Exp Parasitol. 2009 Apr 20. [Epub ahead of print]
Toxoplasma gondii: effects of Artemisia annua L. on susceptibility to infection in experimental models in vitro and in vivo
de Oliveira TS, Oliveira Silva DA, Rostkowska C, Béla SR, Ferro EA, Magalhães PM, Mineo JR.
Laboratory of Immunoparasitology, Institute of Biomedical Sciences, Federal University of Uberlândia, Av. Pará 1720, 38400-902 Uberlândia, MG, Brazil.
Considering that the treatment for toxoplasmosis is based on drugs that show limited efficacy due to their substantial side effects, the purpose of the present study was to evaluate the effects of Artemisia annua on in vitro and in vivoToxoplasma gondii infection. A. annua infusion was prepared from dried herb and tested in human foreskin fibroblasts (HFF) or mice that were infected with the parasite and compared with sulfadiazine treatment. For in vitro experiments, treatment was done on parasite before HFF infection or on cells previously infected with T. gondii and the inhibitory concentration (IC(50)) values for each treatment condition were determined. Viability of HFF cells in the presence of different concentrations of A. annua infusion and sulfadiazine was above 72%, even when the highest concentrations from both treatments were tested. Also, the treatment of T. gondii tachyzoites with A. annua infusion before infection in HFF cells showed a dose-response inhibitory curve that reached up to 75% of inhibition, similarly to the results observed when parasites were treated with sulfadiazine. In vivo experiments with a cystogenic T. gondii strain demonstrated an effective control of infection using A. annua infusion. In conclusion, our results indicate that A. annua infusion is useful to control T. gondii infection, due to its low toxicity and its inhibitory action directly against the parasite, resulting in a well tolerated therapeutic tool.
PMID: 19389400 [PubMed - as supplied by publisher]
Sunday, April 26, 2009
Wednesday, April 22, 2009
Aldolase Is Essential for Energy Production and Bridging Adhesin-Actin Cytoskeletal Interactions during Parasite Invasion of Host Cells
Cell Host Microbe. 2009 Apr 23;5(4):353-64
Aldolase Is Essential for Energy Production and Bridging Adhesin-Actin Cytoskeletal Interactions during Parasite Invasion of Host Cells
Starnes GL, Coincon M, Sygusch J, Sibley LD.
Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63130-1093, USA.
Apicomplexan parasites rely on actin-based motility to drive host cell invasion. Prior in vitro studies implicated aldolase, a tetrameric glycolytic enzyme, in coupling actin filaments to the parasite's surface adhesin microneme protein 2 (MIC2). Here, we test the essentiality of this interaction in host cell invasion. Based on in vitro studies and homology modeling, we generated a series of mutations in Toxoplasma gondii aldolase (TgALD1) that delineated MIC2 tail domain (MIC2t) binding function from its enzyme activity. We tested these mutants by complementing a conditional knockout of TgALD1. Mutations that affected glycolysis also reduced motility. Mutants only affecting binding to MIC2t had no motility phenotype, but were decreased in their efficiency of host cell invasion. Our studies demonstrate that aldolase is not only required for energy production but is also essential for efficient host cell invasion, based on its ability to bridge adhesin-cytoskeleton interactions in the parasite.
PMID: 19380114 [PubMed - in process]
Aldolase Is Essential for Energy Production and Bridging Adhesin-Actin Cytoskeletal Interactions during Parasite Invasion of Host Cells
Starnes GL, Coincon M, Sygusch J, Sibley LD.
Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63130-1093, USA.
Apicomplexan parasites rely on actin-based motility to drive host cell invasion. Prior in vitro studies implicated aldolase, a tetrameric glycolytic enzyme, in coupling actin filaments to the parasite's surface adhesin microneme protein 2 (MIC2). Here, we test the essentiality of this interaction in host cell invasion. Based on in vitro studies and homology modeling, we generated a series of mutations in Toxoplasma gondii aldolase (TgALD1) that delineated MIC2 tail domain (MIC2t) binding function from its enzyme activity. We tested these mutants by complementing a conditional knockout of TgALD1. Mutations that affected glycolysis also reduced motility. Mutants only affecting binding to MIC2t had no motility phenotype, but were decreased in their efficiency of host cell invasion. Our studies demonstrate that aldolase is not only required for energy production but is also essential for efficient host cell invasion, based on its ability to bridge adhesin-cytoskeleton interactions in the parasite.
PMID: 19380114 [PubMed - in process]
Transfection of Eimeria and Toxoplasma using heterologous regulatory sequences
Int J Parasitol. 2009 Apr 17. [Epub ahead of print]
Transfection of Eimeria and Toxoplasma using heterologous regulatory sequences
Zou J, Liu X, Shi T, Huang X, Wang H, Hao L, Yin G, Suo X.
Parasitology Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China.
Eimeriatenella and Toxoplasmagondii are Apicomplexan protozoa and share many similarities in biology and genomics. While the latter parasites are easily cultured in vitro and genetically manipulated, many Eimeria species are difficult to grow in vitro. We hypothesized that molecular tools for the genetic manipulation of T. gondii could be applied to the study of Eimeria parasites. Here we show that three different promoter sequences originating from E. tenella could function effectively not only in other species of the Eimeria genus (histone H4) but also in T. gondii (histone H4, actin and tubulin). Similarly, promoters of the "housekeeping" gene (tubulin) and differentially regulated gene (surface antigen gene, sag1) of T. gondii were effective in driving the expression of the yellow fluorescent protein (YFP) maker gene in E. tenella. The transfection efficiency with heterologous regulatory sequences was similar to that with homologous promoters; while the promoter strength of heterologous vectors is slightly weaker than the homologous vectors in both E. tenella and T. gondii. The results suggest that 5' regulatory sequences are functionally conserved not only among the Eimeria species, but also between T. gondii and E. tenella, and that T. gondii could be used as a novel transfection check system for Eimeria-rooted vectors, accelerating the development of reverse genetics in Eimeria spp.
PMID: 19379753 [PubMed - as supplied by publisher]
Transfection of Eimeria and Toxoplasma using heterologous regulatory sequences
Zou J, Liu X, Shi T, Huang X, Wang H, Hao L, Yin G, Suo X.
Parasitology Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, China.
Eimeriatenella and Toxoplasmagondii are Apicomplexan protozoa and share many similarities in biology and genomics. While the latter parasites are easily cultured in vitro and genetically manipulated, many Eimeria species are difficult to grow in vitro. We hypothesized that molecular tools for the genetic manipulation of T. gondii could be applied to the study of Eimeria parasites. Here we show that three different promoter sequences originating from E. tenella could function effectively not only in other species of the Eimeria genus (histone H4) but also in T. gondii (histone H4, actin and tubulin). Similarly, promoters of the "housekeeping" gene (tubulin) and differentially regulated gene (surface antigen gene, sag1) of T. gondii were effective in driving the expression of the yellow fluorescent protein (YFP) maker gene in E. tenella. The transfection efficiency with heterologous regulatory sequences was similar to that with homologous promoters; while the promoter strength of heterologous vectors is slightly weaker than the homologous vectors in both E. tenella and T. gondii. The results suggest that 5' regulatory sequences are functionally conserved not only among the Eimeria species, but also between T. gondii and E. tenella, and that T. gondii could be used as a novel transfection check system for Eimeria-rooted vectors, accelerating the development of reverse genetics in Eimeria spp.
PMID: 19379753 [PubMed - as supplied by publisher]
Particularities of Mitochondrial Structure in Parasitic Protozoa (Apicomplexa and Kinetoplastida)
Int J Biochem Cell Biol. 2009 Apr 17. [Epub ahead of print]
Particularities of Mitochondrial Structure in Parasitic Protozoa (Apicomplexa and Kinetoplastida)
de Souza W, Attias M, Rodrigues JC.
Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, CCS-Bloco G, Ilha do Fundão, 21941-902, Rio de Janeiro-RJ, Brasil; Diretoria de Programas, Instituto Nacional de Metrologia e Qualidade Industrial-INMETRO.
Without mitochondria, eukaryotic cells would depend entirely on anaerobic glycolysis for ATP generation. This also holds true for Protists, both free-living and parasitic. Parasitic Protists include agents of human and animal diseases that have a huge impact on world populations. In the phylum Apicomplexa, several species of Plasmodium cause malaria, whereas Toxoplasma gondii is a cosmopolite parasite found on all continents. Flagellates of the order Kinetoplastida include the genera Leishmania and Trypanosoma causative agents of human leishmaniasis and (depending on the species) African trypanosomiasis and Chagas disease. Although clearly distinct in many aspects, the members of these two groups bear a single and usually well developed mitochondrion. The single mitochondrion of Apicomplexa has a dense matrix and many cristae with a circular profile. The organelle is even more peculiar in the order kinetoplastida, exhibiting a condensed network of DNA at a specific position, always close to the flagellar basal body. This arrangement is known as Kinetoplast and the name of the Order derived from it. Kinetoplastids also bear glycosomes, peroxisomes that concentrate enzymes of the glycolytic cycle. Mitochondrial volume and activity is maximum when glycosomal is low and vice versa. In both Apicomplexa and Trypanosomatids, mitochondria show particularities that are absent in other eukaryotic organisms. These peculiar features make them an attractive target for therapeutic drugs for the diseases they cause.
PMID: 19379828 [PubMed - as supplied by publisher]
Particularities of Mitochondrial Structure in Parasitic Protozoa (Apicomplexa and Kinetoplastida)
de Souza W, Attias M, Rodrigues JC.
Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, CCS-Bloco G, Ilha do Fundão, 21941-902, Rio de Janeiro-RJ, Brasil; Diretoria de Programas, Instituto Nacional de Metrologia e Qualidade Industrial-INMETRO.
Without mitochondria, eukaryotic cells would depend entirely on anaerobic glycolysis for ATP generation. This also holds true for Protists, both free-living and parasitic. Parasitic Protists include agents of human and animal diseases that have a huge impact on world populations. In the phylum Apicomplexa, several species of Plasmodium cause malaria, whereas Toxoplasma gondii is a cosmopolite parasite found on all continents. Flagellates of the order Kinetoplastida include the genera Leishmania and Trypanosoma causative agents of human leishmaniasis and (depending on the species) African trypanosomiasis and Chagas disease. Although clearly distinct in many aspects, the members of these two groups bear a single and usually well developed mitochondrion. The single mitochondrion of Apicomplexa has a dense matrix and many cristae with a circular profile. The organelle is even more peculiar in the order kinetoplastida, exhibiting a condensed network of DNA at a specific position, always close to the flagellar basal body. This arrangement is known as Kinetoplast and the name of the Order derived from it. Kinetoplastids also bear glycosomes, peroxisomes that concentrate enzymes of the glycolytic cycle. Mitochondrial volume and activity is maximum when glycosomal is low and vice versa. In both Apicomplexa and Trypanosomatids, mitochondria show particularities that are absent in other eukaryotic organisms. These peculiar features make them an attractive target for therapeutic drugs for the diseases they cause.
PMID: 19379828 [PubMed - as supplied by publisher]
Saturday, April 18, 2009
Visualization of Toxoplasma gondii stage conversion by expression of stage-specific dual fluorescent proteins
Parasitology. 2009 Apr 16:1-10. [Epub ahead of print]
Visualization of Toxoplasma gondii stage conversion by expression of stage-specific dual fluorescent proteins
Unno A, Suzuki K, Batanova T, Cha SY, Jang HK, Kitoh K, Takashima Y.
Department of Veterinary Parasitological Diseases, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan.
SUMMARYTo recognize the stage conversion of Toxoplasma gondii between tachyzoite and bradyzoite in live host cells, a transgenic T. gondii line, which expressed stage-specific red and green fluorescence, was constructed. T. gondii PLK strain tachyzoites were stably transformed with genes encoding red fluorescent protein (DsRed Express) and green fluorescent protein (GFP) under the control of tachyzoite-specific SAG1 and bradyzoite-specific BAG1 promoters, respectively. The resulting transgenic parasite was designated PLK/DUAL. When PLK/DUAL was cultured in pH 7.0 medium, the PLK/DUAL zoites expressed red fluorescence, but no detectable levels of green fluorescence were observed. The PLK/DUAL zoites reacted with anti-SAG1 antibody, but not anti-BAG1 antiserum. When PLK/DUAL was cultured under high pH conditions, or in the presence of the p38 MAPK inhibitor SB202190, a small number of zoites expressed green fluorescence and were BAG1 positive. C57BL/6J mice were infected with PLK/DUAL tachyzoites. During the acute and reactivating phase, zoites expressed red fluorescence. However, green fluorescence was not detectable. By contrast, latent cysts expressed green fluorescence. The stage-specific dual fluorescence of PLK/DUAL facilitates identification of the parasitic stage in live cells, with the advantage that fixation or immunostaining is not required.
PMID: 19368740 [PubMed - as supplied by publisher]
Visualization of Toxoplasma gondii stage conversion by expression of stage-specific dual fluorescent proteins
Unno A, Suzuki K, Batanova T, Cha SY, Jang HK, Kitoh K, Takashima Y.
Department of Veterinary Parasitological Diseases, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan.
SUMMARYTo recognize the stage conversion of Toxoplasma gondii between tachyzoite and bradyzoite in live host cells, a transgenic T. gondii line, which expressed stage-specific red and green fluorescence, was constructed. T. gondii PLK strain tachyzoites were stably transformed with genes encoding red fluorescent protein (DsRed Express) and green fluorescent protein (GFP) under the control of tachyzoite-specific SAG1 and bradyzoite-specific BAG1 promoters, respectively. The resulting transgenic parasite was designated PLK/DUAL. When PLK/DUAL was cultured in pH 7.0 medium, the PLK/DUAL zoites expressed red fluorescence, but no detectable levels of green fluorescence were observed. The PLK/DUAL zoites reacted with anti-SAG1 antibody, but not anti-BAG1 antiserum. When PLK/DUAL was cultured under high pH conditions, or in the presence of the p38 MAPK inhibitor SB202190, a small number of zoites expressed green fluorescence and were BAG1 positive. C57BL/6J mice were infected with PLK/DUAL tachyzoites. During the acute and reactivating phase, zoites expressed red fluorescence. However, green fluorescence was not detectable. By contrast, latent cysts expressed green fluorescence. The stage-specific dual fluorescence of PLK/DUAL facilitates identification of the parasitic stage in live cells, with the advantage that fixation or immunostaining is not required.
PMID: 19368740 [PubMed - as supplied by publisher]
Induction of partial protection against infection with Toxoplasma gondii genotype II by DNA vaccination with recombinant chimeric tachyzoite antigens
Vaccine. 2009 Apr 21;27(18):2489-98. Epub 2009 Feb 24
Induction of partial protection against infection with Toxoplasma gondii genotype II by DNA vaccination with recombinant chimeric tachyzoite antigens
Rosenberg C, De Craeye S, Jongert E, Gargano N, Beghetto E, Del Porto P, Vorup-Jensen T, Petersen E.
Department of Infectious Diseases, Aarhus University Hospital, Skejby, Denmark; Biophysical Immunology Laboratory, Institute of Medical Microbiology and Immunology, University of Aarhus, DK-8000 Aarhus C, Denmark.
Infection with the obligate intracellular parasite Toxoplasma gondii is a significant source of parasitic infections worldwide. In adults, infections may often lead to severe retinochoroiditis. Infection of the foetus causes abortion or congenital pathology that may lead to neurological complications. Although several strategies have been suggested for making a vaccine, none is currently available. Here, we investigate the protection conferred by DNA vaccination with two constructs, pcEC2 (MIC2-MIC3-SAG1) and pcEC3 (GRA3-GRA7-M2AP), encoding chimeric proteins containing multiple antigenic sequences from T. gondii. After challenge with a T. gondii genotype II, but not a genotype III strain, a significant decrease in cerebral cyst load was found compared to the controls. The immune protection involved a cell-mediated immune response with the synthesis of the cytokines IFN-? and IL-10. In silico structure analysis and the expression profile of EC2, suggest an association between antigen stability, the degree of protein secondary structure and induction of cellular immune responses. Intracellular protein degradation is an important step in the pathway leading to presentation of antigenic peptides on Major Histocompatibility Complex molecules. We suggest that degradation of this chimeric protein may have contributed to the induction of a cellular immune response via enhanced presentation of antigenic peptides on Major Histocompatibility Complex class I molecules.
PMID: 19368791 [PubMed - in process]
Induction of partial protection against infection with Toxoplasma gondii genotype II by DNA vaccination with recombinant chimeric tachyzoite antigens
Rosenberg C, De Craeye S, Jongert E, Gargano N, Beghetto E, Del Porto P, Vorup-Jensen T, Petersen E.
Department of Infectious Diseases, Aarhus University Hospital, Skejby, Denmark; Biophysical Immunology Laboratory, Institute of Medical Microbiology and Immunology, University of Aarhus, DK-8000 Aarhus C, Denmark.
Infection with the obligate intracellular parasite Toxoplasma gondii is a significant source of parasitic infections worldwide. In adults, infections may often lead to severe retinochoroiditis. Infection of the foetus causes abortion or congenital pathology that may lead to neurological complications. Although several strategies have been suggested for making a vaccine, none is currently available. Here, we investigate the protection conferred by DNA vaccination with two constructs, pcEC2 (MIC2-MIC3-SAG1) and pcEC3 (GRA3-GRA7-M2AP), encoding chimeric proteins containing multiple antigenic sequences from T. gondii. After challenge with a T. gondii genotype II, but not a genotype III strain, a significant decrease in cerebral cyst load was found compared to the controls. The immune protection involved a cell-mediated immune response with the synthesis of the cytokines IFN-? and IL-10. In silico structure analysis and the expression profile of EC2, suggest an association between antigen stability, the degree of protein secondary structure and induction of cellular immune responses. Intracellular protein degradation is an important step in the pathway leading to presentation of antigenic peptides on Major Histocompatibility Complex molecules. We suggest that degradation of this chimeric protein may have contributed to the induction of a cellular immune response via enhanced presentation of antigenic peptides on Major Histocompatibility Complex class I molecules.
PMID: 19368791 [PubMed - in process]
Friday, April 17, 2009
Protective effect of an intranasal SAG1 and MIC4 DNA vaccine in mice
Exp Parasitol. 2009 Apr 11. [Epub ahead of print]
Toxoplasma gondii: Protective effect of an intranasal SAG1 and MIC4 DNA vaccine in mice
Wang H, He S, Yao Y, Cong H, Zhao H, Li T, Zhu XQ.
Department of Parasitology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan, Shandong Province, 250012 P.R. China.
Infections by the intracellular protozoan parasite Toxoplasma gondii are widely prevalent in humans and other animals which can cause severe or lethal toxoplasmosis. So the development of a more effective vaccine is needed urgently. A multiantigenic vaccine against toxoplasmosis was constructed in the present study, which contains two Toxoplasma gondii antigens, SAG1 and MIC4 on the basis of previous immunological and immunization studies. The eukaryotic plasmid pcDNA3.1-SAG1-MIC4, pcDNA3.1-SAG1, pcDNA3.1-MIC4 were constructed first, which can express surface protein SAG1 and microneme protein MIC4 from different stages of Toxoplasma gondii life cycle, and the expression ability of these DNA vaccine in HeLa cells were examined by Western blot. The efficacy of these plasmids with or without co-administration of a plasmid encoding cholera toxin A2/B as a genetic adjuvant by mucosal way to protect BALB/c mice against toxoplasmosis was evaluated. We found these vaccines were able to elicit a significant humoral and cellular immune response in vaccinated mice and they can increase survival rate and prolong the life of mice that were infected by T. gondii especially in the pcDNA3.1-SAG1-MIC4 group. Co-delivery of cholera toxin A2/B further enhanced the potency of multiantigenic DNA vaccine by intranasal route. These results encourage further research towards achieving vaccinal protection against the T. gondii in animals and humans.
PMID: 19366622 [PubMed - as supplied by publisher]
Toxoplasma gondii: Protective effect of an intranasal SAG1 and MIC4 DNA vaccine in mice
Wang H, He S, Yao Y, Cong H, Zhao H, Li T, Zhu XQ.
Department of Parasitology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan, Shandong Province, 250012 P.R. China.
