Wednesday, January 28, 2009

Toxoplasma PRP1 is an ortholog of parafusin (PFUS) in vesicle scaffold assembly in Ca(2+)-regulated exocytosis

Eur J Cell Biol. 2009 Jan 22. [Epub ahead of print]

Toxoplasma PRP1 is an ortholog of parafusin (PFUS) in vesicle scaffold assembly in Ca(2+)-regulated exocytosis

Liu L, Tucker SC, Satir BH.

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx, NY 10461, USA.

The Paramecium tetraurelia protein parafusin (PFUS) and the Toxoplasma gondii protein parafusin-related protein 1 (PRP1) both have two covalent modifications (phosphorylation and phosphoglucosylation) and both are members of the phosphoglucomutase superfamily, associating with secretory vesicle scaffolds in their respective cells. This study tests the hypothesis that PRP1 is a functional ortholog of PFUS, functioning identically in Ca(2+)-regulated exocytosis. Electroporation of fluorescently labeled recombinant His-PRP1 into live Paramecium cells resulted in its localization to docked, dense-core secretory vesicles (DCSVs) in a pattern identical to endogenous PFUS. In tam8 mutants, defective in transport of DCSVs, the fluorescently labeled protein was restricted to the un-transported DCSVs. Specificity of PRP1 localization was demonstrated by electroporating labeled actin or pyruvate kinase, which both failed to localize to either docked or undocked vesicles. In wild-type Paramecium, electroporated His-PRP1 dissociated from DCSVs upon exocytosis, and re-associated as new organelles formed. Mutagenized His-PRP1 species (S146A or S146E) cannot be phosphorylated by P2 calcium-dependent kinase in vitro. Upon electroporation, these molecules remained cytoplasmic and un-associated with DCSVs, while mutation of another PRP1 serine residue (S560A) did neither affect the localization to the DCSVs nor the phosphorylation pattern. Therefore, in this heterologous system, localization, transport and dissociation/re-association of PRP1 substituted for PFUS, supporting the conclusion that the proteins are functional orthologs. The assay also identified a strategic residue S146 within the PFUS ortholog (S138 in PFUS by extrapolation) required for post-translational modification, DCSV scaffold association and for exocytosis.

PMID: 19167775 [PubMed - as supplied by publisher]

Towards New Antifolates Targeting Eukaryotic Opportunistic Infections

Eukaryot Cell. 2009 Jan 23. [Epub ahead of print]

Towards New Antifolates Targeting Eukaryotic Opportunistic Infections

Liu J, Bolstad DB, Bolstad ES, Wright DL, Anderson AC.

Department of Pharmaceutical Sciences, University of Connecticut, 69 N. Eagleville Rd., Storrs, CT 06269.

Trimethoprim, an antifolate commonly prescribed in combination with sulfamethoxazole, potently inhibits several prokaryotic species of dihydrofolate reductase (DHFR). However, several eukaryotic pathogenic organisms are resistant to trimethoprim, preventing its effective use as a therapeutic for those infections. We have been building a program to reengineer trimethoprim to more potently and selectively inhibit eukaryotic species of DHFR as a viable strategy for new drug discovery targeting several opportunistic pathogens. We have developed a series of compounds that exhibit potent and selective inhibition of DHFR from the parasitic protozoa Cryptosporidium and Toxoplasma as well as the fungus, Candida glabrata. A comparison of the structures of DHFR from the fungal species Candida glabrata and Pneumocystis suggests that the compounds may also potently inhibit Pneumocystis DHFR.

PMID: 19168759 [PubMed - as supplied by publisher]

Toxoplasma gondii IWOP-10 2008: One Hundred Years and Counting

Eukaryot Cell. 2009 Jan 23. [Epub ahead of print]

Toxoplasma gondii IWOP-10 2008: One Hundred Years and Counting

Halonen SK, Weiss LM.

Department of Microbiology, Montana State University, Bozeman, MT; and Departments of Pathology and Medicine, Albert Einstein College of Medicine, Bronx, New York.

It has been 100 years since Toxoplasma gondii was initially described in the rodent Ctenodactylus gundi. T. gondii is a ubiquitous, Apicomplexan parasite of warm-blooded animals that can cause several clinical syndromes including encephalitis, chorioretinitis and congenital infection. The association of T. gondii with food and waterborne transmission has resulted in its classification as a National Institute of Allergy and Infectious Diseases (NIAID) Category B priority agent. Due to the extensive repertoire of applicable experimental techniques available for this pathogen it has become a model organism for the study of intracellular pathogens. This review will address information related to recent advances in our understanding of biology of T. gondii presented at the Tenth International Workshop on Opportunistic pathogens and the associated symposium entitled "Centenary Celebration of Toxoplasma Discovery" held at that meeting.

