Dear Colleagues,
Many of you are members of the American Society for Microbiology (ASM) or perhaps are interested in joining for 2009. We are writing to ask you to consider affiliating with Division AA (free-living, symbiotic, and parasitic protists). If you are already affiliated with a Division, you can choose AA as your secondary division. You can easily choose or switch your primary or secondary affiliation on the pulldown menu when you join or renew at http://estore.asm.org/Renew. The cost on line is only $59 for regular members and $39 for postdocs. By increasing our membership, division AA will be entitled to have more symposia at the General Meeting. Your membership also assists ASM's International Affairs which supports microbiology in developing countries, plus you get discounts when you publish in ASM journals! We'd appreciate if you let your colleagues in the lowest income countries know that for them joining ASM is free (click here)!
We want to encourage you to send your students to the ASM General Meeting, and we have been able to provide some travel funds for most students presenting posters. We also anticipate a greater presence of Global Health at the meeting next year, including colloquia on virulence, eradication, and lab research on global health, as well as some basic science on sexual reproduction and evolution of pathogens. We hope to see many of you there!
Thank you
Marilyn Parsons, Chair, Division AA
Kami Kim, Chair-elect
Tuesday, September 30, 2008
Sunday, September 28, 2008
Toxoplasma gondii gene expression is under the control of regulatory pathways acting through chromatin structure
Parasite. 2008 Sep;15(3):206-10
Toxoplasma gondii gene expression is under the control of regulatory pathways acting through chromatin structure
Bougdour A, Sautel CF, Cannella D, Braun L, Hakimi MA
Laboratoire Adaptation et Pathogénie des Micro-organismes, Université Joseph Fourier Grenoble 1, BP 170, F-38042 Grenoble cedex 9, France.
The activity state of a gene is determined by a complex regulatory network of co-acting factors affecting the structure of the chromatin into which the gene is embedded. While significant changes of the transcriptome occur during cell differentiation in apicomplexan parasites, basic mechanisms controlling gene expression are still unknown. Recent studies support and expand the concept of the chromatin environment being key factor for the control of transcriptional activity in these lower eukaryotes organisms. Here, we review recent advances in the field of epigenetic gene regulation in Toxoplasma gondii, the model apicomplexan.
PMID: 18814682 [PubMed - in process]
Toxoplasma gondii gene expression is under the control of regulatory pathways acting through chromatin structure
Bougdour A, Sautel CF, Cannella D, Braun L, Hakimi MA
Laboratoire Adaptation et Pathogénie des Micro-organismes, Université Joseph Fourier Grenoble 1, BP 170, F-38042 Grenoble cedex 9, France.
The activity state of a gene is determined by a complex regulatory network of co-acting factors affecting the structure of the chromatin into which the gene is embedded. While significant changes of the transcriptome occur during cell differentiation in apicomplexan parasites, basic mechanisms controlling gene expression are still unknown. Recent studies support and expand the concept of the chromatin environment being key factor for the control of transcriptional activity in these lower eukaryotes organisms. Here, we review recent advances in the field of epigenetic gene regulation in Toxoplasma gondii, the model apicomplexan.
PMID: 18814682 [PubMed - in process]
Toxoplasma gondii, "new" genotypes and virulence
Parasite. 2008 Sep;15(3):366-71
Toxoplasma gondii, "new" genotypes and virulence
Dardé ML
Faculty of Medicine, Parasitologie-Mycologie, EA3174, National Reference Center and Biological Resource Center for Toxoplasmosis, CHU Dupuytren, 2, av. Martin Luther King, F-87042 Limoges cedex, France. darde@unilim.fr
Toxoplasma gondii has been described as a parasite with a low genetic diversity and a clonal population structure. The three main clonal lineages designated as type I, II or III largely predominate in Europe and North America. But strains not related to these main lineages circulate, notably, in other continents. They possess a shuffled combination of alleles that typify the three clonal types and unique polymorphisms detected by multilocus analysis. The population structure of Toxoplasma in these continents is also characterized by a higher genetic diversity associated with a lower linkage desequilibrium suggesting a role for genetic exchange. Due to their genomic diversity, it is difficult to draw global conclusions about their virulence. However, most of them are virulent in mice at isolation. Several reports also suggest a higher pathogenicity in humans and an association with ocular toxoplasmosis or severe cases of acquired toxoplasmosis in immunocompetent patients.
PMID: 18814708 [PubMed - in process]
Toxoplasma gondii, "new" genotypes and virulence
Dardé ML
Faculty of Medicine, Parasitologie-Mycologie, EA3174, National Reference Center and Biological Resource Center for Toxoplasmosis, CHU Dupuytren, 2, av. Martin Luther King, F-87042 Limoges cedex, France. darde@unilim.fr
Toxoplasma gondii has been described as a parasite with a low genetic diversity and a clonal population structure. The three main clonal lineages designated as type I, II or III largely predominate in Europe and North America. But strains not related to these main lineages circulate, notably, in other continents. They possess a shuffled combination of alleles that typify the three clonal types and unique polymorphisms detected by multilocus analysis. The population structure of Toxoplasma in these continents is also characterized by a higher genetic diversity associated with a lower linkage desequilibrium suggesting a role for genetic exchange. Due to their genomic diversity, it is difficult to draw global conclusions about their virulence. However, most of them are virulent in mice at isolation. Several reports also suggest a higher pathogenicity in humans and an association with ocular toxoplasmosis or severe cases of acquired toxoplasmosis in immunocompetent patients.
PMID: 18814708 [PubMed - in process]
Mucosal immunity in Toxoplasma gondii infection
Parasite. 2008 Sep;15(3):389-95
Mucosal immunity in Toxoplasma gondii infection
Schulthess J, Fourreau D, Darche S, Meresse B, Kasper L, Cerf-Bensussan N, Buzoni-Gatel D
RPPI, Institut Pasteur-INRA, 28, rue du Dr Roux, 75724 Paris cedex 15, France.
Toxoplasma gondii is an intracellular parasite that frequently infects a large spectrum of warm-blooded animals. This parasite induces abortion and establishes both chronic and silent infections, particularly in the brain. Parasite penetration into the host activates a strong anti-parasite immune response. In the present paper, we will discuss the interplay between innate and adaptive immunity that occurs within the infected intestine to clear the parasite and to maintain intestinal homeostasis despite the exacerbation of an inflammatory immune response.
PMID: 18814712 [PubMed - in process]
Mucosal immunity in Toxoplasma gondii infection
Schulthess J, Fourreau D, Darche S, Meresse B, Kasper L, Cerf-Bensussan N, Buzoni-Gatel D
RPPI, Institut Pasteur-INRA, 28, rue du Dr Roux, 75724 Paris cedex 15, France.
Toxoplasma gondii is an intracellular parasite that frequently infects a large spectrum of warm-blooded animals. This parasite induces abortion and establishes both chronic and silent infections, particularly in the brain. Parasite penetration into the host activates a strong anti-parasite immune response. In the present paper, we will discuss the interplay between innate and adaptive immunity that occurs within the infected intestine to clear the parasite and to maintain intestinal homeostasis despite the exacerbation of an inflammatory immune response.
PMID: 18814712 [PubMed - in process]
Structural insights into microneme protein assembly reveal a new mode of EGF domain recognition
EMBO Rep. 2008 Sep 26. [Epub ahead of print]
Structural insights into microneme protein assembly reveal a new mode of EGF domain recognition
Sawmynaden K, Saouros S, Friedrich N, Marchant J, Simpson P, Bleijlevens B, Blackman MJ, Soldati-Favre D, Matthews S.
Division of Molecular Biosciences, Imperial College London, Exhibition Road, South Kensington Campus, London SW7 2AZ, UK.
The obligate intracellular parasite Toxoplasma gondii, a member of the phylum Apicomplexa that includes Plasmodium spp., is one of the most widespread parasites and the causative agent of toxoplasmosis. Adhesive complexes composed of microneme proteins (MICs) are secreted onto the parasite surface from intracellular stores and fulfil crucial roles in host-cell recognition, attachment and penetration. Here, we report the high-resolution solution structure of a complex between two crucial MICs, TgMIC6 and TgMIC1. Furthermore, we identify two analogous interaction sites within separate epidermal growth factor-like (EGF) domains of TgMIC6-EGF2 and EGF3-and confirm that both interactions are functional for the recognition of host cell receptor in the parasite, using immunofluorescence and invasion assays. The nature of this new mode of recognition of the EGF domain and its abundance in apicomplexan surface proteins suggest a more generalized means of constructing functional assemblies by using EGF domains with highly specific receptor-binding properties.
PMID: 18818666 [PubMed - as supplied by publisher]
Structural insights into microneme protein assembly reveal a new mode of EGF domain recognition
Sawmynaden K, Saouros S, Friedrich N, Marchant J, Simpson P, Bleijlevens B, Blackman MJ, Soldati-Favre D, Matthews S.
Division of Molecular Biosciences, Imperial College London, Exhibition Road, South Kensington Campus, London SW7 2AZ, UK.
The obligate intracellular parasite Toxoplasma gondii, a member of the phylum Apicomplexa that includes Plasmodium spp., is one of the most widespread parasites and the causative agent of toxoplasmosis. Adhesive complexes composed of microneme proteins (MICs) are secreted onto the parasite surface from intracellular stores and fulfil crucial roles in host-cell recognition, attachment and penetration. Here, we report the high-resolution solution structure of a complex between two crucial MICs, TgMIC6 and TgMIC1. Furthermore, we identify two analogous interaction sites within separate epidermal growth factor-like (EGF) domains of TgMIC6-EGF2 and EGF3-and confirm that both interactions are functional for the recognition of host cell receptor in the parasite, using immunofluorescence and invasion assays. The nature of this new mode of recognition of the EGF domain and its abundance in apicomplexan surface proteins suggest a more generalized means of constructing functional assemblies by using EGF domains with highly specific receptor-binding properties.
PMID: 18818666 [PubMed - as supplied by publisher]
Friday, September 26, 2008
GRA7 is phosphorylated upon invasion and forms an unexpected association with the rhoptry proteins ROP2 and ROP4
Infect Immun. 2008 Sep 22. [Epub ahead of print]
The Toxoplasma gondii dense granule protein GRA7 is phosphorylated upon invasion and forms an unexpected association with the rhoptry proteins ROP2 and ROP4
Dunn JD, Ravindran S, Kim SK, Boothroyd JC.
Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, CA 94305-5124.