Infections by the intracellular protozoan parasite Toxoplasma gondii are widely prevalent in humans and other animals which can cause severe or lethal toxoplasmosis. So the development of a more effective vaccine is needed urgently. A multiantigenic vaccine against toxoplasmosis was constructed in the present study, which contains two Toxoplasma gondii antigens, SAG1 and MIC4 on the basis of previous immunological and immunization studies. The eukaryotic plasmid pcDNA3.1-SAG1-MIC4, pcDNA3.1-SAG1, pcDNA3.1-MIC4 were constructed first, which can express surface protein SAG1 and microneme protein MIC4 from different stages of Toxoplasma gondii life cycle, and the expression ability of these DNA vaccine in HeLa cells were examined by Western blot. The efficacy of these plasmids with or without co-administration of a plasmid encoding cholera toxin A2/B as a genetic adjuvant by mucosal way to protect BALB/c mice against toxoplasmosis was evaluated. We found these vaccines were able to elicit a significant humoral and cellular immune response in vaccinated mice and they can increase survival rate and prolong the life of mice that were infected by T. gondii especially in the pcDNA3.1-SAG1-MIC4 group. Co-delivery of cholera toxin A2/B further enhanced the potency of multiantigenic DNA vaccine by intranasal route. These results encourage further research towards achieving vaccinal protection against the T. gondii in animals and humans.
PMID: 19366622 [PubMed - as supplied by publisher]
The evolution of the knowledge of cat and dog coccidia
Parasitology. 2009 Apr 14:1-7. [Epub ahead of print]
The evolution of the knowledge of cat and dog coccidia
Dubey JP.
United States Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Animal Parasitic Diseases Laboratory, Building 1001, Beltsville, MD 20705-2350, USA.
SUMMARYBefore the discovery of Toxoplasma gondii as a coccidium of the cat in 1970, cat and dog coccidia were classified in the genus Isospora and considered of little clinical or zoonotic significance. Since 1970, several new (Hammondia sp., Neospora sp.) and previously described species, including Sarcocystis, Besnoitia, and Cryptosporidium have been found as coccidians of cats and dogs with clinical and zoonotic significance. In the present paper I review salient features of the evolution of cat and dog coccidia.
PMID: 19366482 [PubMed - as supplied by publisher]
The evolution of the knowledge of cat and dog coccidia
Dubey JP.
United States Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Animal Parasitic Diseases Laboratory, Building 1001, Beltsville, MD 20705-2350, USA.
SUMMARYBefore the discovery of Toxoplasma gondii as a coccidium of the cat in 1970, cat and dog coccidia were classified in the genus Isospora and considered of little clinical or zoonotic significance. Since 1970, several new (Hammondia sp., Neospora sp.) and previously described species, including Sarcocystis, Besnoitia, and Cryptosporidium have been found as coccidians of cats and dogs with clinical and zoonotic significance. In the present paper I review salient features of the evolution of cat and dog coccidia.
PMID: 19366482 [PubMed - as supplied by publisher]
Thursday, April 16, 2009
Porosome: the secretory portal in cells
Biochemistry. 2009 Apr 13. [Epub ahead of print]
Porosome: the secretory portal in cells
Jena BP.
Porosomes are supramolecular, cup-shaped lipoprotein structures at the cell plasma membrane, where membrane-bound secretory vesicles dock and fuse to release intravesicular contents to the outside during cell secretion. The porosome openings to the outside ranges from 150 nm in diameter in acinar cells of the exocrine pancreas to 12 nm in neurons. In the past decade the composition of the porosome, its structure and dynamics at nm resolution in real time, and its functional reconstitution into artificial lipid membrane, have been demonstrated. Discovery of the universal secretory machinery in cells -the porosome, came as no surprise since porosome-like 'canaliculi' structures for secretion from human platelets, the secretory machinery in single-cell organisms like the secretion apparatus in bacteria and Toxoplasma gondii, and the contractile vacuole in paramecium, have been demonstrated. In this review, the discovery of the porosome complex and the molecular mechanism of its function, and how this information provides a new understanding of cell secretion are discussed.
PMID: 19364126 [PubMed - as supplied by publisher]
Porosome: the secretory portal in cells
Jena BP.
Porosomes are supramolecular, cup-shaped lipoprotein structures at the cell plasma membrane, where membrane-bound secretory vesicles dock and fuse to release intravesicular contents to the outside during cell secretion. The porosome openings to the outside ranges from 150 nm in diameter in acinar cells of the exocrine pancreas to 12 nm in neurons. In the past decade the composition of the porosome, its structure and dynamics at nm resolution in real time, and its functional reconstitution into artificial lipid membrane, have been demonstrated. Discovery of the universal secretory machinery in cells -the porosome, came as no surprise since porosome-like 'canaliculi' structures for secretion from human platelets, the secretory machinery in single-cell organisms like the secretion apparatus in bacteria and Toxoplasma gondii, and the contractile vacuole in paramecium, have been demonstrated. In this review, the discovery of the porosome complex and the molecular mechanism of its function, and how this information provides a new understanding of cell secretion are discussed.
PMID: 19364126 [PubMed - as supplied by publisher]
Friday, April 10, 2009
Identification and functional characterization of cis-regulatory elements in the apicomplexan parasite Toxoplasma
Genome Biol. 2009 Apr 7;10(4):R34. [Epub ahead of print]
Identification and functional characterization of cis-regulatory elements in the apicomplexan parasite Toxoplasma gondii
Mullapudi N, Joseph S, Kissinger JC.
ABSTRACT: BACKGROUND: Toxoplasma gondii is a member of the phylum Apicomplexa; a phylum that consists entirely of parasitic organisms that cause several diseases of veterinary and human importance. Fundamental mechanisms of gene regulation in this group of protistan parasites remain largely uncharacterized. Owing to their medical and veterinary importance, genome sequences are available for several apicomplexan parasites. Their genome sequences reveal an apparent paucity of known transcription factors and the absence of canonical cis-regulatory elements. We have approached the question of gene regulation from a sequence perspective by mining the genomic sequence data to identify putative cis-regulatory elements using a de novo approach. RESULTS: We have identified putative cis-regulatory elements present upstream of functionally related groups of genes and subsequently characterized the function of some of these conserved elements using reporter assays in the parasite. We show a sequence-specific role in gene-expression for 7 out of 8 identified elements. CONCLUSIONS: This work demonstrates the power of pure sequence analysis in the absence of expression data or a priori knowledge of regulatory elements in eukaryotic organisms with compact genomes.
PMID: 19351398 [PubMed - as supplied by publisher]
Identification and functional characterization of cis-regulatory elements in the apicomplexan parasite Toxoplasma gondii
Mullapudi N, Joseph S, Kissinger JC.
ABSTRACT: BACKGROUND: Toxoplasma gondii is a member of the phylum Apicomplexa; a phylum that consists entirely of parasitic organisms that cause several diseases of veterinary and human importance. Fundamental mechanisms of gene regulation in this group of protistan parasites remain largely uncharacterized. Owing to their medical and veterinary importance, genome sequences are available for several apicomplexan parasites. Their genome sequences reveal an apparent paucity of known transcription factors and the absence of canonical cis-regulatory elements. We have approached the question of gene regulation from a sequence perspective by mining the genomic sequence data to identify putative cis-regulatory elements using a de novo approach. RESULTS: We have identified putative cis-regulatory elements present upstream of functionally related groups of genes and subsequently characterized the function of some of these conserved elements using reporter assays in the parasite. We show a sequence-specific role in gene-expression for 7 out of 8 identified elements. CONCLUSIONS: This work demonstrates the power of pure sequence analysis in the absence of expression data or a priori knowledge of regulatory elements in eukaryotic organisms with compact genomes.
PMID: 19351398 [PubMed - as supplied by publisher]
Thursday, April 09, 2009
Drug inhibition of HDAC3 and epigenetic control of differentiation in Apicomplexa parasites
J Exp Med. 2009 Apr 6. [Epub ahead of print]
Drug inhibition of HDAC3 and epigenetic control of differentiation in Apicomplexa parasites
Bougdour A, Maubon D, Baldacci P, Ortet P, Bastien O, Bouillon A, Barale JC, Pelloux H, Ménard R, Hakimi MA.
UMR5163, Laboratoire Adaptation et Pathogénie des Micro-organismes, Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier Grenoble 1, BP 170, 38042 Grenoble, Cedex 09, France.
Plasmodium and Toxoplasma are parasites of major medical importance that belong to the Apicomplexa phylum of protozoa. These parasites transform into various stages during their life cycle and express a specific set of proteins at each stage. Although little is yet known of how gene expression is controlled in Apicomplexa, histone modifications, particularly acetylation, are emerging as key regulators of parasite differentiation and stage conversion. We investigated the anti-Apicomplexa effect of FR235222, a histone deacetylase inhibitor (HDACi). We show that FR235222 is active against a variety of Apicomplexa genera, including Plasmodium and Toxoplasma, and is more potent than other HDACi's such as trichostatin A and the clinically relevant compound pyrimethamine. We identify T. gondii HDAC3 (TgHDAC3) as the target of FR235222 in Toxoplasma tachyzoites and demonstrate the crucial role of the conserved and Apicomplexa HDAC-specific residue TgHDAC3 T99 in the inhibitory activity of the drug. We also show that FR235222 induces differentiation of the tachyzoite (replicative) into the bradyzoite (nonreplicative) stage. Additionally, via its anti-TgHDAC3 activity, FR235222 influences the expression of approximately 370 genes, a third of which are stage-specifically expressed. These results identify FR235222 as a potent HDACi of Apicomplexa, and establish HDAC3 as a central regulator of gene expression and stage conversion in Toxoplasma and, likely, other Apicomplexa.
PMID: 19349466 [PubMed - as supplied by publisher]
Drug inhibition of HDAC3 and epigenetic control of differentiation in Apicomplexa parasites
Bougdour A, Maubon D, Baldacci P, Ortet P, Bastien O, Bouillon A, Barale JC, Pelloux H, Ménard R, Hakimi MA.
UMR5163, Laboratoire Adaptation et Pathogénie des Micro-organismes, Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier Grenoble 1, BP 170, 38042 Grenoble, Cedex 09, France.
Plasmodium and Toxoplasma are parasites of major medical importance that belong to the Apicomplexa phylum of protozoa. These parasites transform into various stages during their life cycle and express a specific set of proteins at each stage. Although little is yet known of how gene expression is controlled in Apicomplexa, histone modifications, particularly acetylation, are emerging as key regulators of parasite differentiation and stage conversion. We investigated the anti-Apicomplexa effect of FR235222, a histone deacetylase inhibitor (HDACi). We show that FR235222 is active against a variety of Apicomplexa genera, including Plasmodium and Toxoplasma, and is more potent than other HDACi's such as trichostatin A and the clinically relevant compound pyrimethamine. We identify T. gondii HDAC3 (TgHDAC3) as the target of FR235222 in Toxoplasma tachyzoites and demonstrate the crucial role of the conserved and Apicomplexa HDAC-specific residue TgHDAC3 T99 in the inhibitory activity of the drug. We also show that FR235222 induces differentiation of the tachyzoite (replicative) into the bradyzoite (nonreplicative) stage. Additionally, via its anti-TgHDAC3 activity, FR235222 influences the expression of approximately 370 genes, a third of which are stage-specifically expressed. These results identify FR235222 as a potent HDACi of Apicomplexa, and establish HDAC3 as a central regulator of gene expression and stage conversion in Toxoplasma and, likely, other Apicomplexa.
PMID: 19349466 [PubMed - as supplied by publisher]
The Ins and Outs of Nuclear Trafficking: Unusual Aspects in Apicomplexans Parasites
DNA Cell Biol. 2009 Apr 6. [Epub ahead of print]
The Ins and Outs of Nuclear Trafficking: Unusual Aspects in Apicomplexans Parasites
Frankel MB, Knoll LJ.
Department of Medical Microbiology and Immunology, University of Wisconsin-Madison , Madison, Wisconsin.
Apicomplexa is a phylum within the kingdom Protista that contains some of the most significant threats to public health. One of the members of this phylum, Toxoplasma gondii, is amenable to molecular genetic analyses allowing for the identification of factors critical for colonization and disease. A pathway found to be important for T. gondii pathogenesis is the Ran network of nuclear trafficking. Bioinformatics analysis of apicomplexan genomes shows that while Ran is well conserved, the key regulators of Ran-Regulator of Chromosome Condensation 1 and Ran GTPase activating protein-are either highly divergent or absent. Likewise, several import and export receptor molecules that are crucial for nuclear transport are either not present or have experienced genetic drift such that they are no longer recognizable by bioinformatics tools. In this minireview we describe the basics of nuclear trafficking and compare components within apicomplexans to defined systems in humans and yeast. A detailed analysis of the nuclear trafficking network in these eukaryotes is required to understand how this potentially unique cellular biological pathway contributes to host-parasite interactions.
PMID: 19348590 [PubMed - as supplied by publisher]
The Ins and Outs of Nuclear Trafficking: Unusual Aspects in Apicomplexans Parasites
Frankel MB, Knoll LJ.
Department of Medical Microbiology and Immunology, University of Wisconsin-Madison , Madison, Wisconsin.
Apicomplexa is a phylum within the kingdom Protista that contains some of the most significant threats to public health. One of the members of this phylum, Toxoplasma gondii, is amenable to molecular genetic analyses allowing for the identification of factors critical for colonization and disease. A pathway found to be important for T. gondii pathogenesis is the Ran network of nuclear trafficking. Bioinformatics analysis of apicomplexan genomes shows that while Ran is well conserved, the key regulators of Ran-Regulator of Chromosome Condensation 1 and Ran GTPase activating protein-are either highly divergent or absent. Likewise, several import and export receptor molecules that are crucial for nuclear transport are either not present or have experienced genetic drift such that they are no longer recognizable by bioinformatics tools. In this minireview we describe the basics of nuclear trafficking and compare components within apicomplexans to defined systems in humans and yeast. A detailed analysis of the nuclear trafficking network in these eukaryotes is required to understand how this potentially unique cellular biological pathway contributes to host-parasite interactions.
PMID: 19348590 [PubMed - as supplied by publisher]
Sunday, April 05, 2009
Apicomplexan Parasites Co-Opt Host Calpains to Facilitate Their Escape from Infected Cells
Science. 2009 Apr 2. [Epub ahead of print]
Apicomplexan Parasites Co-Opt Host Calpains to Facilitate Their Escape from Infected Cells
Chandramohanadas R, Davis PH, Beiting DP, Harbut MB, Darling C, Velmourougane G, Lee MY, Greer PA, Roos DS, Greenbaum DC.
Department of Pharmacology, University of Pennsylvania, Philadelphia, PA 19104, USA.
Apicomplexan parasites, including Plasmodium falciparum and Toxoplasma gondii (the causative agents of malaria and toxoplasmosis, respectively) are responsible for significant morbidity and mortality worldwide. These pathogenic protozoa replicate within an intracellular vacuole inside infected host cells, from which they must escape to initiate a new lytic cycle. Integrating cell biological, pharmacological and genetic approaches, we provide evidence that both Plasmodium and Toxoplasma hijack host cell calpain proteases to facilitate parasite egress. Immunodepletion or inhibition of calpain-1 in hypotonically lysed and resealed erythrocytes prevented the escape of P. falciparum parasites, which was restored by adding purified calpain-1. Similarly, efficient egress of T. gondii from mammalian fibroblasts was blocked by either siRNA-mediated suppression or genetic deletion of calpain activity, and could be restored by genetic complementation.
PMID: 19342550 [PubMed - as supplied by publisher]
Apicomplexan Parasites Co-Opt Host Calpains to Facilitate Their Escape from Infected Cells
Chandramohanadas R, Davis PH, Beiting DP, Harbut MB, Darling C, Velmourougane G, Lee MY, Greer PA, Roos DS, Greenbaum DC.
Department of Pharmacology, University of Pennsylvania, Philadelphia, PA 19104, USA.
Apicomplexan parasites, including Plasmodium falciparum and Toxoplasma gondii (the causative agents of malaria and toxoplasmosis, respectively) are responsible for significant morbidity and mortality worldwide. These pathogenic protozoa replicate within an intracellular vacuole inside infected host cells, from which they must escape to initiate a new lytic cycle. Integrating cell biological, pharmacological and genetic approaches, we provide evidence that both Plasmodium and Toxoplasma hijack host cell calpain proteases to facilitate parasite egress. Immunodepletion or inhibition of calpain-1 in hypotonically lysed and resealed erythrocytes prevented the escape of P. falciparum parasites, which was restored by adding purified calpain-1. Similarly, efficient egress of T. gondii from mammalian fibroblasts was blocked by either siRNA-mediated suppression or genetic deletion of calpain activity, and could be restored by genetic complementation.
PMID: 19342550 [PubMed - as supplied by publisher]
Thursday, April 02, 2009
Severe acquired toxoplasmosis caused by wild cycle of Toxoplasma gondii, French Guiana
Emerg Infect Dis. 2009 Apr;15(4):656-8
Severe acquired toxoplasmosis caused by wild cycle of Toxoplasma gondii, French Guiana
Carme B, Demar M, Ajzenberg D, Dardé ML.
Université des Antilles et de la Guyane, Cayenne, French Guiana.
From 1998 through 2006, 44 cases of severe primary toxoplasmosis were observed in French Guiana in immunocompetent adults. Toxoplasma gondii isolates exhibited an atypical multilocus genotype. Severe disease in humans may result from poor host adaptation to neotropical zoonotic strains of T. gondii circulating in a forest-based cycle.
PMID: 19331765 [PubMed - in process]
Severe acquired toxoplasmosis caused by wild cycle of Toxoplasma gondii, French Guiana
Carme B, Demar M, Ajzenberg D, Dardé ML.
Université des Antilles et de la Guyane, Cayenne, French Guiana.
From 1998 through 2006, 44 cases of severe primary toxoplasmosis were observed in French Guiana in immunocompetent adults. Toxoplasma gondii isolates exhibited an atypical multilocus genotype. Severe disease in humans may result from poor host adaptation to neotropical zoonotic strains of T. gondii circulating in a forest-based cycle.
PMID: 19331765 [PubMed - in process]
Sunday, March 29, 2009
Structural studies of PNP from Toxoplasma gondii
Int J Bioinform Res Appl. 2009;5(2):154-62.
Structural studies of PNP from Toxoplasma gondii
Vivan AL, Caceres RA, Basso LA, Santos DS; Walter F. de Azevedo Jr.
Genetics and Molecular Biology, Federal University of Rio Grande do Sul, Av. Bento Goncalves, 9500, Porto Alegre, RS, 91501-970 Brazil.
Toxoplasmosis is a chronic infection that affects approximately 30% of the human population and is caused by Toxoplasma gondii. Determination of the three dimensional structure of PNP from T. gondii could provide new insights into the purine binding site and sub-strate binding, and could be used for future rational design of new drugs against toxoplasmosis. This work describes the molecular model for three dimensional structure of PNP from T.gondii using, as a template, PNP from Plasmodium falciparum. Molecular dynamics showed that this model is stable during a trajectory of 3 ns.
PMID: 19324601 [PubMed - in process]
Structural studies of PNP from Toxoplasma gondii
Vivan AL, Caceres RA, Basso LA, Santos DS; Walter F. de Azevedo Jr.
Genetics and Molecular Biology, Federal University of Rio Grande do Sul, Av. Bento Goncalves, 9500, Porto Alegre, RS, 91501-970 Brazil.
Toxoplasmosis is a chronic infection that affects approximately 30% of the human population and is caused by Toxoplasma gondii. Determination of the three dimensional structure of PNP from T. gondii could provide new insights into the purine binding site and sub-strate binding, and could be used for future rational design of new drugs against toxoplasmosis. This work describes the molecular model for three dimensional structure of PNP from T.gondii using, as a template, PNP from Plasmodium falciparum. Molecular dynamics showed that this model is stable during a trajectory of 3 ns.
PMID: 19324601 [PubMed - in process]
Waterborne toxoplasmosis - recent developments
Exp Parasitol. 2009 Mar 23. [Epub ahead of print]
Waterborne toxoplasmosis - recent developments
Jones JL, Dubey JP.