PMID: 19168756 [PubMed - as supplied by publisher]

Monday, January 26, 2009

Lack of association between ABO histo-blood groups,secretor and non-secretor phenotypes, and anti-Toxoplasma antibodies among pregnant women

Lack of association between ABO histo-blood groups,secretor and non-secretor phenotypes, and anti-Toxoplasma antibodies among pregnant women from the northwestern region of Sao Paulo State, Brazil

Cinara C. Branda~o de Mattos, Juliana R. Cintra, Ana I.C. Ferreira, Ligia C.J.F. Spegiorin, Katia J. Galisteu, Ricardo L.D. Machado, Luiz C. de Mattos

Arch Med Sci 2008; 4, 3: 254–258

Introduction: Toxoplasma gondii infects humans in several manners including by
the gastrointestinal tract where a-2-L-fucosyltransferase (FUTII) coded by the
FUT2 gene (19q13.3) controls the expression of the ABH glycoconjugate profile.
The presence of functional FUTII defines the secretor phenotype which is
associated with ABO erythrocytic phenotypes. Due to the epidemiological and
clinical importance of T. gondii infection, the aim of this work was to test the
hypothesis that the ABH glycoconjugate profile expressed in the gastrointestinal
tract is associated with the presence of antibodies against this parasite.
Material and methods: A total of 367 pregnant women from the High-Risk Pregnancy
Clinic of the University Hospital de Base in Sa~ o José do Rio Preto were enrolled in
this study. Two blood samples were drawn with only one being mixed with
anticoagulant. ABO erythrocytic phenotyping and detection of anti-T. gondii antibodies
were achieved by the haemagglutination test. Identification of the secretor status
used the PCR-RFLP method.
Results: Differences in the ABO erythrocytic phenotypes (P=0.20) and secretor and nonsecretor
phenotypes (P=0.41), either in isolation or in association, were not statistically
significant with respect to the presence or absence of anti-T. gondii antibodies.
Conclusions: These results suggest that the ABH glycoconjugate profile expressed
in the gastrointestinal tract under regulation of the FUT2 gene is not associated
with anti-T. gondii antibodies.

For article, contact Cinara Mattos:

Saturday, January 24, 2009

EPIC-DB: a proteomics database for studying Apicomplexan organisms

BMC Genomics. 2009 Jan 21;10(1):38. [Epub ahead of print]

EPIC-DB: a proteomics database for studying Apicomplexan organisms

Madrid-Aliste CJ, Dybas JM, Hogue Angeletti R, Weiss LM, Kim K, Simon I, Fiser A.

ABSTRACT: BACKGROUND: High throughput proteomics experiments are useful for analyzing the protein expression of an organism, identifying the correct gene structure of a genome, or locating possible post-translational modifications within proteins. High throughput methods necessitate publicly accessible and easily queried databases for efficiently and logically storing, displaying, and analyzing the large volume of data. Description: EPICDB is a publicly accessible, queryable, relational database that organizes and displays experimental, high throughput proteomics data for Toxoplasma gondii and Cryptosporidium parvum. Along with detailed information on mass spectrometry experiments, the database also provides antibody experimental results and analysis of functional annotations, comparative genomics, and aligned expressed sequence tag (EST) and genomic open reading frame (ORF) sequences. The database contains all available alternative gene datasets for each organism, which comprises a complete theoretical proteome for the respective organism, and all data is referenced to these sequences. The database is structured around clusters of protein sequences, which allows for the evaluation of redundancy, protein prediction discrepancies, and possible splice variants. The database can be expanded to include genomes of other organisms for which proteome-wide experimental data are available. CONCLUSIONS: EPICDB is a comprehensive database of genome-wide T. gondii and C. parvum proteomics data and incorporates many features that allow for the analysis of the entire proteomes and/or annotation of specific protein sequences. EPICDB is complementary to other -genomics- databases of these organisms by offering complete mass spectrometry analysis on a comprehensive set of all available protein sequences.

PMID: 19159464 [PubMed - as supplied by publisher]


J Parasitol. 2009 Jan 22:1. [Epub ahead of print]


Lindsay DS, Dubey JP.

Toxoplasma gondii is now recognized as an important pathogen in costal marine mammals. Oocysts from cat feces are believed to be washed in to seawater and serve as a source of infection via transport hosts. Experimentally it has been demonstrated that T. gondii oocysts can sporulate in seawater and remain infectious for mice for up to 6 mo. The present study examined the long-term survival of T. gondii in seawater (15 ppt NaCl) kept at 4 C or room temperature. Oocysts kept at 4 C for 24 mo were orally infectious for mice, while those kept at room temperature for 24 mo were not.

PMID: 19161250 [PubMed - as supplied by publisher]

Functional aspects of Toll-like receptor/MyD88 signalling during protozoan infection: focus on Toxoplasma

Clin Exp Immunol. 2009 Jan 21. [Epub ahead of print]

Functional aspects of Toll-like receptor/MyD88 signalling during protozoan infection: focus on Toxoplasma gondii

Egan CE, Sukhumavasi W, Butcher BA, Denkers EY.

Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.