The obligate intracellular parasite Toxoplasma gondii infects warm-blooded animals throughout the world and is an opportunistic pathogen of humans. As it invades a host cell, Toxoplasma forms a novel organelle, the parasitophorous vacuole, in which it resides during its intracellular development. The parasite modifies the parasitophorous vacuole and its host cell with numerous proteins delivered from rhoptries and dense granules, secretory organelles unique to the phylum Apicomplexa. For the majority of these proteins, little is known other than their localization. Here we show that the dense granule protein GRA7 is phosphorylated but only in the presence of host cells. Within 10 minutes of invasion, GRA7 is present in strand-like structures in the host cytosol that contain rhoptry proteins. GRA7 strands also contain GRA1 and GRA3. Independently of its phosphorylation state, GRA7 associates with the rhoptry proteins ROP2 and ROP4 in infected host cells. This is the first report of interactions between proteins secreted from rhoptries and dense granules.
PMID: 18809661 [PubMed - as supplied by publisher]
The Toxoplasma gondii dense granule protein GRA7 is phosphorylated upon invasion and forms an unexpected association with the rhoptry proteins ROP2 and ROP4
Dunn JD, Ravindran S, Kim SK, Boothroyd JC.
Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, CA 94305-5124.
The obligate intracellular parasite Toxoplasma gondii infects warm-blooded animals throughout the world and is an opportunistic pathogen of humans. As it invades a host cell, Toxoplasma forms a novel organelle, the parasitophorous vacuole, in which it resides during its intracellular development. The parasite modifies the parasitophorous vacuole and its host cell with numerous proteins delivered from rhoptries and dense granules, secretory organelles unique to the phylum Apicomplexa. For the majority of these proteins, little is known other than their localization. Here we show that the dense granule protein GRA7 is phosphorylated but only in the presence of host cells. Within 10 minutes of invasion, GRA7 is present in strand-like structures in the host cytosol that contain rhoptry proteins. GRA7 strands also contain GRA1 and GRA3. Independently of its phosphorylation state, GRA7 associates with the rhoptry proteins ROP2 and ROP4 in infected host cells. This is the first report of interactions between proteins secreted from rhoptries and dense granules.
PMID: 18809661 [PubMed - as supplied by publisher]
Susceptibility of pregnant women to toxoplasma infection--potential benefits for newborn screening
Ir Med J. 2008 Jul-Aug;101(7):220-1
Susceptibility of pregnant women to toxoplasma infection--potential benefits for newborn screening
Ferguson W, Mayne PD, Lennon B, Butler K, Cafferkey M.
The Rotunda Hospital, Parnell Street, Dublin. wferguson@rotunda.ie
Congenital toxoplasmosis (CT) arises as a result of new acquisition of Toxoplasma infection by a susceptible woman during pregnancy. Early detection of CT through neonatal screening programmes could optimize management and improve infant outcome. This study sought to estimate the prevalence of Toxoplasma susceptibility in pregnant women. As detection of Toxoplasma antibodies in neonatal blood reflects maternal exposure history, maternal antibody seroprevalence was determined using anonymized residual blood from newborn screening cards. A total of 20,252 cards were tested in 1 year. 4,991 (24.6%) cards tested positive for Toxoplasma antibody. Results were stratified by county. Toxoplasma antibody seroprevalence rates of 25% indicated that Toxoplasma infection is common in Ireland and that up to 75% of women remain susceptible to primary infection during pregnancy. This study aimed to a) determine the seroprevalence of Toxoplasma antibody in pregnant women, and hence b) estimate the risk for acquisition of primary toxoplasmosis in pregnancy in order to support an application to fund a pilot newborn screening programme.
Publication Types:
Research Support, Non-U.S. Gov't
PMID: 18807815 [PubMed - in process]
Susceptibility of pregnant women to toxoplasma infection--potential benefits for newborn screening
Ferguson W, Mayne PD, Lennon B, Butler K, Cafferkey M.
The Rotunda Hospital, Parnell Street, Dublin. wferguson@rotunda.ie
Congenital toxoplasmosis (CT) arises as a result of new acquisition of Toxoplasma infection by a susceptible woman during pregnancy. Early detection of CT through neonatal screening programmes could optimize management and improve infant outcome. This study sought to estimate the prevalence of Toxoplasma susceptibility in pregnant women. As detection of Toxoplasma antibodies in neonatal blood reflects maternal exposure history, maternal antibody seroprevalence was determined using anonymized residual blood from newborn screening cards. A total of 20,252 cards were tested in 1 year. 4,991 (24.6%) cards tested positive for Toxoplasma antibody. Results were stratified by county. Toxoplasma antibody seroprevalence rates of 25% indicated that Toxoplasma infection is common in Ireland and that up to 75% of women remain susceptible to primary infection during pregnancy. This study aimed to a) determine the seroprevalence of Toxoplasma antibody in pregnant women, and hence b) estimate the risk for acquisition of primary toxoplasmosis in pregnancy in order to support an application to fund a pilot newborn screening programme.
Publication Types:
Research Support, Non-U.S. Gov't
PMID: 18807815 [PubMed - in process]
Thursday, September 18, 2008
Three-Layer Sandwich Gel Electrophoresis: A Method of Salt Removal and Protein Concentration in Proteome Analysis
J Proteome Res. 2008 Sep 17. [Epub ahead of print]
Three-Layer Sandwich Gel Electrophoresis: A Method of Salt Removal and Protein Concentration in Proteome Analysis
Liu T, Martin AM, Sinai AP, Lynn BC.
Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055, University of Kentucky Mass Spectrometry Facility, Lexington, Kentucky 40505, and Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky, Lexington, Kentucky 40536-0298 bclynn2@uky.edu.
Sample preparation plays a critical role in successful proteomic applications. Features of electrospray mass spectrometry impose limits on the types of buffers, detergents and other reagents that can be used in sample preparation. Unfortunately, many of these mass spectrometry incompatible reagents significantly enhance protein recoveries from complex matrices. This problem prompted our search for a better cleanup protocol. Our data suggest that the Three-layer Sandwich Gel Electrophoresis (TSGE) protocol can solve this problem and provide near quantitative recovery of extremely low concentration proteins from harsh solutions, a feature not available from other cleanup protocols. The hallmark of the TSGE protocol is the combination of the properties of agarose gels (that serve as the matrix to immobilize the proteins of interest) with low- and high-percentage polyacrylamide gels (that serve as the concentration and sealing layers, respectively). By electrophoretically driving the proteins of interest from the agarose matrix into the concentration layer, the TSGE protocol simultaneously concentrates the sample in the concentration layer and provides an environment amenable to downstream buffer exchange and proteolytic digestion. In combination with 2D-LC-MS/MS, the TSGE protocol was evaluated in the analysis of a whole cell extract from the protozoan parasite Toxoplasma gondii. Comparison of our experimental proteomic results with in silico predictions from gene data indicated that TSGE did not bias the protein identification.
PMID: 18795766 [PubMed - as supplied by publisher]
Three-Layer Sandwich Gel Electrophoresis: A Method of Salt Removal and Protein Concentration in Proteome Analysis
Liu T, Martin AM, Sinai AP, Lynn BC.
Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055, University of Kentucky Mass Spectrometry Facility, Lexington, Kentucky 40505, and Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky, Lexington, Kentucky 40536-0298 bclynn2@uky.edu.
Sample preparation plays a critical role in successful proteomic applications. Features of electrospray mass spectrometry impose limits on the types of buffers, detergents and other reagents that can be used in sample preparation. Unfortunately, many of these mass spectrometry incompatible reagents significantly enhance protein recoveries from complex matrices. This problem prompted our search for a better cleanup protocol. Our data suggest that the Three-layer Sandwich Gel Electrophoresis (TSGE) protocol can solve this problem and provide near quantitative recovery of extremely low concentration proteins from harsh solutions, a feature not available from other cleanup protocols. The hallmark of the TSGE protocol is the combination of the properties of agarose gels (that serve as the matrix to immobilize the proteins of interest) with low- and high-percentage polyacrylamide gels (that serve as the concentration and sealing layers, respectively). By electrophoretically driving the proteins of interest from the agarose matrix into the concentration layer, the TSGE protocol simultaneously concentrates the sample in the concentration layer and provides an environment amenable to downstream buffer exchange and proteolytic digestion. In combination with 2D-LC-MS/MS, the TSGE protocol was evaluated in the analysis of a whole cell extract from the protozoan parasite Toxoplasma gondii. Comparison of our experimental proteomic results with in silico predictions from gene data indicated that TSGE did not bias the protein identification.
PMID: 18795766 [PubMed - as supplied by publisher]
Chemogenomics and parasitology: small molecules and cell-based assays to study infectious processes
Comb Chem High Throughput Screen. 2008 Sep;11(8):624-46
Chemogenomics and parasitology: small molecules and cell-based assays to study infectious processes
Muskavitch MA, Barteneva N, Gubbels MJ.
Department of Biology, Boston College, 355 Higgins Hall, 140 Commonwealth Avenue, Chestnut Hill, MA 02467, USA. gubbelsj@bc.edu.
Infectious diseases caused by protozoan parasites - malaria, sleeping sickness, leishmaniasis, Chagas' disease, toxoplasmosis - remain chronic problems for humanity. We lack vaccines and have limited drug options effective against protozoa. Research into anti-protozoan drugs has accelerated with improved in vitro cultivation methods, enhanced genetic accessibility, completed genome sequences for key protozoa, and increased prominence of protozoan diseases on the agendas of well-resourced public figures and foundations. Concurrent advances in high-throughput screening (HTS) technologies and availability of diverse small molecule libraries offer the promise of accelerated discovery of new drug targets and new drugs that will reduce disease burdens imposed on humanity by parasitic protozoa. We provide a status report on HTS technologies in hand and cell-based assays under development for biological investigations and drug discovery directed toward the three best-characterized parasitic protozoa: Trypanosoma brucei, Plasmodium falciparum, and Toxoplasma gondii. We emphasize cell growth assays and new insights into parasite cell biology speeding development of better cell-based assays, useful in primary screens for anti-protozoan drug leads and secondary screens to decipher mechanisms of action of leads identified in growth assays. Small molecules that interfere with specific aspects of protozoan biology, identified in such screens, will be valuable tools for dissecting parasite cell biology and developing anti-protozoan drugs. We discuss potential impacts on drug development of new consortia among academic, corporate, and public partners committed to discovery of new, effective anti-protozoan drugs.
PMID: 18795882 [PubMed - in process]
Chemogenomics and parasitology: small molecules and cell-based assays to study infectious processes
Muskavitch MA, Barteneva N, Gubbels MJ.
Department of Biology, Boston College, 355 Higgins Hall, 140 Commonwealth Avenue, Chestnut Hill, MA 02467, USA. gubbelsj@bc.edu.
