggests that "ROPK" genes are under positive selection, and expression profiling demonstrates that most are differentially expressed between strains and/or during differentiation. Integrating diverse genomic-scale analyses points to ROP38 as likely to be particularly important in parasite biology. Upregulating expression of this previously uncharacterized gene in transgenic parasites dramatically suppresses transcriptional responses in the infected cell. Specifically, parasite ROP38 downregulates host genes associated with MAPK signaling and the control of apoptosis and proliferation. These results highlight the value of integrative genomic approaches in prioritizing candidates for functional validation.

PMID: 20709297 [PubMed - as supplied by publisher]

Molecular characterization of a novel family of cysteine-rich proteins of Toxoplasma gondii and ultrastructural evidence of oocyst wall localization

Int J Parasitol. 2010 Aug 11. [Epub ahead of print]

Molecular characterization of a novel family of cysteine-rich proteins of Toxoplasma gondii and ultrastructural evidence of oocyst wall localization

Possenti A, Cherchi S, Bertuccini L, Pozio E, Dubey JP, Spano F.

Department of Infectious, Parasitic and Immunomediated Diseases, 299 - 00161 Rome, Italy.

Abstract
Among apicomplexan parasites, the coccidia and Cryptosporidium spp. are important pathogens of livestock and humans, and the environmentally resistant stage (oocyst) is essential for their transmission. Little is known of the chemical and molecular composition of the oocyst wall. Currently, the only parasite molecules shown to be involved in oocyst wall formation are the tyrosine-rich proteins gam56, gam82 and gam230 of Eimeria spp. and the cysteine-rich proteins COWP1 and COWP8 of Cryptosporidium parvum. In the present study, we searched the ToxoDB database for the presence of putative Toxoplasma gondii oocyst wall proteins (OWPs) and identified seven candidates, herein named TgOWP1 through TgOWP7, showing homology to the Cryptosporidium COWPs. We analysed a cDNA library from partially sporulated oocysts of T. gondii and cloned the full-length cDNAs encoding TgOWP1, TgOWP2 and TgOWP3, which consist of 499, 462 and 640 amino acids, respectively. The three proteins share 24% sequence identity with each other and a markedly similar overall structure, based on the presence of an N-terminal leader peptide followed by tandem duplications of a six-cysteine amino acid motif closely related to the Type I repeat of COWPs. Using antisera to recombinant TgOWP1, TgOWP2 and TgOWP3, we showed by Western blot that these molecules are expressed in T. gondii oocysts but are not detectable in tachyzoites. The solubilization of TgOWP1-3 strictly depended on the presence of reducing agents, consistent with a likely involvement of these proteins in multimeric complexes mediated by disulfide bridges. Immunofluorescence analysis allowed the localization of TgOWP1, TgOWP2 and TgOWP3 to the oocyst wall. Additionally, using immunoelectron microscopy and the 1G12 monoclonal antibody, TgOWP3 was specifically detected in the outer layer of the oocyst wall, thus representing the first validated molecular marker of this structure in T. gondii.

PMID: 20708619 [PubMed - as supplied by publisher]

A family of intermediate filament-like proteins is sequentially assembled into the cytoskeleton of Toxoplasma gondii

Cell Microbiol. 2010 Aug 2. [Epub ahead of print]

A family of intermediate filament-like proteins is sequentially assembled into the cytoskeleton of Toxoplasma gondii

Anderson-White BR, Ivey FD, Cheng K, Szatanek T, Lorestani A, Beckers CJ, Ferguson DJ, Sahoo N, Gubbels MJ.

Department of Biology, Boston College, Chestnut Hill, MA, USA.

Abstract
The intracellular protozoan parasite Toxoplasma gondii divides by a unique process of internal budding that involves the assembly of two daughter cells within the mother. The cytoskeleton of Toxoplasma, which is composed of microtubules associated with an inner membrane complex (IMC), has an important role in this process. The IMC, which is directly under the plasma membrane, contains a set of flattened membranous sacs lined on the cytoplasmic side by a network of filamentous proteins. This network contains a family of intermediate filament-like proteins or IMC proteins. In order to elucidate the division process, we have characterized a 14-member sub-family of Toxoplasma IMC proteins that share a repeat motif found in proteins associated with the cortical alveoli in all alveolates. By creating fluorescent protein fusion reporters for the family members we determined the spatio-temporal patterns of all 14 IMC proteins through tachyzoite development. This revealed several distinct distribution patterns and some provide the basis for novel structural models such as the assembly of certain family members into the basal complex. Furthermore we identified IMC15 as an early marker of budding and, lastly, the dynamic patterns observed throughout cytokinesis provide a timeline for daughter parasite development and division.