Toll-like receptor (TLR)/MyD88 signalling has emerged as a major pathway of pathogen recognition in the innate immune system. Here, we review recent data that begin to show how this pathway controls the immune response to protozoan infection, with particular emphasis on the opportunistic pathogen Toxoplasma gondii. The various ways that the parasite activates and suppresses TLR/MyD88 signalling defines several key principals that illuminate the complexities of the host-pathogen interaction. We also speculate how TLR/MyD88 signalling might be exploited to provide protection against Toxoplasma, as well as other protozoa and infection in general.

PMID: 19161444 [PubMed - as supplied by publisher]

Thioureides of 2-(phenoxymethyl)benzoic acid 4-R substituted: A novel class of anti-parasitic compounds

Parasitol Int. 2008 Dec 25. [Epub ahead of print]

Thioureides of 2-(phenoxymethyl)benzoic acid 4-R substituted: A novel class of anti-parasitic compounds

Müller J, Limban C, Stadelmann B, Missir AV, Chirita IC, Chifiriuc MC, Nitulescu GM, Hemphill A.

Institute of Parasitology, University of Berne, Länggass-Strasse 122, CH-3012 Berne, Switzerland.

Fifty members of a novel class of antimicrobial compounds, 2-(4-R-phenoxymethyl)benzoic acid thioureides, were synthesized and characterized with respect to their activities against three parasites of human relevance, namely the protozoa Giardia lamblia and Toxoplasma gondii, and the larval (metacestode) stage of the tapeworm Echinococcus multilocularis. To determine the selective toxicity of these compounds, the human colon cancer cell line Caco2 and primary cultures of human foreskin fibroblasts (HFF) were also investigated. The new thioureides were obtained in a three-step-reaction process and subsequently characterized by their physical constants (melting point, solubility). The chemical structures were elucidated by (1)H NMR, (13)C NMR, IR spectral methods and elemental analysis. The analyses confirmed the final and intermediate compound structures and the synthesis. The compounds were then tested on the parasites in vitro. All thioureides, except two compounds with a nitro group, were totally ineffective against Giardia lamblia. 23 compounds inhibited the proliferation of T. gondii, three of them with an IC(50) of approximately 1 muM. The structural integrity of E. multilocularis metacestodes was affected by 22 compounds. In contrast, HFF were not susceptible to any of these thioureides, while Caco2 cells were affected by 17 compounds, two of them inhibiting proliferation with an IC(50) in the micromolar range. Thioureides may thus present a promising class of anti-infective agents.

PMID: 19162220 [PubMed - as supplied by publisher]

Thursday, January 22, 2009

Structural characterization of the bradyzoite surface antigen (BSR4) from Toxoplasma

J Biol Chem. 2009 Jan 20. [Epub ahead of print]

Structural characterization of the bradyzoite surface antigen (BSR4) from Toxoplasma gondii: A unique addition to the SAG1 related superfamily

Crawford J, Grujic O, Bruic E, Czjek M, Grigg ME, Boulanger MJ.

Biochemistry and Microbiology, University of Victoria, Victoria, BC V8W 3P6.

Toxoplasma gondii is an obligate intracellular protozoan parasite that infects nearly one third of the human population. The success of T. gondii is based on its complex life cycle; a lytic tachyzoite form disseminates infection whereas an encysted bradyzoite form establishes a latent, chronic infection. Persistence and transmissibility is central to the survival of the parasite and is, in part, mediated by a family of antigenically distinct SRS (Surface Antigen Glycoprotein (SAG) Related Sequences) adhesins that play a dual role in host cell attachment and host immune evasion. More than 160 members of the SRS family have been identified with only the tachyzoite expressed SAG1 structurally characterized. Here we report the first structural description of the bradyzoite adhesin BSR4 using X-ray crystallography and small angle X-ray scattering (SAXS). The 1.90 A crystal structure of BSR4 reveals an architecture comprised of tandem sandwich domains organized in a head to tail fashion with the N-terminal domain responsible for dimer formation. A restructured topology in BSR4 results in a ligand binding site that is significantly reorganized in both structure and chemistry relative to SAG1, consistent with BSR4 binding a distinct physiological ligand. The SAXS solution structure of BSR4 highlights a potentially important structural role for the interdomain polymorphic linker that imparts significant flexibility that may promote structural adaptation during ligand binding. This study reveals an unexpected level of structural diversity within the SRS superfamily and provides important insight into the role of these virulence factors.

PMID: 19155215 [PubMed - as supplied by publisher]

Host ER-parasitophorous vacuole interaction provides a route of entry for antigen cross-presentation in Toxoplasma gondii-infected dendritic cells

J Exp Med. 2009 Jan 19. [Epub ahead of print]

Host ER-parasitophorous vacuole interaction provides a route of entry for antigen cross-presentation in Toxoplasma gondii-infected dendritic cells

Goldszmid RS, Coppens I, Lev A, Caspar P, Mellman I, Sher A.