Infectious diseases caused by protozoan parasites - malaria, sleeping sickness, leishmaniasis, Chagas' disease, toxoplasmosis - remain chronic problems for humanity. We lack vaccines and have limited drug options effective against protozoa. Research into anti-protozoan drugs has accelerated with improved in vitro cultivation methods, enhanced genetic accessibility, completed genome sequences for key protozoa, and increased prominence of protozoan diseases on the agendas of well-resourced public figures and foundations. Concurrent advances in high-throughput screening (HTS) technologies and availability of diverse small molecule libraries offer the promise of accelerated discovery of new drug targets and new drugs that will reduce disease burdens imposed on humanity by parasitic protozoa. We provide a status report on HTS technologies in hand and cell-based assays under development for biological investigations and drug discovery directed toward the three best-characterized parasitic protozoa: Trypanosoma brucei, Plasmodium falciparum, and Toxoplasma gondii. We emphasize cell growth assays and new insights into parasite cell biology speeding development of better cell-based assays, useful in primary screens for anti-protozoan drug leads and secondary screens to decipher mechanisms of action of leads identified in growth assays. Small molecules that interfere with specific aspects of protozoan biology, identified in such screens, will be valuable tools for dissecting parasite cell biology and developing anti-protozoan drugs. We discuss potential impacts on drug development of new consortia among academic, corporate, and public partners committed to discovery of new, effective anti-protozoan drugs.
PMID: 18795882 [PubMed - in process]
STAT6 signalling is important in CD8(+) T-cell activation and defence against Toxoplasma gondii infection in the brain
Immunology. 2008 Sep 13. [Epub ahead of print]
STAT6 signalling is important in CD8(+) T-cell activation and defence against Toxoplasma gondii infection in the brain
Jin D, Takamoto M, Hu T, Taki S, Sugane K.
Department of Infection and Host Defence, Division of Immunology and Infectious Diseases, Shinshu University Graduate School of Medicine, Matsumoto, Japan.
Signal transducer and activator of transcription (STAT) 6 is a molecule involved in interleukin (IL)-4 and -13 signalling. We investigated the role of STAT6 signalling in Toxoplasma gondii-infected mice using STAT6-deficient (STAT6(-/-)) and wild-type (WT) mice. A significantly larger number of cysts were recovered from the brain in STAT6(-/-) than in WT mice on days 28 and 56 post-infection. CD8(+) T cells in cerebrospinal fluid and spleen stimulated with T. gondii antigen produced higher levels of interferon (IFN)-gamma in WT than in STAT6(-/-) mice. CD8(+) T-cell function, estimated by expression of CD25 and cytotoxic activity, was lower in STAT6(-/-) than in WT mice. Transfer of CD8(+) but not CD4(+) T cells, purified from infected WT mice, into STAT6(-/-) mice successfully prevented formation of cysts in the brain. However, transfer of naïve CD8(+) T cells from WT into STAT6(-/-) mice did not show either activation of CD8(+) T cells or a decrease in the number of cysts in the brain. Transfer of splenic adherent cells from WT into STAT6(-/-) mice induced activation of CD8(+) T cells and decreased the number of cysts in the brain. Expression of CD86 on splenic dendritic cells and IL-12 p40 production were weaker in STAT6(-/-) than in WT mice after T. gondii infection. These results indicate that STAT6 signalling is important in CD8(+) T-cell activation, possibly through regulation of antigen-presenting cells, which could suppress T. gondii infection in the brain.
PMID: 18795973 [PubMed - as supplied by publisher]
STAT6 signalling is important in CD8(+) T-cell activation and defence against Toxoplasma gondii infection in the brain
Jin D, Takamoto M, Hu T, Taki S, Sugane K.
Department of Infection and Host Defence, Division of Immunology and Infectious Diseases, Shinshu University Graduate School of Medicine, Matsumoto, Japan.
Signal transducer and activator of transcription (STAT) 6 is a molecule involved in interleukin (IL)-4 and -13 signalling. We investigated the role of STAT6 signalling in Toxoplasma gondii-infected mice using STAT6-deficient (STAT6(-/-)) and wild-type (WT) mice. A significantly larger number of cysts were recovered from the brain in STAT6(-/-) than in WT mice on days 28 and 56 post-infection. CD8(+) T cells in cerebrospinal fluid and spleen stimulated with T. gondii antigen produced higher levels of interferon (IFN)-gamma in WT than in STAT6(-/-) mice. CD8(+) T-cell function, estimated by expression of CD25 and cytotoxic activity, was lower in STAT6(-/-) than in WT mice. Transfer of CD8(+) but not CD4(+) T cells, purified from infected WT mice, into STAT6(-/-) mice successfully prevented formation of cysts in the brain. However, transfer of naïve CD8(+) T cells from WT into STAT6(-/-) mice did not show either activation of CD8(+) T cells or a decrease in the number of cysts in the brain. Transfer of splenic adherent cells from WT into STAT6(-/-) mice induced activation of CD8(+) T cells and decreased the number of cysts in the brain. Expression of CD86 on splenic dendritic cells and IL-12 p40 production were weaker in STAT6(-/-) than in WT mice after T. gondii infection. These results indicate that STAT6 signalling is important in CD8(+) T-cell activation, possibly through regulation of antigen-presenting cells, which could suppress T. gondii infection in the brain.
PMID: 18795973 [PubMed - as supplied by publisher]
Reporter gene expression in cell culture stages and oocysts of Eimeria nieschulzi
Parasitol Res. 2008 Sep 17. [Epub ahead of print]
Reporter gene expression in cell culture stages and oocysts of Eimeria nieschulzi (Coccidia, Apicomplexa)
Kurth M, Entzeroth R.
Section Molecular Biotechnology, Technische Universität Dresden, 01062, Dresden, Germany, kurth.michael@googlemail.com.
The rat parasite Eimeria nieschulzi is a suitable model for transfection studies and was used as an additional model organism for the genus Eimeria. We describe the transfection of this apicomplexan parasites and the cultivation of transformed stages in cell culture and in vivo. The beta-galactosidase or yellow fluorescent protein was expressed in all parasitic stages up to the second merozoite generation in vitro under control of the heterologous promoter region of Eimeria tenella mic1 gene previously described for E. tenella transfection. Pyrimethamine resistant E. nieschulzi parasites were obtained in vitro after transfection with a plasmid encoding the Toxoplasma gondii dhfr/ts-m2m3 gene. Co-transfection experiments with an YFP-plasmid resulted in pyrimethamine resistant and fluorescent parasitic stages. Infection of rats with transfected E. nieschulzi sporozoites directed to expression of beta-galactosidase or YFP in oocysts. Co-transfection with YFP/DHFR-TS allowed selection of resistant parasites in vivo. Excreted transgenic oocysts showed arrangement of YFP expression which lead to questions about meiotic recombination frequency and mechanisms.
PMID: 18797926 [PubMed - as supplied by publisher]
Reporter gene expression in cell culture stages and oocysts of Eimeria nieschulzi (Coccidia, Apicomplexa)
Kurth M, Entzeroth R.
Section Molecular Biotechnology, Technische Universität Dresden, 01062, Dresden, Germany, kurth.michael@googlemail.com.
The rat parasite Eimeria nieschulzi is a suitable model for transfection studies and was used as an additional model organism for the genus Eimeria. We describe the transfection of this apicomplexan parasites and the cultivation of transformed stages in cell culture and in vivo. The beta-galactosidase or yellow fluorescent protein was expressed in all parasitic stages up to the second merozoite generation in vitro under control of the heterologous promoter region of Eimeria tenella mic1 gene previously described for E. tenella transfection. Pyrimethamine resistant E. nieschulzi parasites were obtained in vitro after transfection with a plasmid encoding the Toxoplasma gondii dhfr/ts-m2m3 gene. Co-transfection experiments with an YFP-plasmid resulted in pyrimethamine resistant and fluorescent parasitic stages. Infection of rats with transfected E. nieschulzi sporozoites directed to expression of beta-galactosidase or YFP in oocysts. Co-transfection with YFP/DHFR-TS allowed selection of resistant parasites in vivo. Excreted transgenic oocysts showed arrangement of YFP expression which lead to questions about meiotic recombination frequency and mechanisms.
PMID: 18797926 [PubMed - as supplied by publisher]
Sunday, September 14, 2008
Hijacking of Host Cellular Functions by the Apicomplexa
Annu Rev Microbiol. 2008 Oct 13;62:471-487
Hijacking of Host Cellular Functions by the Apicomplexa
Plattner F, Soldati-Favre D
Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva CMU, 1211 Geneva 4, Switzerland; email: Fabienne.Plattner@medecine.unige.ch , dominique.soldati-favre@medecine.unige.ch.
Intracellular pathogens such as viruses and bacteria subvert all the major cellular functions of their hosts. Targeted host processes include protein synthesis, membrane trafficking, modulation of gene expression, antigen presentation, and apoptosis. In recent years, it has become evident that protozoan pathogens, including members of the phylum Apicomplexa, also hijack their host cell's functions to access nutrients and to escape cellular defenses and immune responses. These obligate intracellular parasites provide superb illustrations of the subversion of host cell processes such as the recruitment and reorganization of host cell compartments without fusion around the parasitophorous vacuole of Toxoplasma gondii; the export of Plasmodium falciparum proteins on the surface of infected erythrocytes; and the induced transformation of the lymphocytes infected by Theileria parva, which leads to clonal extension.
PMID: 18785844 [PubMed - as supplied by publisher]
Hijacking of Host Cellular Functions by the Apicomplexa
Plattner F, Soldati-Favre D
Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva CMU, 1211 Geneva 4, Switzerland; email: Fabienne.Plattner@medecine.unige.ch , dominique.soldati-favre@medecine.unige.ch.
Intracellular pathogens such as viruses and bacteria subvert all the major cellular functions of their hosts. Targeted host processes include protein synthesis, membrane trafficking, modulation of gene expression, antigen presentation, and apoptosis. In recent years, it has become evident that protozoan pathogens, including members of the phylum Apicomplexa, also hijack their host cell's functions to access nutrients and to escape cellular defenses and immune responses. These obligate intracellular parasites provide superb illustrations of the subversion of host cell processes such as the recruitment and reorganization of host cell compartments without fusion around the parasitophorous vacuole of Toxoplasma gondii; the export of Plasmodium falciparum proteins on the surface of infected erythrocytes; and the induced transformation of the lymphocytes infected by Theileria parva, which leads to clonal extension.