PMID: 20698859 [PubMed - as supplied by publisher]

Identification and Development of Novel Inhibitors of Toxoplasma gondii Enoyl Reductase

J Med Chem. 2010 Aug 10. [Epub ahead of print]

Identification and Development of Novel Inhibitors of Toxoplasma gondii Enoyl Reductase

Tipparaju SK, Muench SP, Mui EJ, Ruzheinikov SN, Lu JZ, Hutson SL, Kirisits MJ, Prigge ST, Roberts CW, Henriquez FL, Kozikowski AP, Rice DW, McLeod RL.

Drug Discovery Program, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois.

Abstract
Toxoplasmosis causes significant morbidity and mortality, and yet available medicines are limited by toxicities and hypersensitivity. Because improved medicines are needed urgently, rational approaches were used to identify novel lead compounds effective against Toxoplasma gondii enoyl reductase (TgENR), a type II fatty acid synthase enzyme essential in parasites but not present in animals. Fifty-three compounds, including three classes that inhibit ENRs, were tested. Six compounds have antiparasite MIC(90)s < 6 muM without toxicity to host cells, three compounds have IC(90)s < 45 nM against recombinant TgENR, and two protect mice. To further understand the mode of inhibition, the cocrystal structure of one of the most promising candidate compounds in complex with TgENR has been determined to 2.7 A. The crystal structure reveals that the aliphatic side chain of compound 19 occupies, as predicted, space made available by replacement of a bulky hydrophobic residue in homologous bacterial ENRs by Ala in TgENR. This provides a paradigm, conceptual foundation, reagents, and lead compounds for future rational development and discovery of improved inhibitors of T. gondii.

PMID: 20698542 [PubMed - as supplied by publisher]

Genome-Wide Survey and Evolutionary Analysis of Trypsin Proteases in Apicomplexan Parasites

Genomics Proteomics Bioinformatics. 2010 Jun;8(2):103-112.

Genome-Wide Survey and Evolutionary Analysis of Trypsin Proteases in Apicomplexan Parasites

Arenas AF, Osorio-Méndez JF, Gutierrez AJ, Gomez-Marin JE.

Grupo de Parasitología Molecular (GEPAMOL), Centro de Investigaciones Biomédicas, Universidad del Quindío, Armenia, Colombia.

Abstract
Apicomplexa are an extremely diverse group of unicellular organisms that infect humans and other animals. Despite the great advances in combating infectious diseases over the past century, these parasites still have a tremendous social and economic burden on human societies, particularly in tropical and subtropical regions of the world. Proteases from apicomplexa have been characterized at the molecular and cellular levels, and central roles have been proposed for proteases in diverse processes. In this work, 16 new genes encoding for trypsin proteases are identified in 8 apicomplexan genomes by a genome-wide survey. Phylogenetic analysis suggests that these genes were gained through both intracellular gene transfer and vertical gene transfer. Identification, characterization and understanding of the evolutionary origin of protease-mediated processes are crucial to increase the knowledge and improve the strategies for the development of novel chemotherapeutic agents and vaccines. Copyright © 2010 Beijing Genomics Institute. Published by Elsevier Ltd. All rights reserved.

PMID: 20691395 [PubMed - as supplied by publisher]

Immunological response of sheep to injections of plasmids encoding Toxoplasma gondii SAG1 and ROP1 genes

Parasite Immunol. 2010 Sep;32(9-10):671-83.

Immunological response of sheep to injections of plasmids encoding Toxoplasma gondii SAG1 and ROP1 genes

Li B, Oledzka G, McFarlane RG, Spellerberg MB, Smith SM, Gelder FB, Kur J, Stankiewicz M.

Lincoln University - Agriculture and Life Sciences Division, Canterbury, New Zealand.