Immunobiology Section, Laboratory of Parasitic Diseases and 2 Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.

Toxoplasma gondii tachyzoites infect host cells by an active invasion process leading to the formation of a specialized compartment, the parasitophorous vacuole (PV). PVs resist fusion with host cell endosomes and lysosomes and are thus distinct from phagosomes. Because the parasite remains sequestered within the PV, it is unclear how T. gondii-derived antigens (Ag's) access the major histocompatibility complex (MHC) class I pathway for presentation to CD8(+) T cells. We demonstrate that recruitment of host endoplasmic reticulum (hER) to the PV in T. gondii-infected dendritic cells (DCs) directly correlates with cross-priming of CD8(+) T cells. Furthermore, we document by immunoelectron microscopy the transfer of hER components into the PV, a process indicative of direct fusion between the two compartments. In strong contrast, no association between hER and phagosomes or Ag presentation activity was observed in DCs containing phagocytosed live or dead parasites. Importantly, cross-presentation of parasite-derived Ag in actively infected cells was blocked when hER retrotranslocation was inhibited, indicating that the hER serves as a conduit for the transport of Ag between the PV and host cytosol. Collectively, these findings demonstrate that pathogen-driven hER-PV interaction can serve as an important mechanism for Ag entry into the MHC class I pathway and CD8(+) T cell cross-priming.

PMID: 19153244 [PubMed - as supplied by publisher]

Monday, January 19, 2009

Repeated secondary loss of adaptin complex genes in the Apicomplexa

Parasitol Int. 2008 Dec 24. [Epub ahead of print]

Repeated secondary loss of adaptin complex genes in the Apicomplexa

Nevin WD, Dacks JB

The Molteno Building, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, UK; Corpus Christi College, Cambridge, CB2 1RH, UK.

The Apicomplexa include parasites of devastating medical and economic consequence. While obviously essential for their parasitic mechanism, the molecular machinery underpinning membrane-trafficking in many apicomplexans is poorly understood. One potentially key set of players, the adaptins, selects cargo for incorporation into trafficking vesicles. Four distinct adaptin (AP) complexes exist in eukaryotes; AP1 and AP3 are involved in transport between the trans-Golgi Network (TGN) and endosomes, AP4 in TGN to cell surface transport, and AP2 in endocytosis from the cell surface. Of particular interest is the involvement of AP1 in Toxoplasma rhoptry biogenesis. The recent completion of several apicomplexan genomes should jump-start molecular parasitological studies and provide systems-level insight into the apicomplexan adaptin machinery. However, many of the encoded adaptin proteins are annotated conservatively and not to the necessary complex or subunit level. Prompted by previous evidence suggesting the lack of AP3 in Plasmodium falciparum, we undertook homology-searching and phylogenetic analysis to produce a rigorously annotated set of adaptin subunits encoded in diverse apicomplexan genomes. We found multiple losses of adaptins across the phylum; in particular Theileria, Babesia, and Cryptosporidium, but surprisingly not Plasmodium, appear to have lost the entirety of the AP3 complex. The losses correlate with a degenerate Golgi body structure and are reminiscent of recently reported secondary losses of additional endocytic components (ESCRTs) in several Apicomplexa. These data may indicate a relaxation of the selective pressure on the apicomplexan endocytic system and, regardless, should greatly facilitate future molecular cell biological investigation of the role of adaptins in these important parasites.

PMID: 19146987 [PubMed - as supplied by publisher]

Friday, January 16, 2009

Effect of artesunate on Toxoplasma gondii: in vitro and in vivo studies

J Egypt Soc Parasitol. 2008 Apr;38(1):185-201

Effect of artesunate on Toxoplasma gondii: in vitro and in vivo studies

El Zawawy LA.

Department of Parasitology, Faculty of Medicine, Alexandria University, Alexandria, Egypt.

The effect of Artesunate (As) on Toxoplasma gondii (T. gondii) in vitro and in vivo was studied. In vitro, tachyzoites of RH strain were exposed to As in a concentration of 2 microg/ml for 72 hours. The assessment of As effect was carried out by studying the viability, infectivity and ultrastructure changes of treated tachyzoites by scanning electron microscope (SEM). In the in vivo study, Swiss albino mice were infected intraperitoneally with tachyzoites of T. gondii RH strain, then orally treated with As in a dose of 200 mg/kg for five successive days. The effect of As was evaluated by the mortality rate and survival time of the treated mice. Parasite burden, viability, infectivity and ultrastructure changes of tachyzoites harvested from the peritoneal cavities of infected treated mice as compared with infected non-treated control mice were also studied. In vitro study demonstrated a significant reduction in viability and infectivity of tachyzoites exposed to As compared with untreated controls. In vivo study, showed that treatment of infected mice with As induced a significant decrease in mortality rate and increase in survival time. There was also a significant reduction in parasite burden in infected treated mice with significant reduction in viability and infectivity of tachyzoites harvested from them as compared with infected non-treated control. SEM showed distortion in tachyzoites' shape, peeling, erosions and discontinuity in areas of surface membrane of treated tachyzoites of both in vitro and in vivo studies. So, As proved an effective and promising drug in treating acute toxoplasmosis.