PMID: 18785844 [PubMed - as supplied by publisher]
Toxoplasma gondii type-II NADH dehydrogenase TgNDH2-I is inhibited by 1-hydroxy-2-alkyl-4(1H)quinolones
Biochim Biophys Acta. 2008 Aug 22. [Epub ahead of print]
The Toxoplasma gondii type-II NADH dehydrogenase TgNDH2-I is inhibited by 1-hydroxy-2-alkyl-4(1H)quinolones
Lin SS, Kerscher S, Saleh A, Brandt U, Groß U, Bohne W.
Institute of Medical Microbiology, University of Göttingen, Kreuzbergring 57, Göttingen D-37075";, Germany.
The apicomplexan parasite Toxoplasma gondii does not possess complex I of the mitochondrial respiratory chain, but has two genes encoding rotenone-insensitive, non-proton pumping type-II NADH dehydrogenases (NDH2s). The absence of such "alternative" NADH dehydrogenases in the human host defines these enzymes as potential drug targets. TgNDH2-I and TgNDH2-II are constitutively expressed in tachyzoites and bradyzoites and are localized to the mitochondrion as shown by epitope tagging. Functional expression of TgNDH2-I in the yeast Yarrowia lipolytica as an internal enzyme, with the active site facing the mitochondrial matrix, permitted growth in the presence of the complex I inhibitor DQA. Bisubstrate kinetics of TgNDH2-I measured within Y. lipolytica mitochondrial membrane preparations were in accordance with a ping-pong mechanism. Using inhibition kinetics we demonstrate here that 1-hydroxy-2-alkyl-4(1)quinolones with long alkyl chains of C(12) (HDQ) and C(14) are high affinity inhibitors for TgNDH2-I, while compounds with shorter side chains (C(5) and C(6)) displayed significantly higher IC(50) values. The efficiency of the various quinolone derivatives to inhibit TgNDH2-I enzyme activity mirrors their inhibitory potency in vivo, suggesting that a long acyl site chain is critical for the inhibitory potential of these compounds.
PMID: 18786503 [PubMed - as supplied by publisher]
The Toxoplasma gondii type-II NADH dehydrogenase TgNDH2-I is inhibited by 1-hydroxy-2-alkyl-4(1H)quinolones
Lin SS, Kerscher S, Saleh A, Brandt U, Groß U, Bohne W.
Institute of Medical Microbiology, University of Göttingen, Kreuzbergring 57, Göttingen D-37075";, Germany.
The apicomplexan parasite Toxoplasma gondii does not possess complex I of the mitochondrial respiratory chain, but has two genes encoding rotenone-insensitive, non-proton pumping type-II NADH dehydrogenases (NDH2s). The absence of such "alternative" NADH dehydrogenases in the human host defines these enzymes as potential drug targets. TgNDH2-I and TgNDH2-II are constitutively expressed in tachyzoites and bradyzoites and are localized to the mitochondrion as shown by epitope tagging. Functional expression of TgNDH2-I in the yeast Yarrowia lipolytica as an internal enzyme, with the active site facing the mitochondrial matrix, permitted growth in the presence of the complex I inhibitor DQA. Bisubstrate kinetics of TgNDH2-I measured within Y. lipolytica mitochondrial membrane preparations were in accordance with a ping-pong mechanism. Using inhibition kinetics we demonstrate here that 1-hydroxy-2-alkyl-4(1)quinolones with long alkyl chains of C(12) (HDQ) and C(14) are high affinity inhibitors for TgNDH2-I, while compounds with shorter side chains (C(5) and C(6)) displayed significantly higher IC(50) values. The efficiency of the various quinolone derivatives to inhibit TgNDH2-I enzyme activity mirrors their inhibitory potency in vivo, suggesting that a long acyl site chain is critical for the inhibitory potential of these compounds.
PMID: 18786503 [PubMed - as supplied by publisher]
Friday, September 12, 2008
Evaluation of a recombinant MIC3 based latex agglutination test for the rapid serodiagnosis of Toxoplasma gondii infection in swines
Vet Parasitol. 2008 Aug 5. [Epub ahead of print]
Evaluation of a recombinant MIC3 based latex agglutination test for the rapid serodiagnosis of Toxoplasma gondii infection in swines
Jiang T, Gong D, Ma LA, Nie H, Zhou Y, Yao B, Zhao J.
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, Hubei Province, PR China; College of Animal Science, Yangtze University, Jingzhou 434025, Hubei Province, PR China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei Province, PR China.
The entire gene encoding microneme protein 3 (MIC3) from Toxoplasma gondii was cloned into the plasmid pGEX-KG and subsequently expressed in Escherichia coli as a glutathione-S-transferase (GST) fusion protein. The recombinant MIC3 (rMIC3) was purified and evaluated in a latex agglutination test (LAT) as the diagnostic antigen for the detection of antibodies to T. gondii in pig sera. The specificity, stability, and reproducibility of the test were examined. No agglutination was found when the sensitized latex beads were mixed with phosphate-buffered saline (PBS), borate-buffered saline (BBS), normal saline, and negative serum samples. There was no cross-reactivity with the standard positive sera of other pathogens. But intense agglutination occurred with T. gondii antibody positive serum samples. In our study, the coincidence rate of tested positive-sera of the LAT with rMIC3-sensitized latex particles and the ELISA with rSAG1 was up to 92.8%, T. gondii specific antibodies were detected by the LAT in all piglets that were experimentally infected with T. gondii tachyzoites from 8 to 42 days after infection. Our results indicated that the rMIC3 based latex agglutination test appears to be suitable for the detection of T. gondii antibodies at the early stage of infection.
PMID: 18783889 [PubMed - as supplied by publisher]
Evaluation of a recombinant MIC3 based latex agglutination test for the rapid serodiagnosis of Toxoplasma gondii infection in swines
Jiang T, Gong D, Ma LA, Nie H, Zhou Y, Yao B, Zhao J.
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, Hubei Province, PR China; College of Animal Science, Yangtze University, Jingzhou 434025, Hubei Province, PR China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei Province, PR China.
The entire gene encoding microneme protein 3 (MIC3) from Toxoplasma gondii was cloned into the plasmid pGEX-KG and subsequently expressed in Escherichia coli as a glutathione-S-transferase (GST) fusion protein. The recombinant MIC3 (rMIC3) was purified and evaluated in a latex agglutination test (LAT) as the diagnostic antigen for the detection of antibodies to T. gondii in pig sera. The specificity, stability, and reproducibility of the test were examined. No agglutination was found when the sensitized latex beads were mixed with phosphate-buffered saline (PBS), borate-buffered saline (BBS), normal saline, and negative serum samples. There was no cross-reactivity with the standard positive sera of other pathogens. But intense agglutination occurred with T. gondii antibody positive serum samples. In our study, the coincidence rate of tested positive-sera of the LAT with rMIC3-sensitized latex particles and the ELISA with rSAG1 was up to 92.8%, T. gondii specific antibodies were detected by the LAT in all piglets that were experimentally infected with T. gondii tachyzoites from 8 to 42 days after infection. Our results indicated that the rMIC3 based latex agglutination test appears to be suitable for the detection of T. gondii antibodies at the early stage of infection.
PMID: 18783889 [PubMed - as supplied by publisher]
Inactive and active states of the interferon-inducible resistance GTPase, Irga6, In Vivo
J Biol Chem. 2008 Sep 10. [Epub ahead of print]
Inactive and active states of the interferon-inducible resistance GTPase, Irga6, In Vivo
Papic N, Hunn JP, Pawlowski N, Zerrahn J, Howard JC.
Department of Cell Genetics, Institute for Genetics, University of Cologne, Cologne 50674.
Irga6, a myristoylated, interferon-inducible member of the Immunity-Related GTPase (IRG) family, contributes to disease resistance against Toxoplasma gondii in mice. Accumulation of Irga6 on the T. gondii parasitophorous vacuole membrane (PVM) is associated with vesiculation and ultimately disruption of the vacuolar membrane in a process that requires an intact GTP-binding domain. The role of the GTP binding domain of Irga6 in pathogen resistance is, however, unclear. We provide evidence that Irga6 in interferon-induced, uninfected cells is predominantly in a GDP-bound state that is maintained by other interferon-induced proteins. However Irga6 that accumulates on the PVM after Toxoplasma infection is in the GTP-bound form. We demonstrate that a monoclonal antibody, 10D7, specifically detects GTP-bound Irga6, and we show that the formation of the 10D7 epitope follows from a GTP-dependent conformational transition of the N-terminus of Irga6, anticipating an important role of the myristoyl group on Irga6 function in vivo.
PMID: 18784077 [PubMed - as supplied by publisher]
Inactive and active states of the interferon-inducible resistance GTPase, Irga6, In Vivo
Papic N, Hunn JP, Pawlowski N, Zerrahn J, Howard JC.
Department of Cell Genetics, Institute for Genetics, University of Cologne, Cologne 50674.
Irga6, a myristoylated, interferon-inducible member of the Immunity-Related GTPase (IRG) family, contributes to disease resistance against Toxoplasma gondii in mice. Accumulation of Irga6 on the T. gondii parasitophorous vacuole membrane (PVM) is associated with vesiculation and ultimately disruption of the vacuolar membrane in a process that requires an intact GTP-binding domain. The role of the GTP binding domain of Irga6 in pathogen resistance is, however, unclear. We provide evidence that Irga6 in interferon-induced, uninfected cells is predominantly in a GDP-bound state that is maintained by other interferon-induced proteins. However Irga6 that accumulates on the PVM after Toxoplasma infection is in the GTP-bound form. We demonstrate that a monoclonal antibody, 10D7, specifically detects GTP-bound Irga6, and we show that the formation of the 10D7 epitope follows from a GTP-dependent conformational transition of the N-terminus of Irga6, anticipating an important role of the myristoyl group on Irga6 function in vivo.
PMID: 18784077 [PubMed - as supplied by publisher]
Bilateral Toxoplasma Retinochoroiditis Simulating Cytomegalovirus Retinitis
Korean J Ophthalmol. 2008 Sep;22(3):197-200
Bilateral Toxoplasma Retinochoroiditis Simulating Cytomegalovirus Retinitis in an Allogeneic Bone Marrow Transplant Patient
Chung H, Kim JG, Choi SH, Lee SY, Yoon YH.
Department of Ophthalmology, Asan Medical Center, University of Ulsan, College of Medicine, Seoul, Korea.