Abstract
Infection with the intracellular protozoan parasite Toxoplasma gondii (T. gondii) causes health problems to both humans and livestock and has a large economic impact worldwide. The immune response in sheep following infection with T. gondii was evaluated using six different combinations of plasmid DNA, recombinant antigen and adjuvant. Sheep were generally vaccinated twice by intramuscular injection with plasmid DNA containing gene sequences for either the surface antigen (SAG1) or the rhoptry protein (ROP1) of T. gondii. Two of the groups injected with plasmid DNA SAG1 were boosted with recombinant protein (SAG1). We investigated the efficacy of including oligodeoxynucleotides (ODN) that contain CG motifs (CpG) and the gene coding for ovine granulocyte-macrophage colony stimulating factor (GM-CSF) as potential adjuvants. Administration of the plasmid encoding the ROP1 gene significantly enhanced both IFN-gamma production from peripheral blood cells when cultured in vitro with Toxoplasma antigen, and ROP1-specific IgG1 and IgG2 antibody levels present in serum. However, injection with SAG1 did not stimulate IFN-gamma production. These results indicate the potential of ROP1, given as plasmid DNA, as a potential vaccine candidate to protect sheep against T. gondii infection.

PMID: 20691019 [PubMed - in process]

Identification of New Pathogens in the Intraocular Fluid of Patients With Uveitis

Am J Ophthalmol. 2010 Aug 4. [Epub ahead of print]

Identification of New Pathogens in the Intraocular Fluid of Patients With Uveitis

de Groot-Mijnes JD, de Visser L, Zuurveen S, Martinus RA, Völker R, Ten Dam-Van Loon NH, de Boer JH, Postma G, de Groot RJ, Van Loon AM, Rothova A.

Department of Virology, University Medical Center Utrecht, Utrecht, The Netherlands; Department of Ophthalmology, University Medical Center Utrecht, Utrecht, The Netherlands.

Abstract
PURPOSE: To determine infectious causes in patients with uveitis of unknown origin by intraocular fluids analysis. DESIGN: Case-control study. METHODS: Ocular fluids from 139 patients suspected of infectious uveitis, but negative for herpes simplex virus, varicella-zoster virus, cytomegalovirus, and Toxoplasma gondii by polymerase chain reaction and/or antibody analysis in intraocular fluids, were assessed for the presence of 18 viruses and 3 bacteria by real-time polymerase chain reaction (PCR). The ocular fluids from 48 patients with uveitis of known etiology or with cataract were included as controls. RESULTS: Positive PCR results were found for Epstein-Barr virus, for rubella virus, and for human herpesvirus 6 each in 1 patient and for human parechovirus in 4 patients. Of the human parechovirus-positive patients, 1 was immunocompromised and had panuveitis. The other 3 patients were immunocompetent and had anterior uveitis, all with corneal involvement. CONCLUSIONS: Human parechovirus might be associated with infectious (kerato)uveitis. Copyright © 2010 Elsevier Inc. All rights reserved.

PMID: 20691420 [PubMed - as supplied by publisher]

Transmission of Toxoplasmosis (Toxoplasma gondii) by Foods

Adv Food Nutr Res. 2010;60C:1-19.

Transmission of Toxoplasmosis (Toxoplasma gondii) by Foods

Pereira KS, Franco RM, Leal DA.

Departamento de Engenharia Bioquímica, Escola de Química, Centro de Tecnologia Bloco E - Sala 203, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil.

Abstract
Protozoan foodborne diseases are generally underrecognized. Toxoplasma gondii is the causative agent of toxoplasmosis, one of the most prevalent parasitic infections to humans and domestic animals. The most likely source of T. gondii occurring through food is the consumption of raw or undercooked meat contaminated with tissue cysts. Sporulated T. gondii oocysts, from the feces of infected cats, present in the environment are a potential source of infection. The ingestion of water contaminated with oocysts and the eating of unwashed raw vegetables or fruits were identified as an important risk factor in most epidemiological studies. This review presents information and data to show the importance of T. gondii transmission by foods. Copyright © 2010 Elsevier Inc. All rights reserved.

PMID: 20691951 [PubMed - as supplied by publisher]

The Absence of IDO Upregulates Type I IFN Production, Resulting in Suppression of Viral Replication in the Retrovirus-Infected Mouse

J Immunol. 2010 Aug 6. [Epub ahead of print]

The Absence of IDO Upregulates Type I IFN Production, Resulting in Suppression of Viral Replication in the Retrovirus-Infected Mouse

Hoshi M, Saito K, Hara A, Taguchi A, Ohtaki H, Tanaka R, Fujigaki H, Osawa Y, Takemura M, Matsunami H, Ito H, Seishima M.

Department of Informative Clinical Medicine and.