PMID: 19143130 [PubMed - in process]

Thursday, January 15, 2009

ROP2: A Virulence Factor with a Protein-Kinase Fold and No Enzymatic Activity

Structure. 2009 Jan;17(1):139-46

ROP2 from Toxoplasma gondii: A Virulence Factor with a Protein-Kinase Fold and No Enzymatic Activity

Labesse G, Gelin M, Bessin Y, Lebrun M, Papoin J, Cerdan R, Arold ST, Dubremetz JF.

Atelier de Bio- et Chimie Informatique Structurale, Centre de Biochimie Structurale, CNRS, UMR5048, Universités Montpellier 1 et 2, F34090 Montpellier, France; INSERM, U554, 29 rue de Navacelles, F34090 Montpellier, France.

The ROP2 protein and its paralogs are important virulence factors secreted into the host cell by the parasite Toxoplasma gondii. Here we describe the crystal structure of a large and soluble domain of mature ROP2, representative of the ROP2-like protein family. This is a structure of a protein-kinase fold that is devoid of catalytic residues and does not bind ATP. Various structural extensions constitute a signature of this protein family and act to maintain the protein kinase in an open conformation. Our ROP2 structure rules out a previous structural model of attachment of ROP2-like proteins to the parasitophorous vacuole membrane. We propose an alternative mode of membrane attachment implicating basic and amphiphatic helices present in the flexible N terminus of ROP2.

PMID: 19141290 [PubMed - in process]

Wednesday, January 14, 2009

Novel components of the Apicomplexan moving junction reveal conserved and coccidia-restricted elements

Cell Microbiol. 2008 Dec 30. [Epub ahead of print]

Novel components of the Apicomplexan moving junction reveal conserved and coccidia-restricted elements

Straub KW, Cheng SJ, Sohn CS, Bradley PJ.

Department of Microbiology, Immunology and Molecular Genetics, University of California - Los Angeles, Los Angeles, CA 90095-1489, USA.

Summary Apicomplexan parasites generally invade their host cells by anchoring the parasite to the host membrane through a structure called the moving junction (MJ). This MJ is also believed to sieve host proteins from the nascent parasitophorous vacuole membrane, which likely protects the pathogen from lysosomal destruction. Previously identified constituents of the Toxoplasma MJ have orthologues in Plasmodium, indicating a conserved structure throughout the Apicomplexa. We report here two novel MJ proteins, RON5 and RON8. While RON5 is conserved in Plasmodium, RON8 appears restricted to the coccidia. RON8, which is likely essential, co-immunoprecipitates RON5 and known MJ proteins from extracellular parasites, indicating that a preformed complex exists within the parasites. Upon secretion, we show that RON8 within the MJ localizes to the cytoplasmic face of the host plasma membrane. To examine interactions between RON8 and the host cell, we expressed RON8 in mammalian cells and show that it targets to its site of action at the periphery in a manner dependent on the C-terminal portion of the protein. The discovery of RON5 and RON8 provides new insight into conserved and unique elements of the MJ, furthering our understanding of how the MJ contributes to the intricate mechanism of Apicomplexan invasion.

PMID: 19134112 [PubMed - as supplied by publisher]

Transmission of Toxoplasma gondii from Infected Dendritic Cells to Natural Killer Cells

Infect Immun. 2009 Jan 12. [Epub ahead of print]

Transmission of Toxoplasma gondii from Infected Dendritic Cells to Natural Killer Cells

Persson CM, Lambert H, Vutova PP, Dellacasa-Lindberg I, Nederby J, Yagita H, Ljunggren HG, Grandien A, Barragan A, Chambers BJ.

Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden; Department of Parasitology, Mycology and Environmental Microbiology, Swedish Institute for Infectious Disease Control, 171 82 Solna, Sweden; Juntendo University School of Medicine, Tokyo, 113-8421, Japan.

The obligate intracellular parasite Toxoplasma gondii (T. gondii) can actively infect any nucleated cell type including cells from the immune system. In the present study, we observed that a large number of natural killer (NK) cells were infected by T. gondii early after i.p. inoculation of parasites into C57BL/6 mice. Interestingly, one mechanism of NK cell infection involved NK cell-mediated targeting of infected dendritic cells (DC). Perforin-dependent killing of infected DC led to active egress of infectious parasites that rapidly infected adjacent effector NK cells. Infected NK cells were not efficiently targeted by other NK cells. These results suggest that rapid transfer of T. gondii from infected DC to effector NK cells may contribute to the parasite's sequestration and shielding from immune recognition shortly after infection.

PMID: 19139191 [PubMed - as supplied by publisher]

Role of autophagy in the host defense against Toxoplasma gondii in astrocytes

Autophagy. 2009 Feb 17;5(2). [Epub ahead of print]

Role of autophagy in the host defense against Toxoplasma gondii in astrocytes

Halonen SK.