A 36-year old female with acute myelogenous leukemia presented with a sudden decrease in vision one month following bone marrow transplantation (BMT). She had been taking multiple immunosuppressants to treat her recently-developed graft-versus-host-disease (GVHD). Visual acuity was 20/60 in her right eye and 20/25 in her left. Ophthalmic examination revealed mild inflammatory reaction in both the anterior chamber and the vitreous of both eyes, as well as densely opaque yellow-white infiltrates with well-demarcated borders in the posterior retina of both eyes. She was originally diagnosed as CMV retinitis, but treatment with ganciclovir failed to improve her ocular condition. Subsequent work-up, including serology and brain MRI, led to a diagnosis of combined ocular and cerebral toxoplasmosis. After 6 weeks of antiparasitic therapy, her retinal lesions became inactive and her cerebral lesions improved. Immunosuppressed patients with necrotizing retinochoroiditis should be suspected of having toxoplasmosis. Accurate differentiation between this condition and CMV, and early intervention with the appropriate treatment may be critical to preserve the best vision.
PMID: 18784451 [PubMed - as supplied by publisher]
Bilateral Toxoplasma Retinochoroiditis Simulating Cytomegalovirus Retinitis in an Allogeneic Bone Marrow Transplant Patient
Chung H, Kim JG, Choi SH, Lee SY, Yoon YH.
Department of Ophthalmology, Asan Medical Center, University of Ulsan, College of Medicine, Seoul, Korea.
A 36-year old female with acute myelogenous leukemia presented with a sudden decrease in vision one month following bone marrow transplantation (BMT). She had been taking multiple immunosuppressants to treat her recently-developed graft-versus-host-disease (GVHD). Visual acuity was 20/60 in her right eye and 20/25 in her left. Ophthalmic examination revealed mild inflammatory reaction in both the anterior chamber and the vitreous of both eyes, as well as densely opaque yellow-white infiltrates with well-demarcated borders in the posterior retina of both eyes. She was originally diagnosed as CMV retinitis, but treatment with ganciclovir failed to improve her ocular condition. Subsequent work-up, including serology and brain MRI, led to a diagnosis of combined ocular and cerebral toxoplasmosis. After 6 weeks of antiparasitic therapy, her retinal lesions became inactive and her cerebral lesions improved. Immunosuppressed patients with necrotizing retinochoroiditis should be suspected of having toxoplasmosis. Accurate differentiation between this condition and CMV, and early intervention with the appropriate treatment may be critical to preserve the best vision.
PMID: 18784451 [PubMed - as supplied by publisher]
Thursday, September 11, 2008
Secondary Mutations Correct Fitness Defects in Toxoplasma gondii with Dinitroaniline Resistance Mutations
Genetics. 2008 Sep 9. [Epub ahead of print]
Secondary Mutations Correct Fitness Defects in Toxoplasma gondii with Dinitroaniline Resistance Mutations
Ma CI, Tran J, Li C, Ganesan L, Wood D, Morrissette NS.
University of California, Irvine.
Dinitroanilines (oryzalin, trifluralin, ethafluralin) disrupt microtubules in protozoa but not vertebrate cells, causing selective death of intracellular Toxoplasma gondii parasites without affecting host cells. Parasites containing alpha1-tubulin point mutations are dinitroaniline resistant but show increased rates of aberrant replication relative to wild type parasites. T. gondii parasites bearing the F52Y mutation were previously demonstrated to spontaneously acquire two distinct intragenic mutations that decrease both resistance levels and replication defects. Parasites bearing the G142S mutation are largely dependent on oryzalin for viable growth in culture. We isolated 46 T. gondii lines that have suppressed microtubule defects associated with the G142S or the F52Y mutations by acquiring secondary mutations. These compensatory mutations were alpha1-tubulin pseudorevertants or extragenic suppressors (the majority alter the beta1-tubulin gene). Many secondary mutations were located in tubulin domains that suggest that they function by destabilizing microtubules. Most strikingly, we identified eight novel mutations that localize to an eight amino acid insert that stabilizes the alpha1-tubulin M loop, including one (P364R) that acts as a compensatory mutation in both F52Y and G142S lines. These lines have reduced dinitroaniline resistance but perform better than parental lines in competition assays, indicating that there is a tradeoff between resistance and replication fitness.
PMID: 18780736 [PubMed - as supplied by publisher]
Secondary Mutations Correct Fitness Defects in Toxoplasma gondii with Dinitroaniline Resistance Mutations
Ma CI, Tran J, Li C, Ganesan L, Wood D, Morrissette NS.
University of California, Irvine.
Dinitroanilines (oryzalin, trifluralin, ethafluralin) disrupt microtubules in protozoa but not vertebrate cells, causing selective death of intracellular Toxoplasma gondii parasites without affecting host cells. Parasites containing alpha1-tubulin point mutations are dinitroaniline resistant but show increased rates of aberrant replication relative to wild type parasites. T. gondii parasites bearing the F52Y mutation were previously demonstrated to spontaneously acquire two distinct intragenic mutations that decrease both resistance levels and replication defects. Parasites bearing the G142S mutation are largely dependent on oryzalin for viable growth in culture. We isolated 46 T. gondii lines that have suppressed microtubule defects associated with the G142S or the F52Y mutations by acquiring secondary mutations. These compensatory mutations were alpha1-tubulin pseudorevertants or extragenic suppressors (the majority alter the beta1-tubulin gene). Many secondary mutations were located in tubulin domains that suggest that they function by destabilizing microtubules. Most strikingly, we identified eight novel mutations that localize to an eight amino acid insert that stabilizes the alpha1-tubulin M loop, including one (P364R) that acts as a compensatory mutation in both F52Y and G142S lines. These lines have reduced dinitroaniline resistance but perform better than parental lines in competition assays, indicating that there is a tradeoff between resistance and replication fitness.
PMID: 18780736 [PubMed - as supplied by publisher]
Securing mucosal borders-migrant monocytes to the rescue
Cell Host Microbe. 2008 Sep 11;4(3):192-4
Securing mucosal borders-migrant monocytes to the rescue
Yap GS, Iwasaki A.
Center for Immunity and Inflammation, Department of Medicine, UMDNJ-New Jersey Medical School, Newark, NJ 07101, USA.
Gr1(+) "inflammatory" monocytes are emerging as key players in the innate immune response to infection and vaccination. In a recent issue of Immunity, Dunay et al. (2008) report a surprisingly crucial role for inflammatory monocytes in mucosal resistance to the parasitic agent Toxoplasma gondii.
PMID: 18779043 [PubMed - in process]
Securing mucosal borders-migrant monocytes to the rescue
Yap GS, Iwasaki A.
Center for Immunity and Inflammation, Department of Medicine, UMDNJ-New Jersey Medical School, Newark, NJ 07101, USA.
Gr1(+) "inflammatory" monocytes are emerging as key players in the innate immune response to infection and vaccination. In a recent issue of Immunity, Dunay et al. (2008) report a surprisingly crucial role for inflammatory monocytes in mucosal resistance to the parasitic agent Toxoplasma gondii.
PMID: 18779043 [PubMed - in process]
Tuesday, September 09, 2008
The immunobiology of the innate response to Toxoplasma
Int J Parasitol. 2008 Aug 22. [Epub ahead of print]
The immunobiology of the innate response to Toxoplasma gondii
Miller CM, Boulter NR, Ikin RJ, Smith NC.
Institute for the Biotechnology of Infectious Diseases, University of Technology, Sydney, P.O. Box 123, Broadway, NSW 2007, Australia.
Toxoplasma gondii is a unique intracellular parasite. It can infect a variety of cells in virtually all warm-blooded animals. It has a worldwide distribution and, overall, around one-third of people are seropositive for the parasite, with essentially the entire human population being at risk of infection. For most people, T. gondii causes asymptomatic infection but the parasite can cause serious disease in the immunocompromised and, if contracted for the first time during pregnancy, can cause spontaneous abortion or congenital defects, which have a substantial emotional, social and economic impact. Toxoplasma gondii provokes one of the most potent innate, pro-inflammatory responses of all infectious disease agents. It is also a supreme manipulator of the immune response so that innate immunity to T. gondii is a delicate balance between the parasite and its host involving a coordinated series of cellular interactions involving enterocytes, neutrophils, dendritic cells, macrophages and natural killer cells. Underpinning these interactions is the regulation of complex molecular reactions involving Toll-like receptors, activation of signalling pathways, cytokine production and activation of anti-microbial effector mechanisms including generation of reactive nitrogen and oxygen intermediates.
PMID: 18775432 [PubMed - as supplied by publisher]
The immunobiology of the innate response to Toxoplasma gondii
Miller CM, Boulter NR, Ikin RJ, Smith NC.
Institute for the Biotechnology of Infectious Diseases, University of Technology, Sydney, P.O. Box 123, Broadway, NSW 2007, Australia.
Toxoplasma gondii is a unique intracellular parasite. It can infect a variety of cells in virtually all warm-blooded animals. It has a worldwide distribution and, overall, around one-third of people are seropositive for the parasite, with essentially the entire human population being at risk of infection. For most people, T. gondii causes asymptomatic infection but the parasite can cause serious disease in the immunocompromised and, if contracted for the first time during pregnancy, can cause spontaneous abortion or congenital defects, which have a substantial emotional, social and economic impact. Toxoplasma gondii provokes one of the most potent innate, pro-inflammatory responses of all infectious disease agents. It is also a supreme manipulator of the immune response so that innate immunity to T. gondii is a delicate balance between the parasite and its host involving a coordinated series of cellular interactions involving enterocytes, neutrophils, dendritic cells, macrophages and natural killer cells. Underpinning these interactions is the regulation of complex molecular reactions involving Toll-like receptors, activation of signalling pathways, cytokine production and activation of anti-microbial effector mechanisms including generation of reactive nitrogen and oxygen intermediates.
PMID: 18775432 [PubMed - as supplied by publisher]
Saturday, September 06, 2008
N9-Substituted 2,4-Diaminoquinazolines: Synthesis and Biological Evaluation of Lipophilic Inhibitors
J Med Chem. 2008 Sep 5. [Epub ahead of print]
N9-Substituted 2,4-Diaminoquinazolines: Synthesis and Biological Evaluation of Lipophilic Inhibitors of Pneumocystis carinii and Toxoplasma gondii Dihydrofolate Reductase
Gangjee A, Adair OO, Pagley M, Queener SF.
Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, Pittsburgh, Pennsylvania 15282, Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana 46202 gangjee@duq.edu.