Abstract
Indoleamine 2,3-dioxygenase, the l-tryptophan-degrading enzyme, plays a key role in the powerful immunomodulatory effects on several different types of cells. Because modulation of IDO activities after viral infection may have great impact on disease progression, we investigated the role of IDO following infection with LP-BM5 murine leukemia virus. We found suppressed BM5 provirus copies and increased type I IFNs in the spleen from IDO knockout (IDO(-/-)) and 1-methyl-d-l-tryptophan-treated mice compared with those from wild-type (WT) mice. Additionally, the number of plasmacytoid dendritic cells in IDO(-/-) mice was higher in the former than in the WT mice. In addition, neutralization of type I IFNs in IDO(-/-) mice resulted in an increase in LP-BM5 viral replication. Moreover, the survival rate of IDO(-/-) mice or 1-methyl-d-l-tryptophan-treated mice infected with LP-BM5 alone or with both Toxoplasma gondii and LP-BM5 was clearly greater than the survival rate of WT mice. To our knowledge, the present study is the first report to observe suppressed virus replication with upregulated type I IFN in IDO(-/-) mice, suggesting that modulation of the IDO pathway may be an effective strategy for treatment of virus infection.

PMID: 20693424 [PubMed - as supplied by publisher]

Comparative Analysis of Stage Specific Gene Regulation of Apicomplexan Parasites: Plasmodium falciparum and Toxoplasma gondii

Infect Disord Drug Targets. 2010 Aug 1;10(4):240-1.

Comparative Analysis of Stage Specific Gene Regulation of Apicomplexan Parasites: Plasmodium falciparum and Toxoplasma gondii

López-Estraño C.

Department of Biology, The University of Memphis, Memphis, TN 38152, USA. cestrano@memphis.edu.

PMID: 20687894 [PubMed - in process]

Biogenesis of the Inner Membrane Complex Is Dependent on Vesicular Transport by the Alveolate Specific GTPase Rab11B

PLoS Pathog. 2010 Jul 29;6(7):e1001029.

Biogenesis of the Inner Membrane Complex Is Dependent on Vesicular Transport by the Alveolate Specific GTPase Rab11B

Agop-Nersesian C, Egarter S, Langsley G, Foth BJ, Ferguson DJ, Meissner M.

Department of Infectiology, Parasitology, University Hospital Heidelberg, Heidelberg, Germany.

Abstract
Apicomplexan parasites belong to a recently recognised group of protozoa referred to as Alveolata. These protists contain membranous sacs (alveoli) beneath the plasma membrane, termed the Inner Membrane Complex (IMC) in the case of Apicomplexa. During parasite replication the IMC is formed de novo within the mother cell in a process described as internal budding. We hypothesized that an alveolate specific factor is involved in the specific transport of vesicles from the Golgi to the IMC and identified the small GTPase Rab11B as an alveolate specific Rab-GTPase that localises to the growing end of the IMC during replication of Toxoplasma gondii. Conditional interference with Rab11B function leads to a profound defect in IMC biogenesis, indicating that Rab11B is required for the transport of Golgi derived vesicles to the nascent IMC of the daughter cell. Curiously, a block in IMC biogenesis did not affect formation of sub-pellicular microtubules, indicating that IMC biogenesis and formation of sub-pellicular microtubules is not mechanistically linked. We propose a model where Rab11B specifically transports vesicles derived from the Golgi to the immature IMC of the growing daughter parasites.

PMID: 20686666 [PubMed - in process]

Transmission dynamics of Toxoplasma gondii along an urban-rural gradient

Theor Popul Biol. 2010 Jun 2. [Epub ahead of print]

Transmission dynamics of Toxoplasma gondii along an urban-rural gradient

Lélu M, Langlais M, Poulle ML, Gilot-Fromont E.

Université de Lyon, F-69000, Lyon; Université Lyon 1; CNRS, UMR5558, Laboratoire de Biométrie et Biologie Evolutive, F-69622, Villeurbanne, France; 2C2A-CERFE, 5 rue de la Héronniére, F-08240 Boult-aux-Bois, France; Université de Reims Champagne-Ardenne, Laboratoire de Parasitologie-Mycologie, EA 3800, UFR de Médecine, IFR 53, 51 rue Cognacq-Jay, F-51096 Reims, France.