Department of Microbiology, Montana State University, Bozeman, Montana, USA.

Autophagy has recently been implicated in the host defense against the intracellular protozoan pathogen, Toxoplasma gondii, a major opportunistic pathogen of the central nervous system in immunosuppressed individuals. In both IFNgamma-activated macrophages and astrocytes, the p47 GTPases traffic to the T. gondii parasitophorous vacuole, followed by vacuolar disruption, parasite killing and clearance of the dead parasites. In macrophages, it is relatively well established that autophagy is involved in parasite elimination and killing. The role of autophagy in parasite elimination in astrocytes, a dominant host cell in the central nervous system, is much less clear. Our studies indicate that in IFNgamma-stimulated astrocytes, autophagy of disrupted vacuoles and/or dead parasites does not occur but rather that degradation of the parasite occurs in the host cytoplasm. However, recent studies indicate autophagy may be involved in the elimination of the degraded parasite material from the astrocyte host cell cytoplasm and suggest that autophagous removal of degraded parasite material may be necessary for survival of the host cell. Delivery of parasite antigen from the cytosol to the endolysosomal compartments in astrocytes is of importance as it suggests a pathway by which astrocytes could present Toxoplasma antigens via the MHC Class II pathway and function as an antigen-presenting cell for the parasite in the brain.

PMID: 19139630 [PubMed - as supplied by publisher]

Saturday, January 10, 2009

Kinetics of systemic cytokine and brain chemokine gene expression in murine toxoplasma infection

J Parasitol. 2008 Dec;94(6):1282-8

Kinetics of systemic cytokine and brain chemokine gene expression in murine toxoplasma infection

Aviles H, Stiles J, O'Donnell P, Orshal J, Leid J, Sonnenfeld G, Monroy F.

Toxoplasma gondii often migrates to the central nervous system in immunocompromised patients, where it induces a severe inflammation referred to as Toxoplasma encephalitis. The mechanisms involved in control of parasite multiplication and prevention of Toxoplasma encephalitis remain unclear. The objective of the present study was to characterize the inflammatory response in the brains of mice during acute T. gondii infection, with emphasis on the expression of chemokine receptors. Susceptible C57BL/6 mice were orally infected with 10 cysts of the low-virulent ME49 strain of T. gondii. Levels of cytokines (TNF-alpha, IFN-gamma, IL-10, IL-6, and IL-12p70) and chemokines (CCL/2MCP-1) were measured in plasma at 5, 10, 15, 20, and 30 days after infection. In addition, the mRNA expression of chemokines (CCL5/RANTES, CCL2/MCP-1, CCL4/MIP-1beta) and chemokine receptors (CCR1, CCR2, CCR5, CCR7, CCR8, CXCR4, and CXR5) were measured in brain tissues at the same time points. Plasma levels of IFN-gamma and CCL2/MCP-1 were highly expressed at day 5, whereas TNF-alpha had a moderate increase at day 5, peaked at day 10, and returned to normal levels by day 30. Plasma levels of IL-10, IL-6, and IL-12p70 were not detected throughout the study. Analyses of mRNA expression of chemokines and chemokine receptors in the brain showed that CCL5/ RANTES, CCR7, CXCR4, and CXCR5 were upregulated, peaking after 10 days of T. gondii infection. IgM-specific antibody levels increased at day 5 and peaked at days 10 and 30, whereas IgG levels increased at day 10 and continued to increase thereafter, reaching maximum levels at day 30 postinfection (PI). Our results suggest that T. gondii infection is controlled at local and systemic levels, and that proinflammatory proteins and their receptors may be acting coordinately to induce stage conversion and prevent parasite multiplication and development of Toxoplasma encephalitis. The early production of IFN-gamma and the delayed expression of CXCR4 and CXCR5 indicate that T. gondii induces an early robust cellular immune response, followed by a strong and sustained antibody-mediated immunity.

PMID: 19127964 [PubMed - in process]

Thursday, January 08, 2009

Toxoplasma gondii strains available at BEIR

Dear colleagues,

A number of Toxoplasma gondii strains from genetic crosses between parental strains II and III are now available at the Biodefense and Emerging Infections Research Resources Repository (BEI Resources). These were deposited by Dr. David Sibley.

Robert Molestina, Ph.D.
Collection/Research Scientist
10801 University Boulevard
Manassas, VA 20110
Phone: (703) 365-2700 (ext. 2628)
Facsimile: (703) 365-2701

Malondialdehyde, Glutathione, and Nitric Oxide Levels in Toxoplasma gondii Seropositive Patients

Korean J Parasitol. 2008 Dec;46(4):293-5. Epub 2008 Dec 20

Malondialdehyde, Glutathione, and Nitric Oxide Levels in Toxoplasma gondii Seropositive Patients

Karaman U, Celik T, Kiran TR, Colak C, Daldal NU.