Division of Parasitic Diseases, National Center for Zoonotic, Vectorborne and Enteric Diseases, Coordinating Center for Infectious Diseases, Centers for Disease Control and Prevention, 4770 Buford Highway, MS: F22, Chamblee, Georgia 30341,USA.
Humans become infected with Toxoplasma gondii mainly by ingesting uncooked meat containing viable tissue cysts or by ingesting food or water contaminated with oocysts from the feces of infected cats. Circumstantial evidence suggests that oocyst-induced infections in humans are clinically more severe than tissue cyst-acquired infections. Until recently, waterborne transmission of T. gondii was considered uncommon, but a large human outbreak linked to contamination of a municipal water reservoir in Canada by wild felids and the widespread infection of marine mammals in the U.S.A. provided reasons to question this view. The present paper examines the possible importance of T. gondii transmission by water.
PMID: 19324041 [PubMed - as supplied by publisher]
Waterborne toxoplasmosis - recent developments
Jones JL, Dubey JP.
Division of Parasitic Diseases, National Center for Zoonotic, Vectorborne and Enteric Diseases, Coordinating Center for Infectious Diseases, Centers for Disease Control and Prevention, 4770 Buford Highway, MS: F22, Chamblee, Georgia 30341,USA.
Humans become infected with Toxoplasma gondii mainly by ingesting uncooked meat containing viable tissue cysts or by ingesting food or water contaminated with oocysts from the feces of infected cats. Circumstantial evidence suggests that oocyst-induced infections in humans are clinically more severe than tissue cyst-acquired infections. Until recently, waterborne transmission of T. gondii was considered uncommon, but a large human outbreak linked to contamination of a municipal water reservoir in Canada by wild felids and the widespread infection of marine mammals in the U.S.A. provided reasons to question this view. The present paper examines the possible importance of T. gondii transmission by water.
PMID: 19324041 [PubMed - as supplied by publisher]
Saturday, March 28, 2009
In vitro effect of TNF-alpha and IFN-gamma in retinal cell infection with Toxoplasma
Invest Ophthalmol Vis Sci. 2009 Apr;50(4):1754-60
In vitro effect of TNF-alpha and IFN-gamma in retinal cell infection with Toxoplasma gondii
Delair E, Creuzet C, Dupouy-Camet J, Roisin MP.
Université Paris Descartes, Faculté de Medicine, Hôpital Cochin, Service d'ophtalmologie, Paris, France. emmanuelle.delair@cch.aphp.fr
PURPOSE: Toxoplasma gondii is an intracellular protozoan parasite and the most common cause of infectious uveitis. This study was conducted to evaluate the in vitro effect of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma in rat retinal cells infected with T. gondii. METHODS: Rat retinal cells, retinal pigment epithelial (RPE) cells, and retinal Müller glial (RMG) cells were in vitro infected with T. gondii RH strain tachyzoites. Cultured cells were stimulated with various concentrations of TNF-alpha and IFN-gamma. The effect of TNF-alpha and IFN-gamma in T. gondii invasion and replication between retinal cells was determined through two different methods: measuring [(3)H]-uracil incorporation and counting infected cells by microscopic examination. RESULTS: Infection by T. gondii was lesser within RPE cells than within RMG cells. IFN-gamma significantly inhibits [(3)H]-uracil incorporation in RMG and RPE cells (respectively, 35%, 83%, and 87% inhibition at 0.1, 1, and 10 ng/mL for RMG cells and 0%, 30%, and 75% for RPE cells). TNF-alpha significantly inhibits [(3)H]-uracil incorporation in RPE cells (23% and 38% inhibition at 1 and 10 ng/mL), but not in RMG cells. These results were confirmed by confocal microscopic data. The percentage of infected cells decreased from 20% to 7% after IFN-gamma stimulation. CONCLUSIONS: Both cytokines IFN-gamma and TNF-alpha inhibited T. gondii replication in the RPE cells, whereas only IFN-gamma had an anti-Toxoplasma activity within the RMG cells. The differences in cytokine response may be the reason that RPE cells are less efficiently infected by T. gondii than are RMG cells.
PMID: 19321794 [PubMed - in process]
In vitro effect of TNF-alpha and IFN-gamma in retinal cell infection with Toxoplasma gondii
Delair E, Creuzet C, Dupouy-Camet J, Roisin MP.
Université Paris Descartes, Faculté de Medicine, Hôpital Cochin, Service d'ophtalmologie, Paris, France. emmanuelle.delair@cch.aphp.fr
PURPOSE: Toxoplasma gondii is an intracellular protozoan parasite and the most common cause of infectious uveitis. This study was conducted to evaluate the in vitro effect of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma in rat retinal cells infected with T. gondii. METHODS: Rat retinal cells, retinal pigment epithelial (RPE) cells, and retinal Müller glial (RMG) cells were in vitro infected with T. gondii RH strain tachyzoites. Cultured cells were stimulated with various concentrations of TNF-alpha and IFN-gamma. The effect of TNF-alpha and IFN-gamma in T. gondii invasion and replication between retinal cells was determined through two different methods: measuring [(3)H]-uracil incorporation and counting infected cells by microscopic examination. RESULTS: Infection by T. gondii was lesser within RPE cells than within RMG cells. IFN-gamma significantly inhibits [(3)H]-uracil incorporation in RMG and RPE cells (respectively, 35%, 83%, and 87% inhibition at 0.1, 1, and 10 ng/mL for RMG cells and 0%, 30%, and 75% for RPE cells). TNF-alpha significantly inhibits [(3)H]-uracil incorporation in RPE cells (23% and 38% inhibition at 1 and 10 ng/mL), but not in RMG cells. These results were confirmed by confocal microscopic data. The percentage of infected cells decreased from 20% to 7% after IFN-gamma stimulation. CONCLUSIONS: Both cytokines IFN-gamma and TNF-alpha inhibited T. gondii replication in the RPE cells, whereas only IFN-gamma had an anti-Toxoplasma activity within the RMG cells. The differences in cytokine response may be the reason that RPE cells are less efficiently infected by T. gondii than are RMG cells.
PMID: 19321794 [PubMed - in process]
Thursday, March 26, 2009
MIC6 associates with aldolase in host cell invasion by Toxoplasma
Parasitol Res. 2009 Mar 24. [Epub ahead of print]
MIC6 associates with aldolase in host cell invasion by Toxoplasma gondii
Zheng B, He A, Gan M, Li Z, He H, Zhan X.
Department of Parasitology, Zhongshan School of Medicine, SunYat-sen University, Guangzhou, 510080, People's Republic of China.
The transmembrane microneme protein MIC6 and its partner MIC1, MIC4 comprise an adhesive complex that play important roles in host cell attachment by the obligate intracellular parasite Toxoplasma gondii. Successful penetration of host cells by T. gondii depends on coordinated interactions between MICs complex and the parasite's cytoskeleton. We have identified that the carboxy-terminal cytoplasmic domain (C domain) of MIC6 interacts with aldolase and the parasite cytoskeleton. Our finding uncovers new features regarding MIC6-aldolase interactions in host cell invasion by T. gondii.
PMID: 19308454 [PubMed - as supplied by publisher]
MIC6 associates with aldolase in host cell invasion by Toxoplasma gondii
Zheng B, He A, Gan M, Li Z, He H, Zhan X.
Department of Parasitology, Zhongshan School of Medicine, SunYat-sen University, Guangzhou, 510080, People's Republic of China.
The transmembrane microneme protein MIC6 and its partner MIC1, MIC4 comprise an adhesive complex that play important roles in host cell attachment by the obligate intracellular parasite Toxoplasma gondii. Successful penetration of host cells by T. gondii depends on coordinated interactions between MICs complex and the parasite's cytoskeleton. We have identified that the carboxy-terminal cytoplasmic domain (C domain) of MIC6 interacts with aldolase and the parasite cytoskeleton. Our finding uncovers new features regarding MIC6-aldolase interactions in host cell invasion by T. gondii.
PMID: 19308454 [PubMed - as supplied by publisher]
Detection of Toxoplasma gondii in cerebrospinal fluid from AIDS patients by nested PCR and rapid identification of type I allele at B1 gene by RFLP
Exp Parasitol. 2009 Mar 21. [Epub ahead of print]
Detection of Toxoplasma gondii in cerebrospinal fluid from AIDS patients by nested PCR and rapid identification of type I allele at B1 gene by RFLP analysis
Alfonso Y, Fraga J, Jiménez N, Fonseca C, Dorta-Contreras AJ, Cox R, Capó V, Bandera F, Pomier O, Ginorio D.
Parasitology Department. Institute of Tropical Medicine Pedro Kourí . PO Box 601. Marianao 13. Ciudad de La Habana. Cuba.
Highly active antiretroviral therapy (HAART) has decreased the incidence of opportunistic infections in the central nervous system (CNS) in AIDS patients. However, toxoplasmic encephalitis (TE) still represents the most common cerebral mass lesion in patients infected with human immunodeficiency virus (HIV). The aim of this study was to evaluate nested PCR-B1 using cerebrospinal fluid (CSF) to detect Toxoplasma gondii DNA for the diagnosis of TE. A total of 114 samples were evaluated, and 33/44 samples from patients with TE were positive by PCR (sensitivity 75%), demonstrating the diagnostic usefulness of PCR technique. PCR B1 products were analyzed by restriction fragment length polymorphism (RFLP) in 30 samples. Only type I allele at B1 was identified in these samples according banding patterns. This is the first report of evaluation of S1-AS1/S2-AS2 set of primers in more than 100 clinical samples as well as the first genotyping study of T. gondii in Cuba.
PMID: 19318095 [PubMed - as supplied by publisher]
Detection of Toxoplasma gondii in cerebrospinal fluid from AIDS patients by nested PCR and rapid identification of type I allele at B1 gene by RFLP analysis
Alfonso Y, Fraga J, Jiménez N, Fonseca C, Dorta-Contreras AJ, Cox R, Capó V, Bandera F, Pomier O, Ginorio D.
Parasitology Department. Institute of Tropical Medicine Pedro Kourí . PO Box 601. Marianao 13. Ciudad de La Habana. Cuba.
Highly active antiretroviral therapy (HAART) has decreased the incidence of opportunistic infections in the central nervous system (CNS) in AIDS patients. However, toxoplasmic encephalitis (TE) still represents the most common cerebral mass lesion in patients infected with human immunodeficiency virus (HIV). The aim of this study was to evaluate nested PCR-B1 using cerebrospinal fluid (CSF) to detect Toxoplasma gondii DNA for the diagnosis of TE. A total of 114 samples were evaluated, and 33/44 samples from patients with TE were positive by PCR (sensitivity 75%), demonstrating the diagnostic usefulness of PCR technique. PCR B1 products were analyzed by restriction fragment length polymorphism (RFLP) in 30 samples. Only type I allele at B1 was identified in these samples according banding patterns. This is the first report of evaluation of S1-AS1/S2-AS2 set of primers in more than 100 clinical samples as well as the first genotyping study of T. gondii in Cuba.
PMID: 19318095 [PubMed - as supplied by publisher]
Laboratory diagnosis of Toxoplasma gondii infection
Int J Med Sci. 2009;6(3):135-6. Epub 2009 Mar 19
Laboratory diagnosis of Toxoplasma gondii infection
Calderaro A, Peruzzi S, Piccolo G, Gorrini C, Montecchini S, Rossi S, Chezzi C, Dettori G.
Department of Pathology and Laboratory Medicine, Section of Microbiology, University of Parma, Parma, Italy.
PMID: 19319234 [PubMed - in process]
Laboratory diagnosis of Toxoplasma gondii infection
Calderaro A, Peruzzi S, Piccolo G, Gorrini C, Montecchini S, Rossi S, Chezzi C, Dettori G.
Department of Pathology and Laboratory Medicine, Section of Microbiology, University of Parma, Parma, Italy.
PMID: 19319234 [PubMed - in process]
Wednesday, March 25, 2009
Intracellular parasitism with Toxoplasma stimulates mTOR-dependent host cell growth despite impaired signalling to S6K1 and 4E-BP1
Cell Microbiol. 2009 Feb 27. [Epub ahead of print]
Intracellular parasitism with Toxoplasma gondii stimulates mammalian-target-of-rapamycin-dependent host cell growth despite impaired signalling to S6K1 and 4E-BP1
Wang Y, Weiss LM, Orlofsky A.
Department of Pathology, Albery Einstein College of Medicine, Bronx, New York 10461, USA.
The Ser/Thr kinase mammalian-target-of-rapamycin (mTOR) is a central regulator of anabolism, growth and proliferation. We investigated the effects of Toxoplasma gondii on host mTOR signalling. Toxoplasma invasion of multiple cell types rapidly induced sustained mTOR activation that was restricted to infected cells, as determined by rapamycin-sensitive phosphorylation of ribosomal protein S6; however, phosphorylation of the growth-associated mTOR substrates 4E-BP1 and S6K1 was not detected. Infected cells still phosphorylated S6K1 and 4E-BP1 in response to insulin, although the S6K1 response was blunted. Parasite-induced S6 phosphorylation was independent of S6K1 and did not require activation of canonical mTOR-inducing pathways mediated by phosphatidylinositol 3-kinase-Akt and ERK. Host mTOR was localized in a vesicular pattern surrounding the parasitophorous vacuole, suggesting potential activation by phosphatidic acid in the vacuolar membrane. In spite of a failure to phosphorylate 4E-BP1 and S6K1, intracellular T. gondii triggered host cell cycle progression in an mTOR-dependent manner and progression of infected cells displayed increased sensitivity to rapamycin. Moreover, normal cell growth was maintained during parasite-induced cell cycle progression, as indicated by total cellular S6 levels. The Toxoplasma-infected cell provides a unique example of non-canonical mTOR activation supporting growth that is independent of signalling through either S6K1 or 4E-BP1.
PMID: 19302577 [PubMed - as supplied by publisher]
Intracellular parasitism with Toxoplasma gondii stimulates mammalian-target-of-rapamycin-dependent host cell growth despite impaired signalling to S6K1 and 4E-BP1
Wang Y, Weiss LM, Orlofsky A.
Department of Pathology, Albery Einstein College of Medicine, Bronx, New York 10461, USA.
The Ser/Thr kinase mammalian-target-of-rapamycin (mTOR) is a central regulator of anabolism, growth and proliferation. We investigated the effects of Toxoplasma gondii on host mTOR signalling. Toxoplasma invasion of multiple cell types rapidly induced sustained mTOR activation that was restricted to infected cells, as determined by rapamycin-sensitive phosphorylation of ribosomal protein S6; however, phosphorylation of the growth-associated mTOR substrates 4E-BP1 and S6K1 was not detected. Infected cells still phosphorylated S6K1 and 4E-BP1 in response to insulin, although the S6K1 response was blunted. Parasite-induced S6 phosphorylation was independent of S6K1 and did not require activation of canonical mTOR-inducing pathways mediated by phosphatidylinositol 3-kinase-Akt and ERK. Host mTOR was localized in a vesicular pattern surrounding the parasitophorous vacuole, suggesting potential activation by phosphatidic acid in the vacuolar membrane. In spite of a failure to phosphorylate 4E-BP1 and S6K1, intracellular T. gondii triggered host cell cycle progression in an mTOR-dependent manner and progression of infected cells displayed increased sensitivity to rapamycin. Moreover, normal cell growth was maintained during parasite-induced cell cycle progression, as indicated by total cellular S6 levels. The Toxoplasma-infected cell provides a unique example of non-canonical mTOR activation supporting growth that is independent of signalling through either S6K1 or 4E-BP1.
PMID: 19302577 [PubMed - as supplied by publisher]
Toxoplasma-induced autophagy: A window into nutritional futile cycles in mammalian cells?
Autophagy. 2009 Apr 9;5(3). [Epub ahead of print]
Toxoplasma-induced autophagy: A window into nutritional futile cycles in mammalian cells?
Orlofsky A.
Department of Pathology, Albert Einstein College of Medicine, Bronx, NY, USA.
The regulation and function of autophagy in response to metabolic signals is not yet well understood. A recent study from our laboratory indicates that an intracellular parasite, Toxoplasma gondii, derives nutritive benefit from the upregulation of host cell autophagy. We discuss this and related findings suggesting that autophagy in infected cells functions as part of a metabolic futile cycle. The hypothesis is presented that endogenous autophagy-based futile cycles may operate in normal mammalian cells, providing a substrate for manipulation by pathogens.
PMID: 19305153 [PubMed - as supplied by publisher]
Toxoplasma-induced autophagy: A window into nutritional futile cycles in mammalian cells?
Orlofsky A.
Department of Pathology, Albert Einstein College of Medicine, Bronx, NY, USA.
The regulation and function of autophagy in response to metabolic signals is not yet well understood. A recent study from our laboratory indicates that an intracellular parasite, Toxoplasma gondii, derives nutritive benefit from the upregulation of host cell autophagy. We discuss this and related findings suggesting that autophagy in infected cells functions as part of a metabolic futile cycle. The hypothesis is presented that endogenous autophagy-based futile cycles may operate in normal mammalian cells, providing a substrate for manipulation by pathogens.
PMID: 19305153 [PubMed - as supplied by publisher]
Tuesday, March 24, 2009
Postdoctoral positions available
POST-DOCTORAL POSITIONS
Post-doctoral fellow positions are available to study transcription, epigenetics, and post-transcriptional control in stress-induced differentiation of Toxoplasma gondii, a protozoan parasite related to malaria that causes opportunistic infections in AIDS patients (see www.sullivanlab.com). This cross-disciplinary research involving parasitology, biochemistry and molecular biology, will be performed in the laboratories of Dr. William Sullivan and Dr. Ronald Wek (Departments of Pharmacology and Biochemistry & Molecular Biology, Indiana University School of Medicine, Indianapolis, USA). Applicants must possess a Ph.D. and have record of productivity as evidenced by publications in international journals. Interested candidates should send their CV and the names/emails of three references electronically to wjsulliv@iupui.edu.
Post-doctoral fellow positions are available to study transcription, epigenetics, and post-transcriptional control in stress-induced differentiation of Toxoplasma gondii, a protozoan parasite related to malaria that causes opportunistic infections in AIDS patients (see www.sullivanlab.com). This cross-disciplinary research involving parasitology, biochemistry and molecular biology, will be performed in the laboratories of Dr. William Sullivan and Dr. Ronald Wek (Departments of Pharmacology and Biochemistry & Molecular Biology, Indiana University School of Medicine, Indianapolis, USA). Applicants must possess a Ph.D. and have record of productivity as evidenced by publications in international journals. Interested candidates should send their CV and the names/emails of three references electronically to wjsulliv@iupui.edu.
Monday, March 23, 2009
Anti-CD25 antibody-mediated depletion of effector T cell populations enhances susceptibility of mice to acute but not chronic Toxoplasma
J Immunol. 2009 Apr 1;182(7):3985-94
Anti-CD25 antibody-mediated depletion of effector T cell populations enhances susceptibility of mice to acute but not chronic Toxoplasma gondii infection
Couper KN, Lanthier PA, Perona-Wright G, Kummer LW, Chen W, Smiley ST, Mohrs M, Johnson LL.
Trudeau Institute, Saranac Lake, NY 12983, USA. kevin.couper@lshtm.ac.uk
Natural regulatory T cells (Tregs) constitutively express the IL-2R alpha-chain (CD25) on their surface. Consequently, administration of anti-CD25 Abs is a commonly used technique to deplete Treg populations in vivo. However, activated effector T cells may also transiently express CD25, and are thus also potential targets for anti-CD25 Abs. In this study using Toxoplasma gondii as a model proinflammatory infection, we have examined the capacity of anti-CD25 Abs to target effector T cell populations during an inflammatory episode, to determine to what extent that this action may modulate the outcome of disease. Anti-CD25 Ab-treated C57BL/6 mice displayed significantly reduced CD4(+) T cell IFN-gamma production during acute T. gondii infection and exhibited reduced weight loss and liver pathology during early acute infection; aspects of infection previously associated with effector CD4(+) T cell responses. In agreement, anti-CD25 Ab administration impaired parasite control and caused mice to succumb to infection during late acute/early chronic stages of infection with elevated tissue parasite burdens. In contrast, anti-CD25 Ab treatment of mice with established chronic infections did not markedly affect brain parasite burdens, suggesting that protective T cell populations do not express CD25 during chronic stages of T. gondii infection. In summary, we have demonstrated that anti-CD25 Abs may directly abrogate effector T cell responses during an inflammatory episode, highlighting important limitations of the use of anti-CD25 Ab administration to examine Treg function during inflammatory settings.