Abstract
Recently, several authors proposed that the availability of intermediate hosts (IH) for definitive hosts (DH) may contribute to determine the dynamics and evolutionary ecology of parasites with facultative complex life cycles. The protozoa Toxoplasma gondii may be transmitted to DH either via predation of infected IH through a complex life cycle (CLC) or directly from contaminated environment through a simple life cycle (SLC). This parasite is also present in contrasting host density environments. We tested the hypothesis that the relative contributions of CLC and SLC along an urban-rural gradient depend on the IH supply. We built and analysed a deterministic model of T. gondii transmission cycle. SLC relative contribution is important only in urban-type environments, i.e., with low predation rate on IH. In contrast, the parasite is predominantly transmitted through CLC in suburban and rural environments. The association of the two cycles enables the parasite to spread in situations of low IH availability and low DH population size for which each cycle alone is insufficient. Copyright © 2010. Published by Elsevier Inc.

PMID: 20685358 [PubMed - as supplied by publisher]

Structure of the micronemal protein 2 (MIC2) A/I domain from Toxoplasma gondii

Protein Sci. 2010 Aug 3. [Epub ahead of print]

Structure of the micronemal protein 2 (MIC2) A/I domain from Toxoplasma gondii

Tonkin ML, Grujic O, Pearce M, Crawford J, Boulanger MJ.

Biochemistry & Microbiology, University of Victoria, Victoria, British Columbia V8W 3P6.

Abstract
Toxoplasma gondii is a widespread zoonotic pathogen capable of causing serious disease in humans and animals. As an obligate intracellular parasite, T. gondii relies on the orchestrated secretion of proteins from its apical complex organelles including the multi-modular, transmembrane micronemal protein 2 (MIC2) that couples recognition of the host cell with cytoskeletal reorganization of the parasite to drive invasion. To probe the basis by which the von Willebrand Factor A (vWA) - Integrin like module of TgMIC2 engages the host cell, we solved the crystal structure of a truncated form of TgMIC2A/I (TgMIC2A/Ic) phased by iodide SIRAS and refined to a resolution of 2.05A. The TgMIC2A/Ic core is organized into a central twisted beta sheet flanked by alpha helices consistent with a canonical vWA fold. A restricted basic patch serves as the putative heparin binding site, but no heparin binding was detected in native gel shift assays. Furthermore, no metal was observed in the metal ion dependent adhesion site (MIDAS). Structural overlays with homologous A/I domains reveal a divergent organization of the MIDAS beta4-alpha4 loop in TgMIC2A/Ic, which is stabilized through the burial of Phe195 into a deep pocket formed by Gly185. Intriguingly, Gly185 appears be unique among A/I domains to TgMIC2A/I suggesting that the divergent loop conformation may also be unique to TgMIC2A/I. Though lacking the C-terminal extension, the TgMIC2A/Ic structure reported here is the first of an A/I domain from an apicomplexan parasite and provides valuable insight into defining the molecular recognition of host cells by these widespread pathogens.

PMID: 20684023 [PubMed - as supplied by publisher]

Toxoplasma gondii infection inhibits the mitochondrial apoptosis through induction of Bcl-2 and HSP70

Parasitol Res. 2010 Aug 3. [Epub ahead of print]

Toxoplasma gondii infection inhibits the mitochondrial apoptosis through induction of Bcl-2 and HSP70

Hwang IY, Quan JH, Ahn MH, Hassan Ahmed HA, Cha GH, Shin DW, Lee YH.

Department of Infection Biology, Research Institute for Medical Science, Chungnam National University School of Medicine, 6 Munhwa-dong, Jung-gu, Daejeon, 301-131, South Korea.

Abstract
Heat-shock protein 70 (HSP70) is highly expressed in Toxoplasma gondii-infected cells. However, the role of this protein is not well understood, especially during apoptosis. This study addresses the mechanism behind the antiapoptotic chaperone activity of HSP70 in Toxoplasma-infected host cells using a human macrophage cell line, THP-1 by Western blot, DNA fragmentation assay, immunoprecipitation, and a caspase-3/7 activity assay based on cleavage of the colorimetric substrate DEVD-pNA. Apoptosis induced by arsenic trioxide (As(2)O(3)) was inhibited in T. gondii-infected THP-1 cells, but not in uninfected cells. Without As(2)O(3) induction of apoptosis, T. gondii infection caused increased expression of Bcl-2 and HSP70, but not caspase-3. However, active form caspase-3 levels were lower in As(2)O(3)-treated infected cells as compared with As(2)O(3)-treated uninfected cells. Bcl-2 expression in As(2)O(3)-treated infected cells was similar to that in cells infected with T. gondii. Translocation of apoptosis-inducing factor (AIF) and release of cytochrome c from mitochondria were inhibited in As(2)O(3)-treated infected cells as compared with As(2)O(3)-treated uninfected cells. Increased parasite loads in Toxoplasma-infected macrophages caused higher HSP70 and Bcl-2 expression in whole-cell extracts and fractionated components, respectively. However, expression of AIF and cytochrome c was unaffected. Toxoplasma dose-dependently inhibited caspase-3 activation, thus revealing an anti-apoptotic parasite activity on cytochrome c-mediated caspase activation in subcellular components. In addition, immunoprecipitation analysis suggested that HSP70 is capable of binding to the pro-apoptotic factors AIF and Apaf-1, but not to cytochrome c or procaspase-9. Taken together, these data demonstrate that T. gondii infection inhibits mitochondrial apoptosis through overproduction of anti-apoptotic Bcl-2 as well as HSP70, which are increased parasite loads dependently.