N9-substituted 2,4-diaminoquinazolines were synthesized and evaluated as inhibitors of Pneumocystis carinii (pc) and Toxoplasma gondii (tg) dihydrofolate reductase (DHFR). Reduction of commercially available 2,4-diamino-6-nitroquinazoline 14 with Raney nickel afforded 2,4,6-triaminoquinazoline 15. Reductive amination of 15 with the appropriate benzaldehydes or naphthaldehydes, followed by N9-alkylation, afforded the target compounds 5- 13. In the 2,5-dimethoxybenzylamino substituted quinazoline analogues, replacement of the N9-CH 3 group of 4 with the N9-C 2H 5 group of 8 resulted in a 9- and 8-fold increase in potency against pcDHFR and tgDHFR, respectively. The N9-C 2H 5 substituted compound 8 was highly potent, with IC 50 values of 9.9 and 3.7 nM against pcDHFR and tgDHFR, respectively. N9-propyl and N9-cyclopropyl methyl substitutions did not afford further increases in potency. This study indicates that the N9-ethyl substitution is optimum for inhibitory activity against pcDHFR and tgDHFR for the 2,4-diaminoquinazolines. Selectivity was unaffected by N9 substitution.
PMID: 18771252 [PubMed - as supplied by publisher]
N9-Substituted 2,4-Diaminoquinazolines: Synthesis and Biological Evaluation of Lipophilic Inhibitors of Pneumocystis carinii and Toxoplasma gondii Dihydrofolate Reductase
Gangjee A, Adair OO, Pagley M, Queener SF.
Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, Pittsburgh, Pennsylvania 15282, Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana 46202 gangjee@duq.edu.
N9-substituted 2,4-diaminoquinazolines were synthesized and evaluated as inhibitors of Pneumocystis carinii (pc) and Toxoplasma gondii (tg) dihydrofolate reductase (DHFR). Reduction of commercially available 2,4-diamino-6-nitroquinazoline 14 with Raney nickel afforded 2,4,6-triaminoquinazoline 15. Reductive amination of 15 with the appropriate benzaldehydes or naphthaldehydes, followed by N9-alkylation, afforded the target compounds 5- 13. In the 2,5-dimethoxybenzylamino substituted quinazoline analogues, replacement of the N9-CH 3 group of 4 with the N9-C 2H 5 group of 8 resulted in a 9- and 8-fold increase in potency against pcDHFR and tgDHFR, respectively. The N9-C 2H 5 substituted compound 8 was highly potent, with IC 50 values of 9.9 and 3.7 nM against pcDHFR and tgDHFR, respectively. N9-propyl and N9-cyclopropyl methyl substitutions did not afford further increases in potency. This study indicates that the N9-ethyl substitution is optimum for inhibitory activity against pcDHFR and tgDHFR for the 2,4-diaminoquinazolines. Selectivity was unaffected by N9 substitution.
PMID: 18771252 [PubMed - as supplied by publisher]
Regulatory interactions between IRG resistance GTPases in the cellular response to Toxoplasma
EMBO J. 2008 Sep 4. [Epub ahead of print]
Regulatory interactions between IRG resistance GTPases in the cellular response to Toxoplasma gondii
Hunn JP, Koenen-Waisman S, Papic N, Schroeder N, Pawlowski N, Lange R, Kaiser F, Zerrahn J, Martens S, Howard JC.
Department of Cell Genetics, Institute for Genetics, University of Cologne, Cologne, Germany.
Members of the immunity-related GTPase (IRG) family are interferon-inducible resistance factors against a broad spectrum of intracellular pathogens including Toxoplasma gondii. The molecular mechanisms governing the function and regulation of the IRG resistance system are largely unknown. We find that IRG proteins function in a system of direct, nucleotide-dependent regulatory interactions between family members. After interferon induction but before infection, the three members of the GMS subfamily of IRG proteins, Irgm1, Irgm2 and Irgm3, which possess an atypical nucleotide-binding site, regulate the intracellular positioning of the conventional GKS subfamily members, Irga6 and Irgb6. Following infection, the normal accumulation of Irga6 protein at the parasitophorous vacuole membrane (PVM) is nucleotide dependent and also depends on the presence of all three GMS proteins. We present evidence that an essential role of the GMS proteins in this response is control of the nucleotide-bound state of the GKS proteins, preventing their GTP-dependent activation before infection. Accumulation of IRG proteins at the PVM has previously been shown to be associated with a block in pathogen replication: our results relate for the first time the enzymatic properties of IRG proteins to their role in pathogen resistance.
PMID: 18772884 [PubMed - as supplied by publisher]
Regulatory interactions between IRG resistance GTPases in the cellular response to Toxoplasma gondii
Hunn JP, Koenen-Waisman S, Papic N, Schroeder N, Pawlowski N, Lange R, Kaiser F, Zerrahn J, Martens S, Howard JC.
Department of Cell Genetics, Institute for Genetics, University of Cologne, Cologne, Germany.
Members of the immunity-related GTPase (IRG) family are interferon-inducible resistance factors against a broad spectrum of intracellular pathogens including Toxoplasma gondii. The molecular mechanisms governing the function and regulation of the IRG resistance system are largely unknown. We find that IRG proteins function in a system of direct, nucleotide-dependent regulatory interactions between family members. After interferon induction but before infection, the three members of the GMS subfamily of IRG proteins, Irgm1, Irgm2 and Irgm3, which possess an atypical nucleotide-binding site, regulate the intracellular positioning of the conventional GKS subfamily members, Irga6 and Irgb6. Following infection, the normal accumulation of Irga6 protein at the parasitophorous vacuole membrane (PVM) is nucleotide dependent and also depends on the presence of all three GMS proteins. We present evidence that an essential role of the GMS proteins in this response is control of the nucleotide-bound state of the GKS proteins, preventing their GTP-dependent activation before infection. Accumulation of IRG proteins at the PVM has previously been shown to be associated with a block in pathogen replication: our results relate for the first time the enzymatic properties of IRG proteins to their role in pathogen resistance.
PMID: 18772884 [PubMed - as supplied by publisher]
Friday, September 05, 2008
IGTP is necessary for Toxoplasma Vacuolar Disruption and Induces Parasite Egression in IFN{gamma} Stimulated Astrocytes
Infect Immun. 2008 Sep 2. [Epub ahead of print]
IGTP is necessary for Toxoplasma Vacuolar Disruption and Induces Parasite Egression in IFN{gamma} Stimulated Astrocytes
Melzer T, Duffy A, Weiss LM, Halonen SK.
Dept. of Microbiology, Montana State University, Bozeman, MT. 59717 U.S.A.; Dept. of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO. 80523; Dept. of Pathology, Albert Einstein College of Medicine, Bronx, NY. 10460.
Toxoplasma gondii is a common central nervous system infection in individuals with immunocompromised immune systems, such as AIDS patients. Interferon-gamma (IFNgamma) is the main cytokine mediating protection against T. gondii. Our previous studies found IFNgamma significantly inhibits T. gondii in astrocytes via an IGTP dependent mechanism. The IGTP-dependent- IFNgamma stimulated inhibition is not understood but recent studies found IGTP induces disruption of the parasitophorous vacuole (PV) in macrophages. In the current study, we have further investigated the mechanism of IFNgamma inhibition and the role of IGTP in the vacuolar disruption in murine astrocytes. Vacuolar disruption was found to be dependent upon IGTP as PV disruption was not observed in IGTP deficient astrocytes (IGTP(-/-)) and PV disruption could be induced in IGTP(-/-) astrocytes transfected with IGTP. Live cell-imaging studies using GFP-IGTP found IGTP is delivered to the PV via host cell ER early after invasion and that IGTP condenses into vesicular-like structures on the vacuole, just prior to PV disruption, suggesting IGTP is involved in PV disruption. Intravacuolar movement of the parasite occurred just prior to PV disruption. In some instances IFNgamma induced parasite egression. Electron microscopy and immunofluorescence studies indicate host cell ER fuses with the PV prior to vacuolar disruption. Based upon these results, we postulate a mechanism by which ER/PV fusion is a crucial event in PV disruption. Fusion of the ER with the PV, releasing calcium into the vacuole may also be the mechanism by which intravacuolar parasite movement and IFNgamma induced parasite egression occurs.
PMID: 18765738 [PubMed - as supplied by publisher]
IGTP is necessary for Toxoplasma Vacuolar Disruption and Induces Parasite Egression in IFN{gamma} Stimulated Astrocytes
Melzer T, Duffy A, Weiss LM, Halonen SK.
Dept. of Microbiology, Montana State University, Bozeman, MT. 59717 U.S.A.; Dept. of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO. 80523; Dept. of Pathology, Albert Einstein College of Medicine, Bronx, NY. 10460.
Toxoplasma gondii is a common central nervous system infection in individuals with immunocompromised immune systems, such as AIDS patients. Interferon-gamma (IFNgamma) is the main cytokine mediating protection against T. gondii. Our previous studies found IFNgamma significantly inhibits T. gondii in astrocytes via an IGTP dependent mechanism. The IGTP-dependent- IFNgamma stimulated inhibition is not understood but recent studies found IGTP induces disruption of the parasitophorous vacuole (PV) in macrophages. In the current study, we have further investigated the mechanism of IFNgamma inhibition and the role of IGTP in the vacuolar disruption in murine astrocytes. Vacuolar disruption was found to be dependent upon IGTP as PV disruption was not observed in IGTP deficient astrocytes (IGTP(-/-)) and PV disruption could be induced in IGTP(-/-) astrocytes transfected with IGTP. Live cell-imaging studies using GFP-IGTP found IGTP is delivered to the PV via host cell ER early after invasion and that IGTP condenses into vesicular-like structures on the vacuole, just prior to PV disruption, suggesting IGTP is involved in PV disruption. Intravacuolar movement of the parasite occurred just prior to PV disruption. In some instances IFNgamma induced parasite egression. Electron microscopy and immunofluorescence studies indicate host cell ER fuses with the PV prior to vacuolar disruption. Based upon these results, we postulate a mechanism by which ER/PV fusion is a crucial event in PV disruption. Fusion of the ER with the PV, releasing calcium into the vacuole may also be the mechanism by which intravacuolar parasite movement and IFNgamma induced parasite egression occurs.
PMID: 18765738 [PubMed - as supplied by publisher]
Diacylglycerol kinases in immune cell function and self-tolerance
Immunol Rev. 2008 Aug;224(1):249-64.
Diacylglycerol kinases in immune cell function and self-tolerance
Zhong XP, Guo R, Zhou H, Liu C, Wan CK.