Inonu University School of Medicine, Department of Parasitology, Malatya, Turkey.

The aim of this study was to investigate the difference in the serum malondialdehyde (MDA), glutathione (GSH), and nitric oxide (NO) levels between normal and T. gondii-infected patients. To this end, MDA, GSH, and NO levels in the sera of 37 seropositive patients and 40 participants in the control group were evaluated. In Toxoplasma ELISA, IgG results of the patient group were 1,013.0 +/- 543.8 in optical density (mean +/- SD). A statistically significant difference was found between patients and the control group in terms of MDA, GSH, and NO levels. A decrease in GSH activity was detected, while MDA and NO levels increased significantly. Consequently, it is suggested that the use of antioxidant vitamins in addition to a parasite treatment shall prove useful. The high infection vs control ratio of MDA and NO levels probably suggests the occurrence as a mechanism of tissue damage in cases of chronic toxoplasmosis. Moreover, it is recommended that the patient levels of MDA, GSH, and NO should be evaluated in toxoplasmosis.

PMID: 19127340 [PubMed - in process]

Interaction between GRA3 and Calcium Modulating Ligand of Host Cell Endoplasmic Reticulum

Korean J Parasitol. 2008 Dec;46(4):209-16. Epub 2008 Dec 20

Interaction between Parasitophorous Vacuolar Membrane-associated GRA3 and Calcium Modulating Ligand of Host Cell Endoplasmic Reticulum in the Parasitism of Toxoplasma gondii

Kim JY, Ahn HJ, Ryu KJ, Nam HW.

Department of Parasitology and Catholic Institute of Parasitic Diseases, Catholic University of Korea, College of Medicine, Seoul 137-701, Korea.

A monoclonal antibody against Toxoplasma gondii of Tg556 clone (Tg556) blotted a 29 kDa protein, which was localized in the dense granules of tachyzoites and secreted into the parasitophorous vacuolar membrane (PVM) after infection to host cells. A cDNA fragment encoding the protein was obtained by screening a T. gondii cDNA expression library with Tg556, and the full-length was completed by 5'-RACE of 2,086 bp containing an open reading frame (ORF) of 669 bp. The ORF encoded a polypeptide of 222 amino acids homologous to the revised GRA3 but not to the first reported one. The polypeptide has 3 hydrophobic moieties of an N-terminal stop transfer sequence and 2 transmembrane domains (TMD) in posterior half of the sequence, a cytoplasmic localization motif after the second TMD and an endoplasmic reticulum (ER) retrival motif in the C-terminal end, which suggests GRA3 as a type III transmembrane protein. With the ORF of GRA3, yeast two-hybrid assay was performed in HeLa cDNA expression library, which resulted in the interaction of GRA3 with calcium modulating ligand (CAMLG), a type II transmembrane protein of ER. The specific binding of GRA3 and CAMLG was confirmed by glutathione S-transferase (GST) pull-down and immunoprecipitation assays. The localities of fluorescence transfectionally expressed from GRA3 and CAMLG plasmids were overlapped completely in HeLa cell cytoplasm. In immunofluorescence assay, GRA3 and CAMLG were shown to be co-localized in the PVM of host cells. Structural binding of PVM-inserted GRA3 to CAMLG of ER suggested the receptor-ligand of ER recruitment to PVM during the parasitism of T. gondii.

PMID: 19127325 [PubMed - in process]

Wednesday, January 07, 2009

The history of Toxoplasma gondii--the first 100 years

J Eukaryot Microbiol. 2008 Nov-Dec;55(6):467-75

The history of Toxoplasma gondii--the first 100 years

Dubey JP.

United States Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Animal Parasitic Diseases Laboratory, Building 1001, Beltsville, Maryland 20705-2350, USA.

In this paper the history of Toxoplasma gondii and toxoplasmosis is reviewed. This protozoan parasite was first discovered in 1908 and named a year later. Its medical importance remained unknown until 1939 when T. gondii was identified in tissues of a congenitally infected infant, and veterinary importance became known when it was found to cause abortion storms in sheep in 1957. The discovery of a T. gondii specific antibody test, Sabin-Feldman dye test in 1948 led to the recognition that T. gondii is a common parasite of warm-blooded hosts with a worldwide distribution. Its life cycle was not discovered until 1970 when it was found that felids are its definitive host and an environmentally resistant stage (oocyst) is excreted in feces of infected cats. The recent discovery of its common infection in certain marine wildlife (sea otters) indicates contamination of our seas with T. gondii oocysts washed from land. Hygiene remains the best preventive measure because currently there is no vaccine to prevent toxoplasmosis in humans.

PMID: 19120791 [PubMed - in process]

Cell-mediated immunity to Toxoplasma gondii develops primarily by local Th1 host immune responses in the absence of parasite replication

J Immunol. 2009 Jan 15;182(2):1069-78

Cell-mediated immunity to Toxoplasma gondii develops primarily by local Th1 host immune responses in the absence of parasite replication

Gigley JP, Fox BA, Bzik DJ.