PMID: 20680337 [PubMed - as supplied by publisher]

Toxoplasma gondii Protease TgSUB1 is Required for Cell Surface Processing of Micronemal Adhesive Complexes and Efficient Adhesion of Tachyzoites

Cell Microbiol. 2010 Jul 30. [Epub ahead of print]

Toxoplasma gondii Protease TgSUB1 is Required for Cell Surface Processing of Micronemal Adhesive Complexes and Efficient Adhesion of Tachyzoites

Lagal V, Binder EM, Huynh MH, Kafsack BF, Harris PK, Diez R, Chen D, Cole RN, Carruthers VB, Kim K.

Departments of Medicine and Microbiology & Immunology, Albert Einstein College of Medicine, Bronx, NY 10461 USA.

Abstract
Abstract Host cell invasion by Toxoplasma gondii is critically dependent upon adhesive proteins secreted from the micronemes. Proteolytic trimming of microneme contents occurs rapidly after their secretion onto the parasite surface and is proposed to regulate adhesive complex activation to enhance binding to host cell receptors. However, the proteases responsible and their exact function are still unknown. In this report, we show that T. gondii tachyzoites lacking the microneme subtilisin protease TgSUB1 have a profound defect in surface processing of secreted microneme proteins. Notably parasites lack protease activity responsible for proteolytic trimming of MIC2, MIC4 and M2AP after release onto the parasite surface. Although complementation with full-length TgSUB1 restores processing, complementation of Deltasub1 parasites with TgSUB1 lacking the GPI anchor (Deltasub1::DeltaGPISUB1) only partially restores microneme protein processing. Loss of TgSUB1 decreases cell attachment and in vitro gliding efficiency leading to lower initial rates of invasion. Deltasub1 andDeltasub1::DeltaGPISUB1 parasites are also less virulent in mice. Thus TgSUB1 is involved in micronemal protein processing and regulation of adhesive properties of macromolecular adhesive complexes involved in host cell invasion.

PMID: 20678172 [PubMed - as supplied by publisher]

Synthesis and evaluation of oryzalin analogs against Toxoplasma

Bioorg Med Chem Lett. 2010 Jul 8. [Epub ahead of print]

Synthesis and evaluation of oryzalin analogs against Toxoplasma gondii

Endeshaw MM, Li C, Leon JD, Yao N, Latibeaudiere K, Premalatha K, Morrissette N, Werbovetz KA.

Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, 500 West 12th Avenue, Columbus, OH 43210-1291, USA.

Abstract
The synthesis and evaluation of 20 dinitroanilines and related compounds against the obligate intracellular parasite Toxoplasmagondii is reported. Using in vitro cultures of parasites in human fibroblasts, we determined that most of these compounds selectively disrupted Toxoplasma microtubules, and several displayed sub-micromolar potency against the parasite. The most potent compound was N(1),N(1)-dipropyl-2,6-dinitro-4-(trifluoromethyl)-1,3-benzenediamine (18b), which displayed an IC(50) value of 36nM against intracellular T. gondii. Based on these data and another recent report [Ma, C.; Tran, J.; Gu, F.; Ochoa, R.; Li, C.; Sept, D.; Werbovetz, K.; Morrissette, N. Antimicrob. AgentsChemother. 2010, 54, 1453], an antimitotic structure-activity relationship for dinitroanilines versus Toxoplasma is presented. Copyright © 2010 Elsevier Ltd. All rights reserved.

PMID: 20675138 [PubMed - as supplied by publisher]