Department of Pediatrics, Duke University Medical Center, Durham, NC 27710, USA. zhong001@mc.duke.edu
Both diacylglycerol (DAG) and phosphatidic acid (PA) are important second messengers involved in signal transduction from many immune cell receptors and can be generated and metabolized through multiple mechanisms. Recent studies indicate that diacylglycerol kinases (DGKs), the enzymes that catalyze phosphorylation of DAG to produce PA, play critical roles in regulating the functions of multiple immune cell lineages. In T cells, two DGK isoforms, alpha and zeta, inhibit DAG-mediated signaling following T-cell receptor engagement and prevent T-cell hyperactivation. DGK alpha and zeta synergistically promote T-cell anergy and are critical for T-cell tolerance. In mast cells, DGKzeta plays differential roles in their activation by promoting degranulation but attenuating cytokine production following engagement of the high affinity receptor for immunoglobulin E. In dendritic cells and macrophages, DGKzeta positively regulates Toll-like receptor-induced proinflammatory cytokine production through its product PA and is critical for host defense against Toxoplasma gondii infection. These studies demonstrate pivotal roles of DGKs in regulating immune cell function by acting both as signal terminator and initiator.
Publication Types:
Research Support, Non-U.S. Gov't
PMID: 18759932 [PubMed - in process]
Diacylglycerol kinases in immune cell function and self-tolerance
Zhong XP, Guo R, Zhou H, Liu C, Wan CK.
Department of Pediatrics, Duke University Medical Center, Durham, NC 27710, USA. zhong001@mc.duke.edu
Both diacylglycerol (DAG) and phosphatidic acid (PA) are important second messengers involved in signal transduction from many immune cell receptors and can be generated and metabolized through multiple mechanisms. Recent studies indicate that diacylglycerol kinases (DGKs), the enzymes that catalyze phosphorylation of DAG to produce PA, play critical roles in regulating the functions of multiple immune cell lineages. In T cells, two DGK isoforms, alpha and zeta, inhibit DAG-mediated signaling following T-cell receptor engagement and prevent T-cell hyperactivation. DGK alpha and zeta synergistically promote T-cell anergy and are critical for T-cell tolerance. In mast cells, DGKzeta plays differential roles in their activation by promoting degranulation but attenuating cytokine production following engagement of the high affinity receptor for immunoglobulin E. In dendritic cells and macrophages, DGKzeta positively regulates Toll-like receptor-induced proinflammatory cytokine production through its product PA and is critical for host defense against Toxoplasma gondii infection. These studies demonstrate pivotal roles of DGKs in regulating immune cell function by acting both as signal terminator and initiator.
Publication Types:
Research Support, Non-U.S. Gov't
PMID: 18759932 [PubMed - in process]
Intervacuolar transport and unique topology of GRA14, a novel dense granule protein in Toxoplasma
Infect Immun. 2008 Sep 2. [Epub ahead of print]
Intervacuolar transport and unique topology of GRA14, a novel dense granule protein in Toxoplasma gondii
Rome ME, Beck JR, Turetzky JM, Webster P, Bradley PJ.
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles CA 90095-1489 USA; Ahmanson Advanced Electron Microscopy and Imaging Center, House Ear Institute, Los Angeles, CA 90057 USA.
Toxoplasma gondii is an obligate intracellular parasite that resides in the cytoplasm of its host in a unique membrane-bound vacuole known as the parasitophorous vacuole (PV). The membrane surrounding the parasite is remodeled by the dense granules, secretory organelles that release an array of proteins into the vacuole and to the parasitophorous vacuole membrane (PVM). Only a small portion of the protein constituents of the dense granules have been identified and little is known regarding their role in infection or how they are trafficked within the infected host cell. In this report, we identify a novel secreted dense granule protein, GRA14, and show that it is targeted to membranous structures within the vacuole known as the intravacuolar network and to the vacuolar membrane surrounding the parasite. We have disrupted GRA14 and exploited the knockout strain to show that GRA14 can be transferred between vacuoles in a co-infection experiment with wild type parasites. We also show that GRA14 has an unexpected topology in the PVM with its C-terminus facing the host cytoplasm and its N-terminus facing the vacuolar lumen. These findings have important implications for both the trafficking of GRA proteins to their ultimate destination and for expectations of functional domains of GRA proteins at the host-parasite interface.
PMID: 18765740 [PubMed - as supplied by publisher]
Intervacuolar transport and unique topology of GRA14, a novel dense granule protein in Toxoplasma gondii
Rome ME, Beck JR, Turetzky JM, Webster P, Bradley PJ.
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles CA 90095-1489 USA; Ahmanson Advanced Electron Microscopy and Imaging Center, House Ear Institute, Los Angeles, CA 90057 USA.
Toxoplasma gondii is an obligate intracellular parasite that resides in the cytoplasm of its host in a unique membrane-bound vacuole known as the parasitophorous vacuole (PV). The membrane surrounding the parasite is remodeled by the dense granules, secretory organelles that release an array of proteins into the vacuole and to the parasitophorous vacuole membrane (PVM). Only a small portion of the protein constituents of the dense granules have been identified and little is known regarding their role in infection or how they are trafficked within the infected host cell. In this report, we identify a novel secreted dense granule protein, GRA14, and show that it is targeted to membranous structures within the vacuole known as the intravacuolar network and to the vacuolar membrane surrounding the parasite. We have disrupted GRA14 and exploited the knockout strain to show that GRA14 can be transferred between vacuoles in a co-infection experiment with wild type parasites. We also show that GRA14 has an unexpected topology in the PVM with its C-terminus facing the host cytoplasm and its N-terminus facing the vacuolar lumen. These findings have important implications for both the trafficking of GRA proteins to their ultimate destination and for expectations of functional domains of GRA proteins at the host-parasite interface.
PMID: 18765740 [PubMed - as supplied by publisher]
Protective role of the RhD molecule against Toxoplasma-induced impairment of reaction times in women
Neuro Endocrinol Lett. 2008 Aug 2;29(4). [Epub ahead of print]
Neurophysiological effect of the Rh factor. Protective role of the RhD molecule against Toxoplasma-induced impairment of reaction times in women
Flegr J, Novotná M, Lindová J, Havlicek J.
Department of Philosophy and History of Natural Science, Charles University, Vinicná 7, Prague 128 44, Czech Republic. flegr@cesnet.cz.
The biological function of RhD protein, a major component of the Rh blood group system, is largely unknown. No phenotypic effect of RhD protein, except its role in hemolytic disease of newborns and protective role against Toxoplasma-induced impairment of reaction times in men, has been described. Here we searched for a protective effect of RhD positivity against Toxoplasma-induced prolongation of reaction times in a set of female students of the Faculty of Science. RhD-positive subjects have been confirmed to be less sensitive to the influence of latent toxoplasmosis on reaction times than Rh-negative subjects. While a protective role of RhD positivity has been demonstrated previously in four populations of men, the present study has shown a similar effect in 226 female students. Our results have also shown that the concentration of testosterone in saliva strongly influences (reduces) reaction times (especially in men) and therefore, this factor should be controlled in future reaction times studies.
PMID: 18766148 [PubMed - as supplied by publisher]
Neurophysiological effect of the Rh factor. Protective role of the RhD molecule against Toxoplasma-induced impairment of reaction times in women
Flegr J, Novotná M, Lindová J, Havlicek J.
Department of Philosophy and History of Natural Science, Charles University, Vinicná 7, Prague 128 44, Czech Republic. flegr@cesnet.cz.
The biological function of RhD protein, a major component of the Rh blood group system, is largely unknown. No phenotypic effect of RhD protein, except its role in hemolytic disease of newborns and protective role against Toxoplasma-induced impairment of reaction times in men, has been described. Here we searched for a protective effect of RhD positivity against Toxoplasma-induced prolongation of reaction times in a set of female students of the Faculty of Science. RhD-positive subjects have been confirmed to be less sensitive to the influence of latent toxoplasmosis on reaction times than Rh-negative subjects. While a protective role of RhD positivity has been demonstrated previously in four populations of men, the present study has shown a similar effect in 226 female students. Our results have also shown that the concentration of testosterone in saliva strongly influences (reduces) reaction times (especially in men) and therefore, this factor should be controlled in future reaction times studies.
PMID: 18766148 [PubMed - as supplied by publisher]
Thursday, September 04, 2008
Functional analysis of key nuclear trafficking components reveals an atypical Ran network required for parasite pathogenesis
Mol Microbiol. 2008 Aug 25. [Epub ahead of print]
Functional analysis of key nuclear trafficking components reveals an atypical Ran network required for parasite pathogenesis
Frankel MB, Knoll LJ.
Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, 1300 University Avenue, Madison, WI 53706, USA.
Protozoan parasites represent major public health challenges. Many aspects of their cell biology are distinct from their animal hosts, providing potential therapeutic targets. Toxoplasma gondii is a protozoan parasite that contains a divergent regulator of chromosome condensation 1 (TgRCC1) that is required for virulence and efficient nuclear trafficking. RCC1 proteins function as a guanine exchange factor for Ras-related nuclear protein (Ran), an abundant GTPase responsible for the majority of nucleocytoplasmic transport. Here we show that while there are dramatic differences from well-conserved RCC1 proteins, TgRCC1 associates with chromatin, interacts with Ran, and complements a mammalian temperature-sensitive RCC1 mutant cell line. During the investigation of TgRCC1, we observed several unprecedented phenotypes for TgRan, despite a high level of sequence conservation. The cellular distribution of TgRan is found throughout the parasite cell, whereas Ran in late branching eukaryotes is predominantly nuclear. Additionally, T. gondii tolerates at least low-level expression of dominant lethal Ran mutants. Wild type parasites expressing dominant negative TgRan grew similar to wild type in standard tissue culture conditions, but were attenuated in serum-starved host cells and mice. These growth characteristics paralleled the TgRCC1 mutant and highlight the importance of the nuclear transport pathway for virulence of eukaryotic pathogens.
PMID: 18761691 [PubMed - as supplied by publisher]
Functional analysis of key nuclear trafficking components reveals an atypical Ran network required for parasite pathogenesis
Frankel MB, Knoll LJ.
Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, 1300 University Avenue, Madison, WI 53706, USA.
Protozoan parasites represent major public health challenges. Many aspects of their cell biology are distinct from their animal hosts, providing potential therapeutic targets. Toxoplasma gondii is a protozoan parasite that contains a divergent regulator of chromosome condensation 1 (TgRCC1) that is required for virulence and efficient nuclear trafficking. RCC1 proteins function as a guanine exchange factor for Ras-related nuclear protein (Ran), an abundant GTPase responsible for the majority of nucleocytoplasmic transport. Here we show that while there are dramatic differences from well-conserved RCC1 proteins, TgRCC1 associates with chromatin, interacts with Ran, and complements a mammalian temperature-sensitive RCC1 mutant cell line. During the investigation of TgRCC1, we observed several unprecedented phenotypes for TgRan, despite a high level of sequence conservation. The cellular distribution of TgRan is found throughout the parasite cell, whereas Ran in late branching eukaryotes is predominantly nuclear. Additionally, T. gondii tolerates at least low-level expression of dominant lethal Ran mutants. Wild type parasites expressing dominant negative TgRan grew similar to wild type in standard tissue culture conditions, but were attenuated in serum-starved host cells and mice. These growth characteristics paralleled the TgRCC1 mutant and highlight the importance of the nuclear transport pathway for virulence of eukaryotic pathogens.