Department of Microbiology and Immunology, Dartmouth Medical School, Lebanon, NH 03756, USA.

A single inoculation of mice with the live, attenuated Toxoplasma gondii uracil auxotroph strain cps1-1 induces long-lasting immunity against lethal challenge with hypervirulent strain RH. The mechanism for this robust immunity in the absence of parasite replication has not been addressed. The mechanism of long-lasting immunity, the importance of route of immunization, cellular recruitment to the site of infection, and local and systemic inflammation were evaluated. Our results show that infection with cps1-1 elicits long-lasting CD8+ T cell- mediated immunity. We show that immunization with cps1-1-infected dendritic cells elicits long-lasting immunity. Intraperitoneal infection with cps1-1 induced a rapid influx of GR1+ neutrophils and two stages of GR1+CD68+ inflammatory monocyte infiltration into the site of inoculation. CD19+ B cells and CD3+ T cells steadily increase for 8 days after infection. CD8+ T cells were rapidly recruited to the site of infection and increased faster than CD4+ T cells. Surprisingly, cps1-1 infection induced high systemic levels of bioactive IL-12p70 and a very low level and transient systemic IFN-gamma. Furthermore, we show significant levels of these inflammatory cytokines were locally produced at the site of cps1-1 inoculation. These findings offer new insight into immunological mechanisms and local host responses to a non-replicating type I parasite infection associated with development of long-lasting immunity to Toxoplasma gondii.

Publication Types:
Research Support, N.I.H., Extramural

PMID: 19124750 [PubMed - in process]

How epigenomics contributes to the understanding of gene regulation in Toxoplasma gondii

J Eukaryot Microbiol. 2008 Nov-Dec;55(6):476-80.

How epigenomics contributes to the understanding of gene regulation in Toxoplasma gondii

Gissot M, Kim K.

Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461, USA.

How apicomplexan parasites regulate their gene expression is poorly understood. The complex life cycle of these parasites implies tight control of gene expression to orchestrate the appropriate expression pattern at the right moment. Recently, several studies have demonstrated the role of epigenetic mechanisms for control of coordinated expression of genes. In this review, we discuss the contribution of epigenomics to the understanding of gene regulation in Toxoplasma gondii. Studying the distribution of modified histones on the genome links chromatin modifications to gene expression or gene repression. In particular, coincident trimethylated lysine 4 on histone H3 (H3K4me3), acetylated lysine 9 on histone H3 (H3K9ac), and acetylated histone H4 (H4ac) mark promoters of actively transcribed genes. However, the presence of these modified histones at some non-expressed genes and other histone modifications at only a subset of active promoters implies the presence of other layers of regulation of chromatin structure in T. gondii. Epigenomics analysis provides a powerful tool to characterize the activation state of genomic loci of T. gondii and possibly of other Apicomplexa including Plasmodium or Cryptosporidium. Further, integration of epigenetic data with expression data and other genome-wide datasets facilitates refinement of genome annotation based upon experimental data.

Publication Types:
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

A pseudouridine synthase homologue is critical to cellular differentiation in Toxoplasma

Eukaryot Cell. 2009 Jan 5. [Epub ahead of print]

A pseudouridine synthase homologue is critical to cellular differentiation in Toxoplasma gondii

Anderson MZ, Brewer J, Singh U, Boothroyd JC.

Deptartment of Genetics, Stanford University School of Medicine, Stanford, CA, 94305-5120; Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, CA 94305-5124.

Toxoplasma gondii is a haploid protozoan parasite infecting about one in seven people in the United States. Key to the worldwide prevalence of T. gondii is its ability to establish a lifelong, chronic infection by evading the immune system and central to this is the developmental switch between the two asexual forms, tachyzoites and bradyzoites. A library of mutants defective in tachyzoite to bradyzoite differentiation (Tbd(-)) was created through insertional mutagenesis. This library contains mutants that, compared to wild-type, are between 20% and 74% as efficient at stage conversion. Two mutants, TBD5 and TBD8, were identified with disruptions in a gene encoding a putative pseudouridine synthase, PUS1. The disruption in TBD8 is in the 5'-end of the PUS1 gene and appears to produce a null allele with a 50% defect in differentiation. This is about the same switch efficiency as obtained with an engineered pus1 deletion mutant (Deltapus1). The insertion in TBD5 is within the PUS1 coding region and this appears to result in a more extreme phenotype of only approximately 10% switch efficiency. Complementation of TBD8 or the Deltapus1 mutant with the genomic PUS1 allele restored wild-type differentiation efficiency. Infection of mice with pus1 mutant strains results in increased mortality during the acute phase and higher cyst burdens during the chronic infection, demonstrating an aberrant differentiation phenotype in vivo due to PUS1 disruption. Our results suggest a surprising and important role for RNA modification in this biological process.

PMID: 19124578 [PubMed - as supplied by publisher]