Publication Types:
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
PMID: 19299696 [PubMed - in process]
Anti-CD25 antibody-mediated depletion of effector T cell populations enhances susceptibility of mice to acute but not chronic Toxoplasma gondii infection
Couper KN, Lanthier PA, Perona-Wright G, Kummer LW, Chen W, Smiley ST, Mohrs M, Johnson LL.
Trudeau Institute, Saranac Lake, NY 12983, USA. kevin.couper@lshtm.ac.uk
Natural regulatory T cells (Tregs) constitutively express the IL-2R alpha-chain (CD25) on their surface. Consequently, administration of anti-CD25 Abs is a commonly used technique to deplete Treg populations in vivo. However, activated effector T cells may also transiently express CD25, and are thus also potential targets for anti-CD25 Abs. In this study using Toxoplasma gondii as a model proinflammatory infection, we have examined the capacity of anti-CD25 Abs to target effector T cell populations during an inflammatory episode, to determine to what extent that this action may modulate the outcome of disease. Anti-CD25 Ab-treated C57BL/6 mice displayed significantly reduced CD4(+) T cell IFN-gamma production during acute T. gondii infection and exhibited reduced weight loss and liver pathology during early acute infection; aspects of infection previously associated with effector CD4(+) T cell responses. In agreement, anti-CD25 Ab administration impaired parasite control and caused mice to succumb to infection during late acute/early chronic stages of infection with elevated tissue parasite burdens. In contrast, anti-CD25 Ab treatment of mice with established chronic infections did not markedly affect brain parasite burdens, suggesting that protective T cell populations do not express CD25 during chronic stages of T. gondii infection. In summary, we have demonstrated that anti-CD25 Abs may directly abrogate effector T cell responses during an inflammatory episode, highlighting important limitations of the use of anti-CD25 Ab administration to examine Treg function during inflammatory settings.
Publication Types:
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
PMID: 19299696 [PubMed - in process]
Thursday, March 19, 2009
Disruption of a mitochondrial MutS DNA repair enzyme homologue confers drug resistance
Mol Microbiol. 2009 Mar 6. [Epub ahead of print]
Disruption of a mitochondrial MutS DNA repair enzyme homologue confers drug resistance in the parasite Toxoplasma gondii
Garrison EM, Arrizabalaga G.
Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Life Sciences South Room 142, Moscow, ID 83844, USA.
MutS homologues (MSHs) are critical components of the eukaryotic mismatch repair machinery. In addition to repairing mismatched DNA, mismatch repair enzymes are known in higher eukaryotes to directly signal cell cycle arrest and apoptosis in response to DNA-damaging agents. Accordingly, mammalian cells lacking certain MSHs are resistant to chemotherapeutic drugs. Interestingly, we have discovered that the disruption of TgMSH-1, an MSH in the pathogenic parasite, Toxoplasma gondii, confers drug resistance. Through a genetic selection for T. gondii mutants resistant to the antiparasitic drug monensin, we have isolated a strain that is resistant not only to monensin but also to salinomycin and the alkylating agent, methylnitrosourea. We have shown that this phenotype is due to the disruption of TgMSH-1 as the multidrug-resistance phenotype is complemented by a wild-type copy of TgMSH-1 and is recapitulated by a directed disruption of this gene in a wild-type strain. We have also shown that, unlike previously described MSHs involved in signalling, TgMSH-1 localizes to the parasite mitochondrion. These results provide the first example of a mitochondrial MSH that is involved in drug sensitivity and implicate the induction of mitochondrial stress as a mode of action of the widely used drug, monensin.
PMID: 19291232 [PubMed - as supplied by publisher]
Disruption of a mitochondrial MutS DNA repair enzyme homologue confers drug resistance in the parasite Toxoplasma gondii
Garrison EM, Arrizabalaga G.
Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Life Sciences South Room 142, Moscow, ID 83844, USA.
MutS homologues (MSHs) are critical components of the eukaryotic mismatch repair machinery. In addition to repairing mismatched DNA, mismatch repair enzymes are known in higher eukaryotes to directly signal cell cycle arrest and apoptosis in response to DNA-damaging agents. Accordingly, mammalian cells lacking certain MSHs are resistant to chemotherapeutic drugs. Interestingly, we have discovered that the disruption of TgMSH-1, an MSH in the pathogenic parasite, Toxoplasma gondii, confers drug resistance. Through a genetic selection for T. gondii mutants resistant to the antiparasitic drug monensin, we have isolated a strain that is resistant not only to monensin but also to salinomycin and the alkylating agent, methylnitrosourea. We have shown that this phenotype is due to the disruption of TgMSH-1 as the multidrug-resistance phenotype is complemented by a wild-type copy of TgMSH-1 and is recapitulated by a directed disruption of this gene in a wild-type strain. We have also shown that, unlike previously described MSHs involved in signalling, TgMSH-1 localizes to the parasite mitochondrion. These results provide the first example of a mitochondrial MSH that is involved in drug sensitivity and implicate the induction of mitochondrial stress as a mode of action of the widely used drug, monensin.
PMID: 19291232 [PubMed - as supplied by publisher]
Tracking Transmission of the Zoonosis Toxoplasma gondii
Adv Parasitol. 2009;68:139-59
Chapter 6 Tracking Transmission of the Zoonosis Toxoplasma gondii
Smith JE.
Toxoplasma gondii is a highly successful parasite that infects many host species and has colonised a wide range of habitats. Review of the parasite's life cycle demonstrates that it has become adapted to exploit multiple routes of transmission through a sexual cycle in the definitive host and asexually, through carnivory, and by vertical transmission. These alternative routes may operate synergistically to enhance transmission, but they might also provide a vehicle for selection leading to partitioning of strains in the environment. Genetic analysis has shown that parasite population structure varies globally. In South America, there is high strain diversity while in North America, Europe and Africa three clonal strain types predominate. This may imply a shift from sexual to asexual transmission. Mapping of the parasite genome has provided a wealth of markers for strain characterisation. Close genotyping of isolates gives evidence of multiple infection and recombination in natural populations and reveals differences in both the distribution and the phenotype of strains. More intensive epidemiological studies are now required to unravel the networks of transmission operating within defined habitats.
PMID: 19289193 [PubMed - in process]
Chapter 6 Tracking Transmission of the Zoonosis Toxoplasma gondii
Smith JE.
Toxoplasma gondii is a highly successful parasite that infects many host species and has colonised a wide range of habitats. Review of the parasite's life cycle demonstrates that it has become adapted to exploit multiple routes of transmission through a sexual cycle in the definitive host and asexually, through carnivory, and by vertical transmission. These alternative routes may operate synergistically to enhance transmission, but they might also provide a vehicle for selection leading to partitioning of strains in the environment. Genetic analysis has shown that parasite population structure varies globally. In South America, there is high strain diversity while in North America, Europe and Africa three clonal strain types predominate. This may imply a shift from sexual to asexual transmission. Mapping of the parasite genome has provided a wealth of markers for strain characterisation. Close genotyping of isolates gives evidence of multiple infection and recombination in natural populations and reveals differences in both the distribution and the phenotype of strains. More intensive epidemiological studies are now required to unravel the networks of transmission operating within defined habitats.
PMID: 19289193 [PubMed - in process]
Destruction and Control of Toxoplasma gondii Tachyzoites Using Gold Nanosphere/Antibody Conjugates
Small. 2009 Mar 16. [Epub ahead of print]
Destruction and Control of Toxoplasma gondii Tachyzoites Using Gold Nanosphere/Antibody Conjugates
Pissuwan D, Valenzuela SM, Miller CM, Killingsworth MC, Cortie MB.
Institute for Nanoscale Technology University of Technology Sydney P.O. Box 123, Broadway, Sydney, NSW 2007 (Australia).
PMID: 19291731 [PubMed - as supplied by publisher]
Destruction and Control of Toxoplasma gondii Tachyzoites Using Gold Nanosphere/Antibody Conjugates
Pissuwan D, Valenzuela SM, Miller CM, Killingsworth MC, Cortie MB.
Institute for Nanoscale Technology University of Technology Sydney P.O. Box 123, Broadway, Sydney, NSW 2007 (Australia).
PMID: 19291731 [PubMed - as supplied by publisher]
Wednesday, March 18, 2009
Host cell entry by apicomplexa parasites requires actin polymerization in the host cell
Cell Host Microbe. 2009 Mar 19;5(3):259-72
Host cell entry by apicomplexa parasites requires actin polymerization in the host cell
Gonzalez V, Combe A, David V, Malmquist NA, Delorme V, Leroy C, Blazquez S, Ménard R, Tardieux I.
Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France; INSERM U567, Université Paris Descartes, CNRS (UMR 8104), 22 rue Méchain, 75014 Paris, France.
Apicomplexa are obligate intracellular parasites that actively invade host cells using their membrane-associated, actin-myosin motor. The current view is that host cell invasion by Apicomplexa requires the formation of a parasite-host cell junction, which has been termed the moving junction, but does not require the active participation of host actin. Using Toxoplasma gondii tachyzoites and Plasmodium berghei sporozoites, we show that host actin participates in parasite entry. Parasites induce the formation of a ring-shaped F-actin structure in the host cell at the parasite-cell junction, which remains stable during parasite entry. The Arp2/3 complex, an actin-nucleating factor, is recruited at the ring structure and is important for parasite entry. We propose that Apicomplexa invasion of host cells requires not only the parasite motor but also de novo polymerization of host actin at the entry site for anchoring the junction on which the parasite pulls to penetrate the host cell.
PMID: 19286135 [PubMed - in process]
Host cell entry by apicomplexa parasites requires actin polymerization in the host cell
Gonzalez V, Combe A, David V, Malmquist NA, Delorme V, Leroy C, Blazquez S, Ménard R, Tardieux I.
Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France; INSERM U567, Université Paris Descartes, CNRS (UMR 8104), 22 rue Méchain, 75014 Paris, France.
Apicomplexa are obligate intracellular parasites that actively invade host cells using their membrane-associated, actin-myosin motor. The current view is that host cell invasion by Apicomplexa requires the formation of a parasite-host cell junction, which has been termed the moving junction, but does not require the active participation of host actin. Using Toxoplasma gondii tachyzoites and Plasmodium berghei sporozoites, we show that host actin participates in parasite entry. Parasites induce the formation of a ring-shaped F-actin structure in the host cell at the parasite-cell junction, which remains stable during parasite entry. The Arp2/3 complex, an actin-nucleating factor, is recruited at the ring structure and is important for parasite entry. We propose that Apicomplexa invasion of host cells requires not only the parasite motor but also de novo polymerization of host actin at the entry site for anchoring the junction on which the parasite pulls to penetrate the host cell.
PMID: 19286135 [PubMed - in process]
Infection and stillbirth
Semin Fetal Neonatal Med. 2009 Mar 11. [Epub ahead of print]
Infection and stillbirth
McClure EM, Goldenberg RL.
Department of Epidemiology, UNC Global School of Public Health, Chapel Hill, North Carolina, USA.
Infection may cause stillbirth by several mechanisms, including direct infection, placental damage, and severe maternal illness. Various organisms have been associated with stillbirth, including many bacteria, viruses, and protozoa. In developed countries, between 10% and 25% of stillbirths may be caused by an infection, whereas in developing countries, which have much higher stillbirth rates, the contribution of infection is much greater. In developed countries, ascending bacterial infection, both before and after membrane rupture, with organisms such as Escherichia coli, group B streptococci, and Ureaplasma urealyticum is usually the most common infectious cause of stillbirth. However, in areas where syphilis is prevalent, up to half of all stillbirths may be caused by this infection alone. Malaria may be an important cause of stillbirth in women infected for the first time in pregnancy. The two most important viral causes of stillbirth are parvovirus and Coxsackie virus, although a number of other viral infections appear to be causal. Toxoplasma gondii, Listeria monocytogenes, and the organisms that cause leptospirosis, Q fever, and Lyme disease have all been implicated as etiologic for stillbirth. In certain developing countries, the stillbirth rate is high and the infection-related component so great that achieving a substantial reduction in stillbirth should be possible by reducing maternal infections. However, because infection-related stillbirth is uncommon in developed countries, and because those that do occur are caused by a wide variety of organisms, reducing this etiologic component of stillbirth much further will be difficult.
PMID: 19285457 [PubMed - as supplied by publisher]
Infection and stillbirth
McClure EM, Goldenberg RL.
Department of Epidemiology, UNC Global School of Public Health, Chapel Hill, North Carolina, USA.
Infection may cause stillbirth by several mechanisms, including direct infection, placental damage, and severe maternal illness. Various organisms have been associated with stillbirth, including many bacteria, viruses, and protozoa. In developed countries, between 10% and 25% of stillbirths may be caused by an infection, whereas in developing countries, which have much higher stillbirth rates, the contribution of infection is much greater. In developed countries, ascending bacterial infection, both before and after membrane rupture, with organisms such as Escherichia coli, group B streptococci, and Ureaplasma urealyticum is usually the most common infectious cause of stillbirth. However, in areas where syphilis is prevalent, up to half of all stillbirths may be caused by this infection alone. Malaria may be an important cause of stillbirth in women infected for the first time in pregnancy. The two most important viral causes of stillbirth are parvovirus and Coxsackie virus, although a number of other viral infections appear to be causal. Toxoplasma gondii, Listeria monocytogenes, and the organisms that cause leptospirosis, Q fever, and Lyme disease have all been implicated as etiologic for stillbirth. In certain developing countries, the stillbirth rate is high and the infection-related component so great that achieving a substantial reduction in stillbirth should be possible by reducing maternal infections. However, because infection-related stillbirth is uncommon in developed countries, and because those that do occur are caused by a wide variety of organisms, reducing this etiologic component of stillbirth much further will be difficult.
PMID: 19285457 [PubMed - as supplied by publisher]
The type II NADH dehydrogenase inhibitor HDQ leads to {Delta}{Psi}m collapse and ATP depletion in Toxoplasma
Eukaryot Cell. 2009 Mar 13. [Epub ahead of print]
The type II NADH dehydrogenase inhibitor HDQ leads to {Delta}{Psi}m collapse and ATP depletion in Toxoplasma gondii
Lin SS, Gross U, Bohne W.
Institute of Medical Microbiology, University of Göttingen, Kreuzbergring 57, Göttingen D-37075; GERMANY.
The apicomplexan parasite Toxoplasma gondii expresses type II NADH dehydrogenases (NDH2s) instead of a canconial complex I at the inner mitochondrial membrane. These non-proton pumping enzymes are considered as promising drug targets, due to their absence in mammalian cells. We recently showed by inhibition kinetics that TgNDH2-I is a target of the quinolone-like compound HDQ, which inhibits T. gondii replication in the nanomolar range. In this study, the cationic fluorescent probes Mitotracker and DiOC6(3) were used to monitor the influence of HDQ on DeltaPsim in T. gondii. Real-time imaging revealed that nanomolar HDQ concentrations led to a DeltaPsim collapse within minutes, which is followed by a severe ATP depletion of 30% after 1 h and 70% after 24 h. The DeltaPsim depolarization was attenuated when substrates for other dehydrogenases which can donate electrons to ubiquinone were added to digitonin-permeabilized cells, or when infected cultures were treated with the F0-ATPase inhibitor oligomycin. A prolonged treatment with sublethal concentrations of HDQ induced differentiation into bradyzoites. This dormant stage is likely to be less dependent on DeltaPsim, since DeltaPsim-positive parasites were found at a significant lower frequency in alkaline pH induced bradyzoites than in tachyzoites. Together, our studies reveal that oxidative phosphorylation is essential for maintaining the ATP level in the fast growing tachyzoite stage and that HDQ interferes with this pathway by inhibiting the electron transport chain at the level of ubiquinone reduction.
PMID: 19286986 [PubMed - as supplied by publisher]
The type II NADH dehydrogenase inhibitor HDQ leads to {Delta}{Psi}m collapse and ATP depletion in Toxoplasma gondii
Lin SS, Gross U, Bohne W.
Institute of Medical Microbiology, University of Göttingen, Kreuzbergring 57, Göttingen D-37075; GERMANY.
The apicomplexan parasite Toxoplasma gondii expresses type II NADH dehydrogenases (NDH2s) instead of a canconial complex I at the inner mitochondrial membrane. These non-proton pumping enzymes are considered as promising drug targets, due to their absence in mammalian cells. We recently showed by inhibition kinetics that TgNDH2-I is a target of the quinolone-like compound HDQ, which inhibits T. gondii replication in the nanomolar range. In this study, the cationic fluorescent probes Mitotracker and DiOC6(3) were used to monitor the influence of HDQ on DeltaPsim in T. gondii. Real-time imaging revealed that nanomolar HDQ concentrations led to a DeltaPsim collapse within minutes, which is followed by a severe ATP depletion of 30% after 1 h and 70% after 24 h. The DeltaPsim depolarization was attenuated when substrates for other dehydrogenases which can donate electrons to ubiquinone were added to digitonin-permeabilized cells, or when infected cultures were treated with the F0-ATPase inhibitor oligomycin. A prolonged treatment with sublethal concentrations of HDQ induced differentiation into bradyzoites. This dormant stage is likely to be less dependent on DeltaPsim, since DeltaPsim-positive parasites were found at a significant lower frequency in alkaline pH induced bradyzoites than in tachyzoites. Together, our studies reveal that oxidative phosphorylation is essential for maintaining the ATP level in the fast growing tachyzoite stage and that HDQ interferes with this pathway by inhibiting the electron transport chain at the level of ubiquinone reduction.
PMID: 19286986 [PubMed - as supplied by publisher]
Evaluation of protective effect of multi-epitope DNA vaccine encoding six antigen segments of Toxoplasma gondii in mice
Parasitol Res. 2009 Mar 14. [Epub ahead of print]
Evaluation of protective effect of multi-epitope DNA vaccine encoding six antigen segments of Toxoplasma gondii in mice
Liu S, Shi L, Cheng YB, Fan GX, Ren HX, Yuan YK.
Department of Immunology and Microbiology, School of Medicine, Xi'an Jiaotong University, Xi'an, 710061, China.
To investigate the vaccine potential of multi-epitope vaccines against toxoplasmosis, a multi-epitope DNA vaccine, eukaryotic plasmid pcDNA3.1/T-ME expressing six antigen segments (SAG1(238-256), SAG1(281-320), GRA1(170-193), GRA4(331-345), GRA4(229-245), and GRA2(171-185)) of Toxoplasma gondii was constructed. We investigated the efficacy of pcDNA3.1/T-ME with or without co-administration of a CpG-oligodeoxynucleotide (CpG-ODN) as an adjuvant to protect mice (BALB/c and C57BL/6) against toxoplasmosis. High survival rates were observed in mice immunized with pcDNA3.1/T-ME when challenged with T. gondii RH strain. Lymphocyte proliferation assays, cytokine, and antibody determinations show that mice immunized with pcDNA3.1/T-ME produced stronger humoral and Th1-type cellular immune responses compared to untreated mice or those immunized with empty plasmids. However, co-immunization with CpG-ODN resulted in impaired immune responses. Our data demonstrates that multi-epitope DNA vaccination is a potential strategy for the control of toxoplasmosis and paves the way for further investigations into producing a multi-epitope anti-T. gondii DNA vaccine.
PMID: 19288132 [PubMed - as supplied by publisher]
Evaluation of protective effect of multi-epitope DNA vaccine encoding six antigen segments of Toxoplasma gondii in mice
Liu S, Shi L, Cheng YB, Fan GX, Ren HX, Yuan YK.
Department of Immunology and Microbiology, School of Medicine, Xi'an Jiaotong University, Xi'an, 710061, China.