PMID: 18761691 [PubMed - as supplied by publisher]
Tuesday, September 02, 2008
Toxoplasma gondii Tic20 is essential for apicoplast protein import
Proc Natl Acad Sci U S A. 2008 Aug 29. [Epub ahead of print]
Toxoplasma gondii Tic20 is essential for apicoplast protein import
van Dooren GG, Tomova C, Agrawal S, Humbel BM, Striepen B.
Center for Tropical and Emerging Global Diseases and.
Apicomplexan parasites harbor a secondary plastid that has lost the ability to photosynthesize yet is essential for the parasite to multiply and cause disease. Bioinformatic analyses predict that 5-10% of all proteins encoded in the parasite genome function within this organelle. However, the mechanisms and molecules that mediate import of such large numbers of cargo proteins across the four membranes surrounding the plastid remain elusive. In this work, we identify a highly diverged member of the Tic20 protein family in Apicomplexa. We demonstrate that Tic20 of Toxoplasma gondii is an integral protein of the innermost plastid membrane. We engineer a conditional null-mutant and show that TgTic20 is essential for parasite growth. To characterize this mutant functionally, we develop several independent biochemical import assays to reveal that loss of TgTic20 leads to severe impairment of apicoplast protein import followed by organelle loss and parasite death. TgTic20 is the first experimentally validated protein import factor identified in apicoplasts. Our studies provide experimental evidence for a common evolutionary origin of import mechanisms across the innermost membranes of primary and secondary plastids.
PMID: 18757752 [PubMed - as supplied by publisher]
Toxoplasma gondii Tic20 is essential for apicoplast protein import
van Dooren GG, Tomova C, Agrawal S, Humbel BM, Striepen B.
Center for Tropical and Emerging Global Diseases and.
Apicomplexan parasites harbor a secondary plastid that has lost the ability to photosynthesize yet is essential for the parasite to multiply and cause disease. Bioinformatic analyses predict that 5-10% of all proteins encoded in the parasite genome function within this organelle. However, the mechanisms and molecules that mediate import of such large numbers of cargo proteins across the four membranes surrounding the plastid remain elusive. In this work, we identify a highly diverged member of the Tic20 protein family in Apicomplexa. We demonstrate that Tic20 of Toxoplasma gondii is an integral protein of the innermost plastid membrane. We engineer a conditional null-mutant and show that TgTic20 is essential for parasite growth. To characterize this mutant functionally, we develop several independent biochemical import assays to reveal that loss of TgTic20 leads to severe impairment of apicoplast protein import followed by organelle loss and parasite death. TgTic20 is the first experimentally validated protein import factor identified in apicoplasts. Our studies provide experimental evidence for a common evolutionary origin of import mechanisms across the innermost membranes of primary and secondary plastids.
PMID: 18757752 [PubMed - as supplied by publisher]
The small ubiquitin-like modifier (SUMO)-conjugating system of Toxoplasma
Int J Parasitol. 2008 Aug 12. [Epub ahead of print]
The small ubiquitin-like modifier (SUMO)-conjugating system of Toxoplasma gondii
Braun L, Cannella D, Pinheiro AM, Kieffer S, Belrhali H, Garin J, Hakimi MA.
Laboratoire Adaptation et Pathogénie des Micro-organismes, Université Joseph Fourier, France; CNRS UMR 5163 - ATIP+ group, France.
SUMOylation, the reversible covalent attachment of small ubiquitin-like modifier (SUMO) peptides has emerged as an important regulator of target protein function. Here we show, by characterization of the Toxoplasma gondii SUMO pathway, that the SUMO conjugation system operates in apicomplexan parasites. A gene encoding the SUMO tag was discovered as were genes encoding the various enzymes required for SUMO processing, ligation and release. Various SUMO conjugates were immuno-detected and by means of a global proteomic-based approach, we identified several T. gondii SUMOylated proteins that reveal many diverse cellular processes in which the modification plays a role. More specifically, SUMO conjugates were seen at the tachyzoite surface in response to signaling generated by host cell contact at the time of invasion. Also, under tissue culture conditions that stimulate bradyzoite differentiation (alkaline pH), we observed the conjugates at the parasitophorous vacuole membrane. The labeling was also at the surface of the mature cysts isolated from parasite-infected mouse brain. Overall, the SUMO conjugation system appears to be a complex and functionally heterogeneous pathway for protein modification in T. gondii with initial data indicating that it is likely to play a putative role in host cell invasion and cyst genesis.
PMID: 18761012 [PubMed - as supplied by publisher]
The small ubiquitin-like modifier (SUMO)-conjugating system of Toxoplasma gondii
Braun L, Cannella D, Pinheiro AM, Kieffer S, Belrhali H, Garin J, Hakimi MA.
Laboratoire Adaptation et Pathogénie des Micro-organismes, Université Joseph Fourier, France; CNRS UMR 5163 - ATIP+ group, France.
SUMOylation, the reversible covalent attachment of small ubiquitin-like modifier (SUMO) peptides has emerged as an important regulator of target protein function. Here we show, by characterization of the Toxoplasma gondii SUMO pathway, that the SUMO conjugation system operates in apicomplexan parasites. A gene encoding the SUMO tag was discovered as were genes encoding the various enzymes required for SUMO processing, ligation and release. Various SUMO conjugates were immuno-detected and by means of a global proteomic-based approach, we identified several T. gondii SUMOylated proteins that reveal many diverse cellular processes in which the modification plays a role. More specifically, SUMO conjugates were seen at the tachyzoite surface in response to signaling generated by host cell contact at the time of invasion. Also, under tissue culture conditions that stimulate bradyzoite differentiation (alkaline pH), we observed the conjugates at the parasitophorous vacuole membrane. The labeling was also at the surface of the mature cysts isolated from parasite-infected mouse brain. Overall, the SUMO conjugation system appears to be a complex and functionally heterogeneous pathway for protein modification in T. gondii with initial data indicating that it is likely to play a putative role in host cell invasion and cyst genesis.
PMID: 18761012 [PubMed - as supplied by publisher]
A recombinant pseudorabies virus expressing TgSAG1 protects against challenge with the virulent Toxoplasma
Microbes Infect. 2008 Aug 12. [Epub ahead of print]
A recombinant pseudorabies virus expressing TgSAG1 protects against challenge with the virulent Toxoplasma gondii RH strain and pseudorabies in BALB/c mice
Liu Q, Gao S, Jiang L, Shang L, Men J, Wang Z, Zhai Y, Xia Z, Hu R, Zhang X, Zhu XQ.
Laboratory of Parasitology, Veterinary Institute, Academy of Military Medical Sciences, Changchun 130062, Jilin Province, China.
The major immunodominant surface antigen 1 (TgSAG1) of invasive tachyzoites is a vaccine candidate antigen for Toxoplasma gondii. In this study, we developed a recombinant pseudorabies virus (PRV) expressing TgSAG1 (rPRV/SAG1) based on the PRV vaccine strain Bartha K-61 by homologous recombination, in which partial PK and gG genes were deleted. The growth assay of rPRV/SAG1 showed that the recombinant virus can replicate in vitro as efficiently as PRV Bartha K-61, demonstrating that insertion of the TgSAG1 gene in the PK and gG locus of PRV does not affect the replication of PRV. All mice vaccinated with rPRV/SAG1 developed a high level of specific antibody responses against T. gondii lysate antigen (TLA), a strong increase of the splenocyte proliferative response, and significant levels of IFN-gamma and IL-2 production. Furthermore, the immunization of mice with rPRV/SAG1 elicited strong cytotoxic T lymphocyte (CTL) responses in vitro. These results demonstrate that rPRV/SAG1 could induce significant humoral and cellular Th1 immune responses. Moreover, rPVR/SAG1 immunization induced partial protection (60%) against a lethal challenge with the highly virulent T. gondii RH strain, and neutralizing antibodies against PRV in a BALB/c mouse model. These results suggest that expression of protective antigens of T. gondii in PRV Bartha K-61 is a novel approach towards the development of a vaccine against both animal toxoplasmosis and pseudorabies.
PMID: 18761418 [PubMed - as supplied by publisher]
A recombinant pseudorabies virus expressing TgSAG1 protects against challenge with the virulent Toxoplasma gondii RH strain and pseudorabies in BALB/c mice
Liu Q, Gao S, Jiang L, Shang L, Men J, Wang Z, Zhai Y, Xia Z, Hu R, Zhang X, Zhu XQ.
Laboratory of Parasitology, Veterinary Institute, Academy of Military Medical Sciences, Changchun 130062, Jilin Province, China.
The major immunodominant surface antigen 1 (TgSAG1) of invasive tachyzoites is a vaccine candidate antigen for Toxoplasma gondii. In this study, we developed a recombinant pseudorabies virus (PRV) expressing TgSAG1 (rPRV/SAG1) based on the PRV vaccine strain Bartha K-61 by homologous recombination, in which partial PK and gG genes were deleted. The growth assay of rPRV/SAG1 showed that the recombinant virus can replicate in vitro as efficiently as PRV Bartha K-61, demonstrating that insertion of the TgSAG1 gene in the PK and gG locus of PRV does not affect the replication of PRV. All mice vaccinated with rPRV/SAG1 developed a high level of specific antibody responses against T. gondii lysate antigen (TLA), a strong increase of the splenocyte proliferative response, and significant levels of IFN-gamma and IL-2 production. Furthermore, the immunization of mice with rPRV/SAG1 elicited strong cytotoxic T lymphocyte (CTL) responses in vitro. These results demonstrate that rPRV/SAG1 could induce significant humoral and cellular Th1 immune responses. Moreover, rPVR/SAG1 immunization induced partial protection (60%) against a lethal challenge with the highly virulent T. gondii RH strain, and neutralizing antibodies against PRV in a BALB/c mouse model. These results suggest that expression of protective antigens of T. gondii in PRV Bartha K-61 is a novel approach towards the development of a vaccine against both animal toxoplasmosis and pseudorabies.
PMID: 18761418 [PubMed - as supplied by publisher]
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