To investigate the vaccine potential of multi-epitope vaccines against toxoplasmosis, a multi-epitope DNA vaccine, eukaryotic plasmid pcDNA3.1/T-ME expressing six antigen segments (SAG1(238-256), SAG1(281-320), GRA1(170-193), GRA4(331-345), GRA4(229-245), and GRA2(171-185)) of Toxoplasma gondii was constructed. We investigated the efficacy of pcDNA3.1/T-ME with or without co-administration of a CpG-oligodeoxynucleotide (CpG-ODN) as an adjuvant to protect mice (BALB/c and C57BL/6) against toxoplasmosis. High survival rates were observed in mice immunized with pcDNA3.1/T-ME when challenged with T. gondii RH strain. Lymphocyte proliferation assays, cytokine, and antibody determinations show that mice immunized with pcDNA3.1/T-ME produced stronger humoral and Th1-type cellular immune responses compared to untreated mice or those immunized with empty plasmids. However, co-immunization with CpG-ODN resulted in impaired immune responses. Our data demonstrates that multi-epitope DNA vaccination is a potential strategy for the control of toxoplasmosis and paves the way for further investigations into producing a multi-epitope anti-T. gondii DNA vaccine.
PMID: 19288132 [PubMed - as supplied by publisher]
Thursday, March 12, 2009
A unique dual activity amino acid hydroxylase in Toxoplasma gondii
PLoS ONE. 2009;4(3):e4801. Epub 2009 Mar 11
A unique dual activity amino acid hydroxylase in Toxoplasma gondii
Gaskell EA, Smith JE, Pinney JW, Westhead DR, McConkey GA.
Institute of Integrative and Comparative Biology, University of Leeds, Leeds, United Kingdom.
The genome of the protozoan parasite Toxoplasma gondii was found to contain two genes encoding tyrosine hydroxylase; that produces L-DOPA. The encoded enzymes metabolize phenylalanine as well as tyrosine with substrate preference for tyrosine. Thus the enzymes catabolize phenylalanine to tyrosine and tyrosine to L-DOPA. The catalytic domain descriptive of this class of enzymes is conserved with the parasite enzyme and exhibits similar kinetic properties to metazoan tyrosine hydroxylases, but contains a unique N-terminal extension with a signal sequence motif. One of the genes, TgAaaH1, is constitutively expressed while the other gene, TgAaaH2, is induced during formation of the bradyzoites of the cyst stages of the life cycle. This is the first description of an aromatic amino acid hydroxylase in an apicomplexan parasite. Extensive searching of apicomplexan genome sequences revealed an ortholog in Neospora caninum but not in Eimeria, Cryptosporidium, Theileria, or Plasmodium. Possible role(s) of these bi-functional enzymes during host infection are discussed.
Publication Types:
Research Support, Non-U.S. Gov't
PMID: 19277211 [PubMed - in process]
A unique dual activity amino acid hydroxylase in Toxoplasma gondii
Gaskell EA, Smith JE, Pinney JW, Westhead DR, McConkey GA.
Institute of Integrative and Comparative Biology, University of Leeds, Leeds, United Kingdom.
The genome of the protozoan parasite Toxoplasma gondii was found to contain two genes encoding tyrosine hydroxylase; that produces L-DOPA. The encoded enzymes metabolize phenylalanine as well as tyrosine with substrate preference for tyrosine. Thus the enzymes catabolize phenylalanine to tyrosine and tyrosine to L-DOPA. The catalytic domain descriptive of this class of enzymes is conserved with the parasite enzyme and exhibits similar kinetic properties to metazoan tyrosine hydroxylases, but contains a unique N-terminal extension with a signal sequence motif. One of the genes, TgAaaH1, is constitutively expressed while the other gene, TgAaaH2, is induced during formation of the bradyzoites of the cyst stages of the life cycle. This is the first description of an aromatic amino acid hydroxylase in an apicomplexan parasite. Extensive searching of apicomplexan genome sequences revealed an ortholog in Neospora caninum but not in Eimeria, Cryptosporidium, Theileria, or Plasmodium. Possible role(s) of these bi-functional enzymes during host infection are discussed.
Publication Types:
Research Support, Non-U.S. Gov't
PMID: 19277211 [PubMed - in process]
Wednesday, March 11, 2009
A hypothesis for the evolution of nuclear-encoded, plastid-targeted glyceraldehyde-3-phosphate dehydrogenase genes in "chromalveolate" members
PLoS ONE. 2009;4(3):e4737. Epub 2009 Mar 9
A hypothesis for the evolution of nuclear-encoded, plastid-targeted glyceraldehyde-3-phosphate dehydrogenase genes in "chromalveolate" members
Takishita K, Yamaguchi H, Maruyama T, Inagaki Y.
Japan Agency for Marine-Earth Science and Technology, Yokosuka, Kanagawa, Japan. takishitak@jamstec.go.jp
Eukaryotes bearing red alga-derived plastids--photosynthetic alveolates (dinoflagellates plus the apicomplexan Toxoplasma gondii plus the chromerid Chromera velia), photosynthetic stramenopiles, haptophytes, and cryptophytes--possess unique plastid-targeted glyceraldehyde-3-phosphate dehydrogenases (henceforth designated as "GapC1"). Pioneering phylogenetic studies have indicated a single origin of the GapC1 enzymes in eukaryotic evolution, but there are two potential idiosyncrasies in the GapC1 phylogeny: Firstly, the GapC1 tree topology is apparently inconsistent with the organismal relationship among the "GapC1-containing" groups. Secondly, four stramenopile GapC1 homologues are consistently paraphyletic in previously published studies, although these organisms have been widely accepted as monophyletic. For a closer examination of the above issues, in this study GapC1 gene sampling was improved by determining/identifying nine stramenopile and two cryptophyte genes. Phylogenetic analyses of our GapC1 dataset, which is particularly rich in the stramenopile homologues, prompt us to propose a new scenario that assumes multiple, lateral GapC1 gene transfer events to explain the incongruity between the GapC1 phylogeny and the organismal relationships amongst the "GapC1-containing" groups. Under our new scenario, GapC1 genes uniquely found in photosynthetic alveolates, photosynthetic stramenopiles, haptophytes, and cryptopyhytes are not necessarily a character vertically inherited from a common ancestor.
Publication Types:
Research Support, Non-U.S. Gov't
PMID: 19270733 [PubMed - in process]
A hypothesis for the evolution of nuclear-encoded, plastid-targeted glyceraldehyde-3-phosphate dehydrogenase genes in "chromalveolate" members
Takishita K, Yamaguchi H, Maruyama T, Inagaki Y.
Japan Agency for Marine-Earth Science and Technology, Yokosuka, Kanagawa, Japan. takishitak@jamstec.go.jp
Eukaryotes bearing red alga-derived plastids--photosynthetic alveolates (dinoflagellates plus the apicomplexan Toxoplasma gondii plus the chromerid Chromera velia), photosynthetic stramenopiles, haptophytes, and cryptophytes--possess unique plastid-targeted glyceraldehyde-3-phosphate dehydrogenases (henceforth designated as "GapC1"). Pioneering phylogenetic studies have indicated a single origin of the GapC1 enzymes in eukaryotic evolution, but there are two potential idiosyncrasies in the GapC1 phylogeny: Firstly, the GapC1 tree topology is apparently inconsistent with the organismal relationship among the "GapC1-containing" groups. Secondly, four stramenopile GapC1 homologues are consistently paraphyletic in previously published studies, although these organisms have been widely accepted as monophyletic. For a closer examination of the above issues, in this study GapC1 gene sampling was improved by determining/identifying nine stramenopile and two cryptophyte genes. Phylogenetic analyses of our GapC1 dataset, which is particularly rich in the stramenopile homologues, prompt us to propose a new scenario that assumes multiple, lateral GapC1 gene transfer events to explain the incongruity between the GapC1 phylogeny and the organismal relationships amongst the "GapC1-containing" groups. Under our new scenario, GapC1 genes uniquely found in photosynthetic alveolates, photosynthetic stramenopiles, haptophytes, and cryptopyhytes are not necessarily a character vertically inherited from a common ancestor.
Publication Types:
Research Support, Non-U.S. Gov't
PMID: 19270733 [PubMed - in process]
Molecular diagnosis of Toxoplasma gondii infection in cerebrospinal fluid from AIDS patients
Cerebrospinal Fluid Res. 2009 Mar 6;6(1):2. [Epub ahead of print]
Molecular diagnosis of Toxoplasma gondii infection in cerebrospinal fluid from AIDS patients
Alfonso Y, Fraga J, Fonseca C, Jimenez N, Pinillos T, Dorta-Contreras AJ, Cox R, Capo V, Pomier O, Bandera F, Ginorio D.
ABSTRACT: BACKGROUND: Toxoplasmic encephalitis (TE) is one of the most common opportunistic infections in immunocompromised patients. In Cuba, despite the highly active antiretroviral therapy, TE is still the most important cause of cerebral mass lesions in patients infected with the human immunodeficiency virus (HIV). The detection of Toxoplasma gondii by PCR may facilitate the diagnosis and follow-up of TE in acquired immunodeficiency syndrome (AIDS) patients by direct identification of parasite DNA in clinical samples. The aim of the present study was to evaluate a rapid PCR method using the B1 gene to detect T. gondii in cerebrospinal fluid (CSF) samples from patients with suspected TE. METHODS: CSF samples from AIDS and HIV-negative patients were analyzed. Patients were divided into two groups according to the Centre for Disease Control and Prevention (CDC) criteria for AIDS-related TE: AIDS patients with suspected neurotoxoplasmosis and AIDS and HIV-negative patients with other confirmed neurological diseases but no suspicions of TE. Predictive values, diagnostic accuracy, sensitivity and specificity of the PCR B1 method were calculated. RESULTS: The results obtained from 190 patients showed that this assay has a good sensitivity and specificity (83.3% and 95.7%, respectively) for the diagnosis of TE in AIDS patients. CONCLUSIONS: PCR using the B1 gene and B22/B23 set of primers is a single, rapid and reliable method that may be valuable for discrimination between toxoplasmosis and other central nervous system (CNS) diseases.
PMID: 19267913 [PubMed - as supplied by publisher]
Molecular diagnosis of Toxoplasma gondii infection in cerebrospinal fluid from AIDS patients
Alfonso Y, Fraga J, Fonseca C, Jimenez N, Pinillos T, Dorta-Contreras AJ, Cox R, Capo V, Pomier O, Bandera F, Ginorio D.
ABSTRACT: BACKGROUND: Toxoplasmic encephalitis (TE) is one of the most common opportunistic infections in immunocompromised patients. In Cuba, despite the highly active antiretroviral therapy, TE is still the most important cause of cerebral mass lesions in patients infected with the human immunodeficiency virus (HIV). The detection of Toxoplasma gondii by PCR may facilitate the diagnosis and follow-up of TE in acquired immunodeficiency syndrome (AIDS) patients by direct identification of parasite DNA in clinical samples. The aim of the present study was to evaluate a rapid PCR method using the B1 gene to detect T. gondii in cerebrospinal fluid (CSF) samples from patients with suspected TE. METHODS: CSF samples from AIDS and HIV-negative patients were analyzed. Patients were divided into two groups according to the Centre for Disease Control and Prevention (CDC) criteria for AIDS-related TE: AIDS patients with suspected neurotoxoplasmosis and AIDS and HIV-negative patients with other confirmed neurological diseases but no suspicions of TE. Predictive values, diagnostic accuracy, sensitivity and specificity of the PCR B1 method were calculated. RESULTS: The results obtained from 190 patients showed that this assay has a good sensitivity and specificity (83.3% and 95.7%, respectively) for the diagnosis of TE in AIDS patients. CONCLUSIONS: PCR using the B1 gene and B22/B23 set of primers is a single, rapid and reliable method that may be valuable for discrimination between toxoplasmosis and other central nervous system (CNS) diseases.
PMID: 19267913 [PubMed - as supplied by publisher]
Monday, March 09, 2009
Selection at a Single Locus Leads to Widespread Expansion of Toxoplasma gondii Lineages That Are Virulent in Mice
PLoS Genet. 2009 Mar;5(3):e1000404. Epub 2009 Mar 6
Selection at a Single Locus Leads to Widespread Expansion of Toxoplasma gondii Lineages That Are Virulent in Mice
Khan A, Taylor S, Ajioka JW, Rosenthal BM, Sibley LD.
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, United States of America.
Pathogenicity differences among laboratory isolates of the dominant clonal North American and European lineages of Toxoplasma gondii are largely controlled by polymorphisms and expression differences in rhoptry secretory proteins (ROPs). However, the extent to which such differences control virulence in natural isolates of T. gondii, including those from more diverse genetic backgrounds, is uncertain. We elucidated the evolutionary history and functional consequences of diversification in the serine/threonine kinase ROP18, a major virulence determinant in the mouse model. We characterized the extent of sequence polymorphism and the evolutionary forces acting on ROP18 and several antigen-encoding genes within a large collection of natural isolates, comparing them to housekeeping genes and introns. Surprisingly, despite substantial genetic diversity between lineages, we identified just three principal alleles of ROP18, which had very ancient ancestry compared to other sampled loci. Expression and allelic differences between these three alleles of ROP18 accounted for much of the variation in acute mouse virulence among natural isolates. While the avirulent type III allele was the most ancient, intermediate virulent (type II) and highly virulent (type I) lineages predominated and showed evidence of strong selective pressure. Out-group comparison indicated that historical loss of an upstream regulatory element increased ROP18 expression, exposing it to newfound diversifying selection, resulting in greatly enhanced virulence in the mouse model and expansion of new lineages. Population sweeps are evident in many genomes, yet their causes and evolutionary histories are rarely known. Our results establish that up-regulation of expression and selection at ROP18 in T. gondii has resulted in three distinct alleles with widely different levels of acute virulence in the mouse model. Preservation of all three alleles in the wild indicates they are likely adaptations for different niches. Our findings demonstrate that sweeping changes in population structure can result from alterations in a single gene.
PMID: 19266027 [PubMed - in process]
Selection at a Single Locus Leads to Widespread Expansion of Toxoplasma gondii Lineages That Are Virulent in Mice
Khan A, Taylor S, Ajioka JW, Rosenthal BM, Sibley LD.
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, United States of America.
Pathogenicity differences among laboratory isolates of the dominant clonal North American and European lineages of Toxoplasma gondii are largely controlled by polymorphisms and expression differences in rhoptry secretory proteins (ROPs). However, the extent to which such differences control virulence in natural isolates of T. gondii, including those from more diverse genetic backgrounds, is uncertain. We elucidated the evolutionary history and functional consequences of diversification in the serine/threonine kinase ROP18, a major virulence determinant in the mouse model. We characterized the extent of sequence polymorphism and the evolutionary forces acting on ROP18 and several antigen-encoding genes within a large collection of natural isolates, comparing them to housekeeping genes and introns. Surprisingly, despite substantial genetic diversity between lineages, we identified just three principal alleles of ROP18, which had very ancient ancestry compared to other sampled loci. Expression and allelic differences between these three alleles of ROP18 accounted for much of the variation in acute mouse virulence among natural isolates. While the avirulent type III allele was the most ancient, intermediate virulent (type II) and highly virulent (type I) lineages predominated and showed evidence of strong selective pressure. Out-group comparison indicated that historical loss of an upstream regulatory element increased ROP18 expression, exposing it to newfound diversifying selection, resulting in greatly enhanced virulence in the mouse model and expansion of new lineages. Population sweeps are evident in many genomes, yet their causes and evolutionary histories are rarely known. Our results establish that up-regulation of expression and selection at ROP18 in T. gondii has resulted in three distinct alleles with widely different levels of acute virulence in the mouse model. Preservation of all three alleles in the wild indicates they are likely adaptations for different niches. Our findings demonstrate that sweeping changes in population structure can result from alterations in a single gene.
PMID: 19266027 [PubMed - in process]
Genotype of 88 Toxoplasma gondii Isolates Associated with Toxoplasmosis in Immunocompromised Patients and Correlation with Clinical Findings
J Infect Dis. 2009 Mar 5. [Epub ahead of print]
Genotype of 88 Toxoplasma gondii Isolates Associated with Toxoplasmosis in Immunocompromised Patients and Correlation with Clinical Findings
Ajzenberg D, Yera H, Marty P, Paris L, Dalle F, Menotti J, Aubert D, Franck J, Bessières MH, Quinio D, Pelloux H, Delhaes L, Desbois N, Thulliez P, Robert-Gangneux F, Kauffmann-Lacroix C, Pujol S, Rabodonirina M, Bougnoux ME, Cuisenier B, Duhamel C, Duong TH, Filisetti D, Flori P, Gay-Andrieu F, Pratlong F, Nevez G, Totet A, Carme B, Bonnabau H, Dardé ML, Villena I.
Centre Hospitalier Universitaire, Amiens, 2Centre Hospitalier Universitaire, Bordeaux, 3Centre Hospitalier Universitaire, Brest, 4Centre Hospitalier Universitaire, Caen, 5Centre Hospitalier Universitaire, Dijon, 6Centre Hospitalier Universitaire, Grenoble, 7Centre Hospitalier Universitaire, Lille, 8Laboratoire de Parasitologie-Mycologie, EA 3174, Faculté de Médecine, Université de Limoges, and 9Centre National de Référence Toxoplasmose, Centre Hospitalier Universitaire, and 10Unité Fonctionnelle de Recherche Clinique et Biostatistique UFRCB, Centre Hospitalier Universitaire, Limoges, Université de Limoges, Limoges, 11Hospices civils de Lyon, Hôpital de la Croix-Rousse, Lyon, 12Centre Hospitalier Universitaire, Marseille, 13Centre Hospitalier Universitaire, Montpellier, 14Centre Hospitalier Universitaire, Nantes, 15Centre Hospitalier Universitaire, Nice, 16Institut de Puériculture, 17Hôpital Cochin and 18Hôpital Necker-Enfants Malades, 19Hôpital Pitié-Salpêtrière, and 20Hôpital Saint-Louis, Assistance Publique-Hôpitaux de Paris, Paris, 21Centre Hospitalier Universitaire, Poitiers, 22Centre Hospitalier Universitaire, Reims, 23Centre Hospitalier Universitaire, Rennes, 24Centre Hospitalier Universitaire, Saint-Etienne, 25Hôpitaux Universitaires de Strasbourg, Strasbourg, 26Centre Hospitalier Universitaire, Toulouse, 27Centre Hospitalier Universitaire, Tours, France; 28Centre Hospitalier Général, Cayenne, French Guiana; 29Centre Hospitalier Universitaire, Fort de France, Martinique.
We report the genotyping analysis of Toxoplasma gondii isolates in samples collected from 88 immunocompromised patients, along with clinical and epidemiological data. Most of these samples were collected in France during the current decade by the Toxoplasma Biological Resource Center. Lack of specific anti-Toxoplasma treatment, pulmonary toxoplasmosis, and involvement of multiple organs were the 3 main risk factors associated with death for this patient group. Genotyping results with 6 microsatellite markers showed that type II isolates were predominant among patients who acquired toxoplasmic infection in Europe. Non-type II isolates included 13 different genotypes and were mainly collected from patients who acquired toxoplasmosis outside Europe. Type III was the second most common genotype recovered from patients, whereas type I was rare in our population. Three nonarchetypal genotypes were repeatedly recovered from different patients who acquired the infection in sub-Saharan Africa (genotypes Africa 1 and Africa 2) and in the French West Indies (genotype Caribbean 1). The distribution of genotypes (type II vs. non-type II) was not significantly different when patients were stratified by underlying cause of immunosuppression, site of infection, or outcome. We conclude that in immunocompromised patients, host factors are much more involved than parasite factors in patients' resistance or susceptibility to toxoplasmosis.
PMID: 19265484 [PubMed - as supplied by publisher]
Genotype of 88 Toxoplasma gondii Isolates Associated with Toxoplasmosis in Immunocompromised Patients and Correlation with Clinical Findings
Ajzenberg D, Yera H, Marty P, Paris L, Dalle F, Menotti J, Aubert D, Franck J, Bessières MH, Quinio D, Pelloux H, Delhaes L, Desbois N, Thulliez P, Robert-Gangneux F, Kauffmann-Lacroix C, Pujol S, Rabodonirina M, Bougnoux ME, Cuisenier B, Duhamel C, Duong TH, Filisetti D, Flori P, Gay-Andrieu F, Pratlong F, Nevez G, Totet A, Carme B, Bonnabau H, Dardé ML, Villena I.
Centre Hospitalier Universitaire, Amiens, 2Centre Hospitalier Universitaire, Bordeaux, 3Centre Hospitalier Universitaire, Brest, 4Centre Hospitalier Universitaire, Caen, 5Centre Hospitalier Universitaire, Dijon, 6Centre Hospitalier Universitaire, Grenoble, 7Centre Hospitalier Universitaire, Lille, 8Laboratoire de Parasitologie-Mycologie, EA 3174, Faculté de Médecine, Université de Limoges, and 9Centre National de Référence Toxoplasmose, Centre Hospitalier Universitaire, and 10Unité Fonctionnelle de Recherche Clinique et Biostatistique UFRCB, Centre Hospitalier Universitaire, Limoges, Université de Limoges, Limoges, 11Hospices civils de Lyon, Hôpital de la Croix-Rousse, Lyon, 12Centre Hospitalier Universitaire, Marseille, 13Centre Hospitalier Universitaire, Montpellier, 14Centre Hospitalier Universitaire, Nantes, 15Centre Hospitalier Universitaire, Nice, 16Institut de Puériculture, 17Hôpital Cochin and 18Hôpital Necker-Enfants Malades, 19Hôpital Pitié-Salpêtrière, and 20Hôpital Saint-Louis, Assistance Publique-Hôpitaux de Paris, Paris, 21Centre Hospitalier Universitaire, Poitiers, 22Centre Hospitalier Universitaire, Reims, 23Centre Hospitalier Universitaire, Rennes, 24Centre Hospitalier Universitaire, Saint-Etienne, 25Hôpitaux Universitaires de Strasbourg, Strasbourg, 26Centre Hospitalier Universitaire, Toulouse, 27Centre Hospitalier Universitaire, Tours, France; 28Centre Hospitalier Général, Cayenne, French Guiana; 29Centre Hospitalier Universitaire, Fort de France, Martinique.
We report the genotyping analysis of Toxoplasma gondii isolates in samples collected from 88 immunocompromised patients, along with clinical and epidemiological data. Most of these samples were collected in France during the current decade by the Toxoplasma Biological Resource Center. Lack of specific anti-Toxoplasma treatment, pulmonary toxoplasmosis, and involvement of multiple organs were the 3 main risk factors associated with death for this patient group. Genotyping results with 6 microsatellite markers showed that type II isolates were predominant among patients who acquired toxoplasmic infection in Europe. Non-type II isolates included 13 different genotypes and were mainly collected from patients who acquired toxoplasmosis outside Europe. Type III was the second most common genotype recovered from patients, whereas type I was rare in our population. Three nonarchetypal genotypes were repeatedly recovered from different patients who acquired the infection in sub-Saharan Africa (genotypes Africa 1 and Africa 2) and in the French West Indies (genotype Caribbean 1). The distribution of genotypes (type II vs. non-type II) was not significantly different when patients were stratified by underlying cause of immunosuppression, site of infection, or outcome. We conclude that in immunocompromised patients, host factors are much more involved than parasite factors in patients' resistance or susceptibility to toxoplasmosis.
PMID: 19265484 [PubMed - as supplied by publisher]
Virulent Toxoplasma gondii Evade Immunity-Related GTPase-Mediated Parasite Vacuole Disruption within Primed Macrophages
J Immunol. 2009 Mar 15;182(6):3775-81
Virulent Toxoplasma gondii Evade Immunity-Related GTPase-Mediated Parasite Vacuole Disruption within Primed Macrophages
Zhao Y, Ferguson DJ, Wilson DC, Howard JC, Sibley LD, Yap GS.
Department of Medicine and Center for Immunity and Inflammation, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, NJ 07101.
Cytokine-activated macrophages restrain the replication of intracellular parasites and disrupt the integrity of vacuolar pathogens. In this study, we show that inducible nitric oxide synthase and the immunity-related GTPase (IRG) family member Irgm3, respectively, are required for the ability of in vivo primed macrophages to restrain the growth of Toxoplasma gondii and to destroy the parasite's intracellular niche. Remarkably, virulent Type I strains of T. gondii evade IRG-dependent vacuolar disruption, while remaining susceptible to iNOS-dependent restriction. The ability of virulent T. gondii to escape killing by macrophages is controlled at the level of the individual vacuole and is associated with differential permissiveness for association of the IRG proteins Irga6 (IIGP1) and Irgb6 (TGTP) to the vacuolar membrane. Surprisingly, expression of the Type I ROP-18 virulence determinant in an avirulent strain did not confer the evasive phenotype. These results pinpoint evasion of vacuolar disruption by IRG proteins as a new determinant of pathogen virulence.
PMID: 19265156 [PubMed - in process]
Virulent Toxoplasma gondii Evade Immunity-Related GTPase-Mediated Parasite Vacuole Disruption within Primed Macrophages
Zhao Y, Ferguson DJ, Wilson DC, Howard JC, Sibley LD, Yap GS.
Department of Medicine and Center for Immunity and Inflammation, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, NJ 07101.
Cytokine-activated macrophages restrain the replication of intracellular parasites and disrupt the integrity of vacuolar pathogens. In this study, we show that inducible nitric oxide synthase and the immunity-related GTPase (IRG) family member Irgm3, respectively, are required for the ability of in vivo primed macrophages to restrain the growth of Toxoplasma gondii and to destroy the parasite's intracellular niche. Remarkably, virulent Type I strains of T. gondii evade IRG-dependent vacuolar disruption, while remaining susceptible to iNOS-dependent restriction. The ability of virulent T. gondii to escape killing by macrophages is controlled at the level of the individual vacuole and is associated with differential permissiveness for association of the IRG proteins Irga6 (IIGP1) and Irgb6 (TGTP) to the vacuolar membrane. Surprisingly, expression of the Type I ROP-18 virulence determinant in an avirulent strain did not confer the evasive phenotype. These results pinpoint evasion of vacuolar disruption by IRG proteins as a new determinant of pathogen virulence.
PMID: 19265156 [PubMed - in process]
Friday, March 06, 2009
A4D12 monoclonal antibody recognizes a new linear epitope from SAG2A Toxoplasma gondii tachyzoites, identified by phage display bioselection
Immunobiology. 2009 Mar 2. [Epub ahead of print]
A4D12 monoclonal antibody recognizes a new linear epitope from SAG2A Toxoplasma gondii tachyzoites, identified by phage display bioselection
Cunha-Júnior JP, Silva DA, Silva NM, Souza MA, Souza GR, Prudencio CR, Pirovani CP, Cezar M Cascardo J, Barbosa BF, Goulart LR, Mineo JR.
Laboratory of Immunoparasitology, Institute of Biomedical Sciences, Federal University of Uberlândia, Brazil.
Toxoplasma gondii surface is coated by closely related antigens that belong to SRS (SAG-1 related sequences) superfamily. Two tachyzoite-specific SRS antigens, SAG1 and SAG2, are immunodominant proteins that apparently modulate the virulence of infection by inducing the host immune response against tachyzoites during the acute phase. In this study, we described a conformationally insensitive monoclonal antibody (A4D12mAb) that recognizes a linear epitope shared by two isoforms of p22 that is expressed in the surface of T. gondii tachyzoites. By using phage display approach and production of recombinant proteins, we clearly demonstrated that the A4D12mAb recognizes an epitope within C-terminal region of SAG2A. This mAb reacts with both T. gondii genotypes (I and II) but not with a closely related parasite, Neospora caninum. Also, the pretreatment of tachyzoites with A4D12 mAb did not inhibit T. gondii infection, suggesting that the epitope herein mapped is not crucial for tachyzoite invasion. However, a panel of human T. gondii positive sera showed significant degree of inhibition of A4D12 mAb reactivity against T. gondii native antigens, indicating that both A4D12 mAb and human sera recognize an overlapping immunodominant epitope within C-terminal region of SAG2A. To our knowledge, this is the first evidence using bioselection by phage display that identifies a T. gondii linear epitope recognized by a mAb specific to SAG2A. In conclusion, the results here presented add a new piece of information concerning T. gondii SAG2A molecule, emphasizing two dissimilar biological roles of this molecule, particularly for A4D12 epitope, suggesting that these characteristics may be important for parasite survival, since it is part of parasite components able to induce a strong immune response enough to allow host survival and establish long-term chronic infection.
PMID: 19261354 [PubMed - as supplied by publisher]
A4D12 monoclonal antibody recognizes a new linear epitope from SAG2A Toxoplasma gondii tachyzoites, identified by phage display bioselection
Cunha-Júnior JP, Silva DA, Silva NM, Souza MA, Souza GR, Prudencio CR, Pirovani CP, Cezar M Cascardo J, Barbosa BF, Goulart LR, Mineo JR.
Laboratory of Immunoparasitology, Institute of Biomedical Sciences, Federal University of Uberlândia, Brazil.
Toxoplasma gondii surface is coated by closely related antigens that belong to SRS (SAG-1 related sequences) superfamily. Two tachyzoite-specific SRS antigens, SAG1 and SAG2, are immunodominant proteins that apparently modulate the virulence of infection by inducing the host immune response against tachyzoites during the acute phase. In this study, we described a conformationally insensitive monoclonal antibody (A4D12mAb) that recognizes a linear epitope shared by two isoforms of p22 that is expressed in the surface of T. gondii tachyzoites. By using phage display approach and production of recombinant proteins, we clearly demonstrated that the A4D12mAb recognizes an epitope within C-terminal region of SAG2A. This mAb reacts with both T. gondii genotypes (I and II) but not with a closely related parasite, Neospora caninum. Also, the pretreatment of tachyzoites with A4D12 mAb did not inhibit T. gondii infection, suggesting that the epitope herein mapped is not crucial for tachyzoite invasion. However, a panel of human T. gondii positive sera showed significant degree of inhibition of A4D12 mAb reactivity against T. gondii native antigens, indicating that both A4D12 mAb and human sera recognize an overlapping immunodominant epitope within C-terminal region of SAG2A. To our knowledge, this is the first evidence using bioselection by phage display that identifies a T. gondii linear epitope recognized by a mAb specific to SAG2A. In conclusion, the results here presented add a new piece of information concerning T. gondii SAG2A molecule, emphasizing two dissimilar biological roles of this molecule, particularly for A4D12 epitope, suggesting that these characteristics may be important for parasite survival, since it is part of parasite components able to induce a strong immune response enough to allow host survival and establish long-term chronic infection.
PMID: 19261354 [PubMed - as supplied by publisher]
Thursday, March 05, 2009
Development of forward genetics in Toxoplasma gondii
Int J Parasitol. 2009 Feb 27. [Epub ahead of print]
Development of forward genetics in Toxoplasma gondii
Sibley LD.
Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Ave. St. Louis MO 63110 USA.
The development of forward genetics as a functional system in Toxoplasma gondii spanned more than three decades from the mid-1970s until now. The initial demonstration of experimental genetics relied on chemically-induced drug resistant mutants that were crossed by co-infecting cats, collecting oocysts, sporulating and hatching progeny in vitro. To capitalize on this, genetic markers were employed to develop linkage maps by tracking inheritance through experimental crosses. In all, three generations of genetic maps were developed to define the chromosomes, estimate recombination rates, and provide a system for linkage analysis. Ultimately this genetic map would become the foundation for the assembly of the T. gondii genome, which was derived from whole genome shotgun sequencing, into a chromosome-centric view. Finally, application of forward genetics to multigenic biological traits showed the potential to map and identify specific genes that control complex phenotypes including virulence.
PMID: 19254720 [PubMed - as supplied by publisher]
Development of forward genetics in Toxoplasma gondii
Sibley LD.
Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Ave. St. Louis MO 63110 USA.
The development of forward genetics as a functional system in Toxoplasma gondii spanned more than three decades from the mid-1970s until now. The initial demonstration of experimental genetics relied on chemically-induced drug resistant mutants that were crossed by co-infecting cats, collecting oocysts, sporulating and hatching progeny in vitro. To capitalize on this, genetic markers were employed to develop linkage maps by tracking inheritance through experimental crosses. In all, three generations of genetic maps were developed to define the chromosomes, estimate recombination rates, and provide a system for linkage analysis. Ultimately this genetic map would become the foundation for the assembly of the T. gondii genome, which was derived from whole genome shotgun sequencing, into a chromosome-centric view. Finally, application of forward genetics to multigenic biological traits showed the potential to map and identify specific genes that control complex phenotypes including virulence.
PMID: 19254720 [PubMed - as supplied by publisher]
AZITHROMYCIN REDUCES OCULAR INFECTION DURING CONGENITAL TRANSMISSION OF TOXOPLASMOSIS IN THE Calomys Callosus MODEL
J Parasitol. 2009 Mar 2:1. [Epub ahead of print]
AZITHROMYCIN REDUCES OCULAR INFECTION DURING CONGENITAL TRANSMISSION OF TOXOPLASMOSIS IN THE Calomys Callosus MODEL
Lopes CD, Silva NM, Ferro EA, Sousa RA, Firmino ML, Bernardes ES, Roque-Barreira MC, Pena JD.
Toxoplasma gondii is a widely distributed obligatory intracellular parasite that causes severe disease to the fetus when transmitted during pregnancy. Drugs used to avoid congenital transmission have shown side effects and their efficacy is controversial. The most widely used drug for the treatment of acute toxoplasmosis during pregnancy is the association between pyrimethamine and sulfadiazine, which has several side effects. In this work we tested the efficacy of azithromycin in reducing congenital transmission of Toxoplasma in the rodent Calomys callosus. Females of C. callosus were inoculated perorally with 20 cysts of ME49 strain of T. gondii on the day of fertilization and fetuses were collected from the 15th to the 19th day of gestation. Azithromycin (300mg/kg) or association with pyrimethamine (100 or 50 mg/Kg) and sulfadiazine (100 or 75mg/kg) and folinic acid (15mg/kg) (SPAf) or vehicle was administered orally in different days after infection. Brain and ocular tissues were removed and processed for immunohistochemistry using a polyclonal antibody against T. gondii, or processed for parasite DNA quantification. Toxoplasma gondii was detected in the brains of all females and fetuses' eyes when treated with SPAf. On the other hand, in females treated with azithromycin, there was a reduction of T. gondii in the brains of mothers and no parasites were detected in eyes of fetuses, indicating that azithromycin may represent an alternative treatment for toxoplasmosis during pregnancy.
PMID: 19254072 [PubMed - as supplied by publisher]
AZITHROMYCIN REDUCES OCULAR INFECTION DURING CONGENITAL TRANSMISSION OF TOXOPLASMOSIS IN THE Calomys Callosus MODEL
Lopes CD, Silva NM, Ferro EA, Sousa RA, Firmino ML, Bernardes ES, Roque-Barreira MC, Pena JD.
Toxoplasma gondii is a widely distributed obligatory intracellular parasite that causes severe disease to the fetus when transmitted during pregnancy. Drugs used to avoid congenital transmission have shown side effects and their efficacy is controversial. The most widely used drug for the treatment of acute toxoplasmosis during pregnancy is the association between pyrimethamine and sulfadiazine, which has several side effects. In this work we tested the efficacy of azithromycin in reducing congenital transmission of Toxoplasma in the rodent Calomys callosus. Females of C. callosus were inoculated perorally with 20 cysts of ME49 strain of T. gondii on the day of fertilization and fetuses were collected from the 15th to the 19th day of gestation. Azithromycin (300mg/kg) or association with pyrimethamine (100 or 50 mg/Kg) and sulfadiazine (100 or 75mg/kg) and folinic acid (15mg/kg) (SPAf) or vehicle was administered orally in different days after infection. Brain and ocular tissues were removed and processed for immunohistochemistry using a polyclonal antibody against T. gondii, or processed for parasite DNA quantification. Toxoplasma gondii was detected in the brains of all females and fetuses' eyes when treated with SPAf. On the other hand, in females treated with azithromycin, there was a reduction of T. gondii in the brains of mothers and no parasites were detected in eyes of fetuses, indicating that azithromycin may represent an alternative treatment for toxoplasmosis during pregnancy.
PMID: 19254072 [PubMed - as supplied by publisher]
Tuesday, March 03, 2009
The role played by electron microscopy in advancing our understanding of Toxoplasma gondii and other apicomplexans
Int J Parasitol. 2009 Feb 25. [Epub ahead of print]
The role played by electron microscopy in advancing our understanding of Toxoplasma gondii and other apicomplexans
Dubremetz JF, Ferguson DJ.
UMR CNRS 5235, Bt 24, CC 107, Université de Montpellier 2, Place Eugène Bataillon, 34095 Montpellier cedex 05 France.
In many ways the history of the discovery of the life cycle of Toxoplasma gondii and the development of biological electron microscopy progressed in parallel through the 1950s and 1960s. Although Toxoplasma was discovered in 1908, it was only in the 1950s that the extent of the infection in humans and domestic animals was realised and work was undertaken to elucidate its life cycle (reviewed elsewhere in this edition). The development of ultrastructural techniques and their application to biological systems including Toxoplasma developed over the same period. This resulted in a synergistic effect with the re-classification of previously unrelated parasites within a single phylum, the Apicomplexa, which was based on the ultrastructural appearances of the infectious stages. This review will describe the central role played by electron microscopy and Toxoplasma in the developments associated with this progress.
PMID: 19249305 [PubMed - as supplied by publisher]
The role played by electron microscopy in advancing our understanding of Toxoplasma gondii and other apicomplexans
Dubremetz JF, Ferguson DJ.
UMR CNRS 5235, Bt 24, CC 107, Université de Montpellier 2, Place Eugène Bataillon, 34095 Montpellier cedex 05 France.
In many ways the history of the discovery of the life cycle of Toxoplasma gondii and the development of biological electron microscopy progressed in parallel through the 1950s and 1960s. Although Toxoplasma was discovered in 1908, it was only in the 1950s that the extent of the infection in humans and domestic animals was realised and work was undertaken to elucidate its life cycle (reviewed elsewhere in this edition). The development of ultrastructural techniques and their application to biological systems including Toxoplasma developed over the same period. This resulted in a synergistic effect with the re-classification of previously unrelated parasites within a single phylum, the Apicomplexa, which was based on the ultrastructural appearances of the infectious stages. This review will describe the central role played by electron microscopy and Toxoplasma in the developments associated with this progress.
PMID: 19249305 [PubMed - as supplied by publisher]
The early years of Toxoplasma research: What's past is prologue
Int J Parasitol. 2009 Feb 26. [Epub ahead of print]
The early years of Toxoplasma research: What's past is prologue
Morrissette NS, Ajioka JW.
Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine CA, 92697 USA.
In the century since the first description of Toxoplasma gondii, history and circumstance have led scientists to define this organism in diverse contexts. From its discovery by researchers shaped by early 20th century versions of the germ theory to its more recent roles as an important globally distributed pathogen and a model apicomplexan, our definitions of Toxoplasma are as much a reflection of our frame of reference as they are an absolute definition of this organism. Although these transformations act as portals for new avenues of investigation, the essential questions that inform current research are founded in the work of early investigators who studied Toxoplasma.
PMID: 19250939 [PubMed - as supplied by publisher]
The early years of Toxoplasma research: What's past is prologue
Morrissette NS, Ajioka JW.
Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine CA, 92697 USA.
In the century since the first description of Toxoplasma gondii, history and circumstance have led scientists to define this organism in diverse contexts. From its discovery by researchers shaped by early 20th century versions of the germ theory to its more recent roles as an important globally distributed pathogen and a model apicomplexan, our definitions of Toxoplasma are as much a reflection of our frame of reference as they are an absolute definition of this organism. Although these transformations act as portals for new avenues of investigation, the essential questions that inform current research are founded in the work of early investigators who studied Toxoplasma.
PMID: 19250939 [PubMed - as supplied by publisher]
Saturday, February 28, 2009
Export of a Toxoplasma gondii Rhoptry Neck Protein Complex at the Host Cell Membrane to Form the Moving Junction during Invasion
PLoS Pathog. 2009 Feb;5(2):e1000309. Epub 2009 Feb 27
Export of a Toxoplasma gondii Rhoptry Neck Protein Complex at the Host Cell Membrane to Form the Moving Junction during Invasion
Besteiro S, Michelin A, Poncet J, Dubremetz JF, Lebrun M.
UMR 5235 CNRS, Université de Montpellier 2, Montpelier, France.
One of the most conserved features of the invasion process in Apicomplexa parasites is the formation of a moving junction (MJ) between the apex of the parasite and the host cell membrane that moves along the parasite and serves as support to propel it inside the host cell. The MJ was, up to a recent period, completely unknown at the molecular level. Recently, proteins originated from two distinct post-Golgi specialised secretory organelles, the micronemes (for AMA1) and the neck of the rhoptries (for RON2/RON4/RON5 proteins), have been shown to form a complex. AMA1 and RON4 in particular, have been localised to the MJ during invasion. Using biochemical approaches, we have identified RON8 as an additional member of the complex. We also demonstrated that all RON proteins are present at the MJ during invasion. Using metabolic labelling and immunoprecipitation, we showed that RON2 and AMA1 were able to interact in the absence of the other members. We also discovered that all MJ proteins are subjected to proteolytic maturation during trafficking to their respective organelles and that they could associate as non-mature forms in vitro. Finally, whereas AMA1 has previously been shown to be inserted into the parasite membrane upon secretion, we demonstrated, using differential permeabilization and loading of RON-specific antibodies into the host cell, that the RON complex is targeted to the host cell membrane, where RON4/5/8 remain associated with the cytoplasmic face. Globally, these results point toward a model of MJ organization where the parasite would be secreting and inserting interacting components on either side of the MJ, both at the host and at its own plasma membranes.
PMID: 19247437 [PubMed - in process]
Export of a Toxoplasma gondii Rhoptry Neck Protein Complex at the Host Cell Membrane to Form the Moving Junction during Invasion
Besteiro S, Michelin A, Poncet J, Dubremetz JF, Lebrun M.
UMR 5235 CNRS, Université de Montpellier 2, Montpelier, France.
One of the most conserved features of the invasion process in Apicomplexa parasites is the formation of a moving junction (MJ) between the apex of the parasite and the host cell membrane that moves along the parasite and serves as support to propel it inside the host cell. The MJ was, up to a recent period, completely unknown at the molecular level. Recently, proteins originated from two distinct post-Golgi specialised secretory organelles, the micronemes (for AMA1) and the neck of the rhoptries (for RON2/RON4/RON5 proteins), have been shown to form a complex. AMA1 and RON4 in particular, have been localised to the MJ during invasion. Using biochemical approaches, we have identified RON8 as an additional member of the complex. We also demonstrated that all RON proteins are present at the MJ during invasion. Using metabolic labelling and immunoprecipitation, we showed that RON2 and AMA1 were able to interact in the absence of the other members. We also discovered that all MJ proteins are subjected to proteolytic maturation during trafficking to their respective organelles and that they could associate as non-mature forms in vitro. Finally, whereas AMA1 has previously been shown to be inserted into the parasite membrane upon secretion, we demonstrated, using differential permeabilization and loading of RON-specific antibodies into the host cell, that the RON complex is targeted to the host cell membrane, where RON4/5/8 remain associated with the cytoplasmic face. Globally, these results point toward a model of MJ organization where the parasite would be secreting and inserting interacting components on either side of the MJ, both at the host and at its own plasma membranes.
PMID: 19247437 [PubMed - in process]
Manipulative parasites in the world of veterinary science: Implications for epidemiology and pathology
Vet J. 2009 Feb 23. [Epub ahead of print]
Manipulative parasites in the world of veterinary science: Implications for epidemiology and pathology
Lagrue C, Poulin R.
Department of Zoology, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand.
One of the most complex and least understood transmission strategies displayed by pathogenic parasites is that of manipulation of host behaviour. A wide variety of parasites alter their host's behaviour, including species of medical and veterinary importance, such as Diplostomum spathaceum, Echinococcus spp. and Toxoplasma gondii. The manipulative ability of these parasites has implications for pathology and transmission dynamics. Domestic animals are hosts for manipulative pathogens, either by being the target host and acquiring the parasite as a result of vector-host manipulation, or by having their behaviour changed by manipulative parasites. This review uses several well-known pathogens to demonstrate how host manipulation by parasites is potentially important in epidemiology.
PMID: 19243982 [PubMed - as supplied by publisher]
Manipulative parasites in the world of veterinary science: Implications for epidemiology and pathology
Lagrue C, Poulin R.
Department of Zoology, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand.
One of the most complex and least understood transmission strategies displayed by pathogenic parasites is that of manipulation of host behaviour. A wide variety of parasites alter their host's behaviour, including species of medical and veterinary importance, such as Diplostomum spathaceum, Echinococcus spp. and Toxoplasma gondii. The manipulative ability of these parasites has implications for pathology and transmission dynamics. Domestic animals are hosts for manipulative pathogens, either by being the target host and acquiring the parasite as a result of vector-host manipulation, or by having their behaviour changed by manipulative parasites. This review uses several well-known pathogens to demonstrate how host manipulation by parasites is potentially important in epidemiology.
PMID: 19243982 [PubMed - as supplied by publisher]
Thursday, February 26, 2009
Guanidine hydrochloride- and urea- induced unfolding of Toxoplasma gondii Ferredoxin- NADP+ reductase
J Biochem. 2009 Feb 23. [Epub ahead of print]
Guanidine hydrochloride- and urea- induced unfolding of Toxoplasma gondii Ferredoxin- NADP+ reductase: stabilization of a functionally inactive holo-intermediate.
Singh K, Bhakuni V.
Division of Molecular and Structural Biology Central Drug Research Institute Lucknow- 226 001, India.
Usually during the folding/unfolding of flavoproteins an apo-intermediate is stabilized before global unfolding of the enzymes occurs. However, stabilization of a holo-intermediate has also been reported for a few flavoproteins. We have studied the unfolding of Toxoplasma gondii Ferredoxin- NADP(+) reductase (TgFNR) using GdnHCl and urea. A functionally inactive holo-intermediate of the enzyme was found to be stabilized during this unfolding process. The intermediate species had cofactor FAD bound to it, but it showed free movement due to which the stabilized intermediates were functionally inactive. The native TgFNR behaves cooperatively with the two structural domains interacting strongly with each other. The denaturants GdnHCl and urea, at low concentrations were found to interact selectively with the NADP(+)- binding domain of TgFNR and induce structural modifications in it. These selective modifications in the protein molecule lead to loss of interactions between two domains and the enzyme behaved non-cooperatively resulting in stabilization of an intermediate species. Significant differences in the structural properties of the GdnHCl- and urea-stabilized holo-intermediates of TgFNR were observed. Comparison of the unfolding pathway of TgFNR (a plant-type FNR) with that of FprA (a GR-type FNR) demonstrates that they follow very different pathways of unfolding.
PMID: 19237441 [PubMed - as supplied by publisher]
Guanidine hydrochloride- and urea- induced unfolding of Toxoplasma gondii Ferredoxin- NADP+ reductase: stabilization of a functionally inactive holo-intermediate.
Singh K, Bhakuni V.
Division of Molecular and Structural Biology Central Drug Research Institute Lucknow- 226 001, India.
Usually during the folding/unfolding of flavoproteins an apo-intermediate is stabilized before global unfolding of the enzymes occurs. However, stabilization of a holo-intermediate has also been reported for a few flavoproteins. We have studied the unfolding of Toxoplasma gondii Ferredoxin- NADP(+) reductase (TgFNR) using GdnHCl and urea. A functionally inactive holo-intermediate of the enzyme was found to be stabilized during this unfolding process. The intermediate species had cofactor FAD bound to it, but it showed free movement due to which the stabilized intermediates were functionally inactive. The native TgFNR behaves cooperatively with the two structural domains interacting strongly with each other. The denaturants GdnHCl and urea, at low concentrations were found to interact selectively with the NADP(+)- binding domain of TgFNR and induce structural modifications in it. These selective modifications in the protein molecule lead to loss of interactions between two domains and the enzyme behaved non-cooperatively resulting in stabilization of an intermediate species. Significant differences in the structural properties of the GdnHCl- and urea-stabilized holo-intermediates of TgFNR were observed. Comparison of the unfolding pathway of TgFNR (a plant-type FNR) with that of FprA (a GR-type FNR) demonstrates that they follow very different pathways of unfolding.
PMID: 19237441 [PubMed - as supplied by publisher]
Friday, February 20, 2009
GRA2 and ROP1 recombinant antigens as potential markers for detection of Toxoplasma gondii-specific immunoglobulin G in human with acute toxoplasmosis
Clin Vaccine Immunol. 2009 Feb 18. [Epub ahead of print]
GRA2 and ROP1 recombinant antigens as potential markers for detection of Toxoplasma gondii-specific immunoglobulin G in human with acute toxoplasmosis
Holec-Gasior L, Kur J, Hiszczynska-Sawicka E.
Gdask University of Technology, Chemical Faculty, Department of Microbiology, Gdask, POLAND.
A goal of the current study was to evaluate serological applications of Toxoplasma gondii GRA2 and ROP1 antigens. Soluble recombinant GRA2 and ROP1 antigens as fusion proteins containing six histidyl residues at N- and C-terminal were obtained using an Escherichia coli expression system. Purification by a one-step metal affinity chromatography allowed recovery of milligram amounts of pure recombinant proteins per litre of culture. The usefulness of these antigens for diagnosis of human infections was tested on 167 serum samples obtained during routine diagnostic tests. A panel of 37 sera from patients with acute toxoplasmosis was compared to a panel of 90 sera from individuals with past infection. The results indicate that both GRA2 and ROP1 recombinant antigens detected antibodies more frequently from acute (100% and 94.6%, respectively) than from chronic infections (22.5% and 15.5% respectively). These results suggest that IgG antibodies against GRA2 and ROP1 antigens are produced during the acute stage of toxoplasmosis but are uncommon in the chronic phase of the infection. Hence, these recombinant proteins can be used as specific molecular markers to differentiate between acute and chronic infections.
PMID: 19225074 [PubMed - as supplied by publisher]
GRA2 and ROP1 recombinant antigens as potential markers for detection of Toxoplasma gondii-specific immunoglobulin G in human with acute toxoplasmosis
Holec-Gasior L, Kur J, Hiszczynska-Sawicka E.
Gdask University of Technology, Chemical Faculty, Department of Microbiology, Gdask, POLAND.
A goal of the current study was to evaluate serological applications of Toxoplasma gondii GRA2 and ROP1 antigens. Soluble recombinant GRA2 and ROP1 antigens as fusion proteins containing six histidyl residues at N- and C-terminal were obtained using an Escherichia coli expression system. Purification by a one-step metal affinity chromatography allowed recovery of milligram amounts of pure recombinant proteins per litre of culture. The usefulness of these antigens for diagnosis of human infections was tested on 167 serum samples obtained during routine diagnostic tests. A panel of 37 sera from patients with acute toxoplasmosis was compared to a panel of 90 sera from individuals with past infection. The results indicate that both GRA2 and ROP1 recombinant antigens detected antibodies more frequently from acute (100% and 94.6%, respectively) than from chronic infections (22.5% and 15.5% respectively). These results suggest that IgG antibodies against GRA2 and ROP1 antigens are produced during the acute stage of toxoplasmosis but are uncommon in the chronic phase of the infection. Hence, these recombinant proteins can be used as specific molecular markers to differentiate between acute and chronic infections.
PMID: 19225074 [PubMed - as supplied by publisher]
Wednesday, February 18, 2009
A Dynamin Is Required for the Biogenesis of Secretory Organelles in Toxoplasma gondii
Curr Biol. 2009 Feb 11. [Epub ahead of print]
A Dynamin Is Required for the Biogenesis of Secretory Organelles in Toxoplasma gondii
Breinich MS, Ferguson DJ, Foth BJ, van Dooren GG, Lebrun M, Quon DV, Striepen B, Bradley PJ, Frischknecht F, Carruthers VB, Meissner M.
Hygiene Institute, Department of Parasitology, Heidelberg University School of Medicine, Heidelberg 69120, Germany.
BACKGROUND: Apicomplexans contain only a core set of factors involved in vesicular traffic. Yet these obligate intracellular parasites evolved a set of unique secretory organelles (micronemes, rhoptries, and dense granules) that are required for invasion and modulation of the host cell. Apicomplexa replicate by budding from or within a single mother cell, and secretory organelles are synthesized de novo at the final stage of division. To date, the molecular basis for their biogenesis is unknown. RESULTS: We demonstrate that the apicomplexan dynamin-related protein B (DrpB) belongs to an alveolate specific family of dynamins that is expanded in ciliates. DrpB accumulates in a cytoplasmic region close to the Golgi that breaks up during replication and reforms after assembly of the daughter cells. Conditional ablation of DrpB function results in mature daughter parasites that are devoid of micronemes and rhoptries. In the absence of these organelles, invasion-related secretory proteins are mistargeted to the constitutive secretory pathway. Mutant parasites are able to replicate but are unable to escape from or invade into host cells. CONCLUSIONS: DrpB is the essential mechanoenzyme for the biogenesis of secretory organelles in Apicomplexa. We suggest that DrpB is required during replication to generate vesicles for the regulated secretory pathway that form the unique secretory organelles. Our study supports a role of an alveolate-specific dynamin that was required for the evolution of novel, secretory organelles. In the case of Apicomplexa, these organelles further evolved to enable a parasitic lifestyle.
PMID: 19217293 [PubMed - as supplied by publisher]
A Dynamin Is Required for the Biogenesis of Secretory Organelles in Toxoplasma gondii
Breinich MS, Ferguson DJ, Foth BJ, van Dooren GG, Lebrun M, Quon DV, Striepen B, Bradley PJ, Frischknecht F, Carruthers VB, Meissner M.
Hygiene Institute, Department of Parasitology, Heidelberg University School of Medicine, Heidelberg 69120, Germany.
BACKGROUND: Apicomplexans contain only a core set of factors involved in vesicular traffic. Yet these obligate intracellular parasites evolved a set of unique secretory organelles (micronemes, rhoptries, and dense granules) that are required for invasion and modulation of the host cell. Apicomplexa replicate by budding from or within a single mother cell, and secretory organelles are synthesized de novo at the final stage of division. To date, the molecular basis for their biogenesis is unknown. RESULTS: We demonstrate that the apicomplexan dynamin-related protein B (DrpB) belongs to an alveolate specific family of dynamins that is expanded in ciliates. DrpB accumulates in a cytoplasmic region close to the Golgi that breaks up during replication and reforms after assembly of the daughter cells. Conditional ablation of DrpB function results in mature daughter parasites that are devoid of micronemes and rhoptries. In the absence of these organelles, invasion-related secretory proteins are mistargeted to the constitutive secretory pathway. Mutant parasites are able to replicate but are unable to escape from or invade into host cells. CONCLUSIONS: DrpB is the essential mechanoenzyme for the biogenesis of secretory organelles in Apicomplexa. We suggest that DrpB is required during replication to generate vesicles for the regulated secretory pathway that form the unique secretory organelles. Our study supports a role of an alveolate-specific dynamin that was required for the evolution of novel, secretory organelles. In the case of Apicomplexa, these organelles further evolved to enable a parasitic lifestyle.
PMID: 19217293 [PubMed - as supplied by publisher]
A Novel Dynamin-Related Protein Has Been Recruited for Apicoplast Fission in Toxoplasma gondii
Curr Biol. 2009 Feb 11. [Epub ahead of print]
A Novel Dynamin-Related Protein Has Been Recruited for Apicoplast Fission in Toxoplasma gondii
van Dooren GG, Reiff SB, Tomova C, Meissner M, Humbel BM, Striepen B.
Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA 30602, USA.
Background: Apicomplexan parasites cause numerous important human diseases, including malaria and toxoplasmosis. Apicomplexa belong to the Alveolata, a group that also includes ciliates and dinoflagellates. Apicomplexa retain a plastid organelle (the apicoplast) that was derived from an endosymbiotic relationship between the alveolate ancestor and a red alga. Apicoplasts are essential for parasite growth and must correctly divide and segregate into daughter cells upon cytokinesis. Apicoplast division depends on association with the mitotic spindle, although little is known about the molecular machinery involved in this process. Apicoplasts lack the conserved machinery that divides chloroplasts in plants and red algae, suggesting that these mechanisms are unique. Results: Here, we demonstrate that a dynamin-related protein in Toxoplasma gondii (TgDrpA) localizes to punctate regions on the apicoplast surface. We generate a conditional dominant-negative TgDrpA cell line to disrupt TgDrpA functions and demonstrate that TgDrpA is essential for parasite growth and apicoplast biogenesis. Fluorescence recovery after photobleaching and time-lapse imaging studies provide evidence for a direct role for TgDrpA in apicoplast fission. Conclusions: Our data suggest that DrpA was likely recruited from the alveolate ancestor to function in fission of the symbiont and ultimately replaced the conserved division machinery of that symbiont.
PMID: 19217294 [PubMed - as supplied by publisher]
A Novel Dynamin-Related Protein Has Been Recruited for Apicoplast Fission in Toxoplasma gondii
van Dooren GG, Reiff SB, Tomova C, Meissner M, Humbel BM, Striepen B.
Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA 30602, USA.
Background: Apicomplexan parasites cause numerous important human diseases, including malaria and toxoplasmosis. Apicomplexa belong to the Alveolata, a group that also includes ciliates and dinoflagellates. Apicomplexa retain a plastid organelle (the apicoplast) that was derived from an endosymbiotic relationship between the alveolate ancestor and a red alga. Apicoplasts are essential for parasite growth and must correctly divide and segregate into daughter cells upon cytokinesis. Apicoplast division depends on association with the mitotic spindle, although little is known about the molecular machinery involved in this process. Apicoplasts lack the conserved machinery that divides chloroplasts in plants and red algae, suggesting that these mechanisms are unique. Results: Here, we demonstrate that a dynamin-related protein in Toxoplasma gondii (TgDrpA) localizes to punctate regions on the apicoplast surface. We generate a conditional dominant-negative TgDrpA cell line to disrupt TgDrpA functions and demonstrate that TgDrpA is essential for parasite growth and apicoplast biogenesis. Fluorescence recovery after photobleaching and time-lapse imaging studies provide evidence for a direct role for TgDrpA in apicoplast fission. Conclusions: Our data suggest that DrpA was likely recruited from the alveolate ancestor to function in fission of the symbiont and ultimately replaced the conserved division machinery of that symbiont.
PMID: 19217294 [PubMed - as supplied by publisher]
Toxoplasma gondii: congenital transmission in a hamster model
Exp Parasitol. 2009 Feb 12. [Epub ahead of print]
Toxoplasma gondii: congenital transmission in a hamster model
Freyre A, Fialho CG, Bigatti LE, Araujo FA, Falcón JD, Mendez J, González M.
Laboratorio de Toxoplasmosis, Departamento de Parasitologi a, Facultad de Veterinaria, Montevideo, Uruguay.
The objective of the research was to test the hamster for a model of transmission of congenital toxoplasmosis. A non invasive method for the diagnosis of pregnancy in hamsters was designed, with a specificity and a sensitivity of 70.2 and 94.7%, respectively (n=168). Of 32 females with a chronic toxoplasma infection, 3 transmitted Toxoplasma congenitally during their first pregnancy, but not during the subsequent pregnancy. Congenital transmission rates of infections initiated during pregnancy with 2 stages of 2 strains of Toxoplasma were in the range of 33 to 100% of the 76 females inoculated . Only 1 of 17 females transmitted the parasite exclusively via milk. It was concluded that the hamster is a promising species for a model of transmission of congenital toxoplasmosis.
PMID: 19217907 [PubMed - as supplied by publisher]
Toxoplasma gondii: congenital transmission in a hamster model
Freyre A, Fialho CG, Bigatti LE, Araujo FA, Falcón JD, Mendez J, González M.
Laboratorio de Toxoplasmosis, Departamento de Parasitologi a, Facultad de Veterinaria, Montevideo, Uruguay.
The objective of the research was to test the hamster for a model of transmission of congenital toxoplasmosis. A non invasive method for the diagnosis of pregnancy in hamsters was designed, with a specificity and a sensitivity of 70.2 and 94.7%, respectively (n=168). Of 32 females with a chronic toxoplasma infection, 3 transmitted Toxoplasma congenitally during their first pregnancy, but not during the subsequent pregnancy. Congenital transmission rates of infections initiated during pregnancy with 2 stages of 2 strains of Toxoplasma were in the range of 33 to 100% of the 76 females inoculated . Only 1 of 17 females transmitted the parasite exclusively via milk. It was concluded that the hamster is a promising species for a model of transmission of congenital toxoplasmosis.
PMID: 19217907 [PubMed - as supplied by publisher]
Toxoplasmosis: a history of clinical observations
Int J Parasitol. 2009 Feb 12. [Epub ahead of print]
Toxoplasmosis: a history of clinical observations
Weiss LM, Dubey JP.
Departments of Medicine (Division of Infectious Diseases) and Pathology (Division of Parasitology),Albert Einstein College of Medicine,,1300 Morris Park Avenue, Forchheimer 504, Bronx, NY 10461, USA.
It has been 100 years since Toxoplasma gondii was initially described in Tunis by Nicolle and Manceaux (1908) in the tissues of the gundi (Ctenodoactylus gundi) and in Brazil by Splendore (1908) in the tissues of a rabbit. Toxoplasma gondii is a ubiquitous, Apicomplexan parasite of warm-blooded animals that can cause several clinical syndromes including encephalitis, chorioretinitis, congenital infection and neonatal mortality. Fifteen years after the description of T. gondii by Nicolle and Manceaux a fatal case of toxoplasmosis in a child was reported by Janků. In 1939 Wolf, Cowen and Paige were the first to conclusively identify T. gondii as a cause of human disease. This review examines the clinical manifestations of infection with T. gondii and the history of the discovery of these manifestations.
PMID: 19217908 [PubMed - as supplied by publisher]
Toxoplasmosis: a history of clinical observations
Weiss LM, Dubey JP.
Departments of Medicine (Division of Infectious Diseases) and Pathology (Division of Parasitology),Albert Einstein College of Medicine,,1300 Morris Park Avenue, Forchheimer 504, Bronx, NY 10461, USA.
It has been 100 years since Toxoplasma gondii was initially described in Tunis by Nicolle and Manceaux (1908) in the tissues of the gundi (Ctenodoactylus gundi) and in Brazil by Splendore (1908) in the tissues of a rabbit. Toxoplasma gondii is a ubiquitous, Apicomplexan parasite of warm-blooded animals that can cause several clinical syndromes including encephalitis, chorioretinitis, congenital infection and neonatal mortality. Fifteen years after the description of T. gondii by Nicolle and Manceaux a fatal case of toxoplasmosis in a child was reported by Janků. In 1939 Wolf, Cowen and Paige were the first to conclusively identify T. gondii as a cause of human disease. This review examines the clinical manifestations of infection with T. gondii and the history of the discovery of these manifestations.
PMID: 19217908 [PubMed - as supplied by publisher]
Sexual recombination punctuated by outbreaks and clonal expansions predicts Toxoplasma gondii population genetics
Int J Parasitol. 2009 Feb 12. [Epub ahead of print]
Sexual recombination punctuated by outbreaks and clonal expansions predicts Toxoplasma gondii population genetics
Grigg ME, Sundar N.
Molecular Parasitology Unit, Laboratory of Parasitic Diseases, NIAID, NIH, Bethesda MD, 20815, USA.
The cosmopolitan parasitic pathogen Toxoplasma gondii is capable of infecting essentially any warm-blooded vertebrate worldwide, including most birds and mammals, and establishes chronic infections in one-third of the globe's human population. The success of this highly prevalent zoonosis is largely the result of its ability to propagate both sexually and clonally. Frequent genetic exchanges via sexual recombination among extant parasite lineages that mix in the definitive felid host produces new lines that emerge to expand the parasite's host range and cause outbreaks. Highly successful lines spread clonally via carnivorism and in some cases sweep to pandemic levels. The extent to which sexual reproduction versus clonal expansion shapes Toxoplasma's current, global population genetic structure is the central question this review will attempt to answer.
PMID: 19217909 [PubMed - as supplied by publisher]
Sexual recombination punctuated by outbreaks and clonal expansions predicts Toxoplasma gondii population genetics
Grigg ME, Sundar N.
Molecular Parasitology Unit, Laboratory of Parasitic Diseases, NIAID, NIH, Bethesda MD, 20815, USA.
The cosmopolitan parasitic pathogen Toxoplasma gondii is capable of infecting essentially any warm-blooded vertebrate worldwide, including most birds and mammals, and establishes chronic infections in one-third of the globe's human population. The success of this highly prevalent zoonosis is largely the result of its ability to propagate both sexually and clonally. Frequent genetic exchanges via sexual recombination among extant parasite lineages that mix in the definitive felid host produces new lines that emerge to expand the parasite's host range and cause outbreaks. Highly successful lines spread clonally via carnivorism and in some cases sweep to pandemic levels. The extent to which sexual reproduction versus clonal expansion shapes Toxoplasma's current, global population genetic structure is the central question this review will attempt to answer.
PMID: 19217909 [PubMed - as supplied by publisher]
Efficient gene replacements in Toxoplasma gondii strains deficient for nonhomologous end-joining
Eukaryot Cell. 2009 Feb 13. [Epub ahead of print]
Efficient gene replacements in Toxoplasma gondii strains deficient for nonhomologous end-joining
Fox BA, Ristuccia JG, Gigley JP, Bzik DJ.
Department of Microbiology and Immunology, Dartmouth Medical School, 1 Medical Center Drive, Lebanon, NH 03756 USA.
A high frequency of nonhomologous recombination has hampered gene targeting approaches in the model Apicomplexan parasite Toxoplasma gondii. To address whether the nonhomologous end-joining (NHEJ) DNA repair pathway could be disrupted in this obligate intracellular parasite, putative KU proteins were identified and a predicted KU80 gene was deleted. The efficiency of gene targeting via double cross-over homologous recombination at several genetic loci was found to be greater than 97% of the total transformants in KU80 knockouts. Gene replacement efficiency was markedly increased (300 to 400-fold) in KU80 knockouts compared to wild-type strains. Target DNA flanks of only approximately 500 bp were found to be sufficient for efficient gene replacements in KU80 knockouts. KU80 knockouts stably retained a normal growth rate in vitro and the high virulence phenotype of Type I strains, but exhibited an increased sensitivity to double-strand DNA breaks induced by treatment with phleomycin or gamma-irradiation. Collectively, these results revealed that a significant KU-dependent NHEJ DNA repair pathway was present in Toxoplasma gondii. Integration essentially occurs only at the homologous targeted sites in the KU80 knockout background, making this genetic background an efficient host for gene targeting to speed post-genome functional analysis and genetic dissection of parasite biology.
PMID: 19218423 [PubMed - as supplied by publisher]
Efficient gene replacements in Toxoplasma gondii strains deficient for nonhomologous end-joining
Fox BA, Ristuccia JG, Gigley JP, Bzik DJ.
Department of Microbiology and Immunology, Dartmouth Medical School, 1 Medical Center Drive, Lebanon, NH 03756 USA.
A high frequency of nonhomologous recombination has hampered gene targeting approaches in the model Apicomplexan parasite Toxoplasma gondii. To address whether the nonhomologous end-joining (NHEJ) DNA repair pathway could be disrupted in this obligate intracellular parasite, putative KU proteins were identified and a predicted KU80 gene was deleted. The efficiency of gene targeting via double cross-over homologous recombination at several genetic loci was found to be greater than 97% of the total transformants in KU80 knockouts. Gene replacement efficiency was markedly increased (300 to 400-fold) in KU80 knockouts compared to wild-type strains. Target DNA flanks of only approximately 500 bp were found to be sufficient for efficient gene replacements in KU80 knockouts. KU80 knockouts stably retained a normal growth rate in vitro and the high virulence phenotype of Type I strains, but exhibited an increased sensitivity to double-strand DNA breaks induced by treatment with phleomycin or gamma-irradiation. Collectively, these results revealed that a significant KU-dependent NHEJ DNA repair pathway was present in Toxoplasma gondii. Integration essentially occurs only at the homologous targeted sites in the KU80 knockout background, making this genetic background an efficient host for gene targeting to speed post-genome functional analysis and genetic dissection of parasite biology.
PMID: 19218423 [PubMed - as supplied by publisher]
Tagging of endogenous genes in a Toxoplasma gondii strain lacking Ku80
Eukaryot Cell. 2009 Feb 13. [Epub ahead of print]
Tagging of endogenous genes in a Toxoplasma gondii strain lacking Ku80
Huynh MH, Carruthers VB.
Department of Microbiology and Immunology, University of Michigan School of Medicine, 1150 W. Medical Center Dr., Ann Arbor, MI 48109 USA.
As with other organisms with a completed genome sequence, opportunities for performing large scale studies, such as expression and localization, on Toxoplasma gondii are now much more feasible. We present a system for tagging genes endogenously with yellow fluorescent protein (YFP) in a Deltaku80 strain. Ku80 is involved in DNA strand repair and non-homologous DNA end-joining; previous studies in other organisms have shown that in its absence, random integration is eliminated, allowing the insertion of constructs with homologous sequences into the proper loci. We generated a vector consisting of YFP and a DHFR-TS selectable marker. The YFP is preceded by a LIC (ligation-independent cloning) cassette, which allows the insertion of PCR products containing complementary LIC sequences. We demonstrate that the Deltaku80 strain is more effective and efficient in integrating the YFP-tagged genes into the correct locus compared to the wild-type strain RH. We then selected several hypothetical proteins that were identified by a proteomic screen of excreted-secreted antigens (ESA) and that displayed microarray expression profiles similar to known micronemal proteins, with the thought that these could potentially be new proteins with roles in cell invasion. We localized these hypothetical proteins by YFP fluorescence and show expression by immunoblotting. Our findings demonstrate that the combination of the Deltaku80 strain and the pYFP.LIC constructs reduces both the time and cost required to determine localization of a new gene of interest. This should open the opportunity for performing larger scale studies of novel T. gondii genes.
PMID: 19218426 [PubMed - as supplied by publisher]
Tagging of endogenous genes in a Toxoplasma gondii strain lacking Ku80
Huynh MH, Carruthers VB.
Department of Microbiology and Immunology, University of Michigan School of Medicine, 1150 W. Medical Center Dr., Ann Arbor, MI 48109 USA.
As with other organisms with a completed genome sequence, opportunities for performing large scale studies, such as expression and localization, on Toxoplasma gondii are now much more feasible. We present a system for tagging genes endogenously with yellow fluorescent protein (YFP) in a Deltaku80 strain. Ku80 is involved in DNA strand repair and non-homologous DNA end-joining; previous studies in other organisms have shown that in its absence, random integration is eliminated, allowing the insertion of constructs with homologous sequences into the proper loci. We generated a vector consisting of YFP and a DHFR-TS selectable marker. The YFP is preceded by a LIC (ligation-independent cloning) cassette, which allows the insertion of PCR products containing complementary LIC sequences. We demonstrate that the Deltaku80 strain is more effective and efficient in integrating the YFP-tagged genes into the correct locus compared to the wild-type strain RH. We then selected several hypothetical proteins that were identified by a proteomic screen of excreted-secreted antigens (ESA) and that displayed microarray expression profiles similar to known micronemal proteins, with the thought that these could potentially be new proteins with roles in cell invasion. We localized these hypothetical proteins by YFP fluorescence and show expression by immunoblotting. Our findings demonstrate that the combination of the Deltaku80 strain and the pYFP.LIC constructs reduces both the time and cost required to determine localization of a new gene of interest. This should open the opportunity for performing larger scale studies of novel T. gondii genes.
PMID: 19218426 [PubMed - as supplied by publisher]
Comparison of indirect fluorescent antibody test and modified agglutination test
Parasitol Res. 2009 Feb 17. [Epub ahead of print]
Comparison of indirect fluorescent antibody test and modified agglutination test for detecting Toxoplasma gondii immunoglobulin G antibodies in dog and cat
Macrì G, Sala M, Linder AM, Pettirossi N, Scarpulla M.
Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana (IZS LT), Rome, Italy, gladia.macri@izslt.it.
The present study describes the comparison between a modified agglutination test (MAT) and the indirect fluorescent antibody test (IFAT) for the detection of Toxoplasma specific IgG antibodies in dog and cat sera. MAT showed an "almost perfect" agreement with IFAT in detecting positive and negative results in cat sera, where as only a "substantial" agreement was observed in dog sera due to false negative results. Differences relative to sample dilution were recorded and serological titres obtained by MAT and IFAT are not directly comparable in cat and dog sera.
PMID: 19221795 [PubMed - as supplied by publisher]
Comparison of indirect fluorescent antibody test and modified agglutination test for detecting Toxoplasma gondii immunoglobulin G antibodies in dog and cat
Macrì G, Sala M, Linder AM, Pettirossi N, Scarpulla M.
Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana (IZS LT), Rome, Italy, gladia.macri@izslt.it.
The present study describes the comparison between a modified agglutination test (MAT) and the indirect fluorescent antibody test (IFAT) for the detection of Toxoplasma specific IgG antibodies in dog and cat sera. MAT showed an "almost perfect" agreement with IFAT in detecting positive and negative results in cat sera, where as only a "substantial" agreement was observed in dog sera due to false negative results. Differences relative to sample dilution were recorded and serological titres obtained by MAT and IFAT are not directly comparable in cat and dog sera.
PMID: 19221795 [PubMed - as supplied by publisher]
Friday, February 13, 2009
Neuropsychiatric disease and Toxoplasma gondii infection
Neuroimmunomodulation. 2009;16(2):122-33. Epub 2009 Feb 11
Neuropsychiatric disease and Toxoplasma gondii infection
Henriquez SA, Brett R, Alexander J, Pratt J, Roberts CW.
Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow, UK.
Toxoplasma gondii infects approximately 30% of the world's population, but causes overt clinical symptoms in only a small proportion of people. In recent years, the ability of the parasite to manipulate the behaviour of infected mice and rats and alter personality attributes of humans has been reported. Furthermore, a number of studies have now suggested T. gondii infection as a risk factor for the development of schizophrenia and depression in humans. As T. gondii forms cysts that are located in various anatomical sites including the brain during a chronic infection, it is well placed anatomically to mediate these effects directly. The T. gondii genome is known to contain 2 aromatic amino acid hydroxylases that potentially could directly affect dopamine and/or serotonin biosynthesis. However, stimulation of the immune response has also recently been associated with mood and behavioural alterations in humans, and compounds designed to alter mood, such as fluoxetine, have been demonstrated to alter aspects of immune function. Herein, the evidence for T.-gondii-induced behavioural changes relevant to schizophrenia and depression is reviewed. Potential mechanisms responsible for these changes in behaviour including the role of tryptophan metabolism and the hypothalamic-pituitary-adrenal axis are discussed. Copyright (c) 2009 S. Karger AG, Basel.
PMID: 19212132 [PubMed - in process]
Neuropsychiatric disease and Toxoplasma gondii infection
Henriquez SA, Brett R, Alexander J, Pratt J, Roberts CW.
Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow, UK.
Toxoplasma gondii infects approximately 30% of the world's population, but causes overt clinical symptoms in only a small proportion of people. In recent years, the ability of the parasite to manipulate the behaviour of infected mice and rats and alter personality attributes of humans has been reported. Furthermore, a number of studies have now suggested T. gondii infection as a risk factor for the development of schizophrenia and depression in humans. As T. gondii forms cysts that are located in various anatomical sites including the brain during a chronic infection, it is well placed anatomically to mediate these effects directly. The T. gondii genome is known to contain 2 aromatic amino acid hydroxylases that potentially could directly affect dopamine and/or serotonin biosynthesis. However, stimulation of the immune response has also recently been associated with mood and behavioural alterations in humans, and compounds designed to alter mood, such as fluoxetine, have been demonstrated to alter aspects of immune function. Herein, the evidence for T.-gondii-induced behavioural changes relevant to schizophrenia and depression is reviewed. Potential mechanisms responsible for these changes in behaviour including the role of tryptophan metabolism and the hypothalamic-pituitary-adrenal axis are discussed. Copyright (c) 2009 S. Karger AG, Basel.
PMID: 19212132 [PubMed - in process]
Wednesday, February 11, 2009
The Toxoplasma gondii-Shuttling Function of Dendritic Cells is Linked to the Parasite Genotype
Infect Immun. 2009 Feb 9. [Epub ahead of print]
The Toxoplasma gondii-Shuttling Function of Dendritic Cells is Linked to the Parasite Genotype
Lambert H, Vutova PP, Adams WC, Loré K, Barragan A.
Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, SE-141 86 Stockholm, Sweden; Department of Parasitology, Mycology and Environmental Microbiology, and Department of Virology, Swedish Institute for Infectious Disease Control, SE-171 82 Stockholm, Sweden.
Following intestinal invasion, the processes leading to systemic dissemination of the obligate intracellular protozoan Toxoplasma gondii remain poorly understood. Recently, tachyzoites representative of type I, II and III T. gondii populations were shown to differ with respect to their ability to transmigrate across cellular barriers. In this process of active parasite motility, type I strains exhibit a superior migratory capacity than the type II and type III strains. Data also suggest that tachyzoites rely on migrating dendritic cells (DC) as shuttling leukocytes to disseminate in tissue, e.g. the brain where cysts develop. In this study, T. gondii tachyzoites sampled from the three populations were allowed to infect primary human blood DC, murine intestinal DC or in vitro-derived DC, and compared for different phenotypic traits. All three archetypical lineages of T. gondii induced a hypermigratory phenotype in DC shortly after infection in vitro. Type II (and III) strains induced higher migratory frequency and intensity in DC compared to type I. Additionally, adoptive transfer of infected DC favored dissemination of type II and type III parasites compared to type I parasites in syngeneic mice. Type II parasites exhibited stronger intracellular association to both CD11c(+) DC and other leukocytes in vivo compared to type I. Altogether, these findings suggest that infected DC contribute to parasite propagation in a strain type specific manner, and that the parasite genotype (type II) most frequently associated with toxoplasmosis in humans efficiently exploits DC migration for parasite dissemination.
PMID: 19204091 [PubMed - as supplied by publisher]
The Toxoplasma gondii-Shuttling Function of Dendritic Cells is Linked to the Parasite Genotype
Lambert H, Vutova PP, Adams WC, Loré K, Barragan A.
Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, SE-141 86 Stockholm, Sweden; Department of Parasitology, Mycology and Environmental Microbiology, and Department of Virology, Swedish Institute for Infectious Disease Control, SE-171 82 Stockholm, Sweden.
Following intestinal invasion, the processes leading to systemic dissemination of the obligate intracellular protozoan Toxoplasma gondii remain poorly understood. Recently, tachyzoites representative of type I, II and III T. gondii populations were shown to differ with respect to their ability to transmigrate across cellular barriers. In this process of active parasite motility, type I strains exhibit a superior migratory capacity than the type II and type III strains. Data also suggest that tachyzoites rely on migrating dendritic cells (DC) as shuttling leukocytes to disseminate in tissue, e.g. the brain where cysts develop. In this study, T. gondii tachyzoites sampled from the three populations were allowed to infect primary human blood DC, murine intestinal DC or in vitro-derived DC, and compared for different phenotypic traits. All three archetypical lineages of T. gondii induced a hypermigratory phenotype in DC shortly after infection in vitro. Type II (and III) strains induced higher migratory frequency and intensity in DC compared to type I. Additionally, adoptive transfer of infected DC favored dissemination of type II and type III parasites compared to type I parasites in syngeneic mice. Type II parasites exhibited stronger intracellular association to both CD11c(+) DC and other leukocytes in vivo compared to type I. Altogether, these findings suggest that infected DC contribute to parasite propagation in a strain type specific manner, and that the parasite genotype (type II) most frequently associated with toxoplasmosis in humans efficiently exploits DC migration for parasite dissemination.
PMID: 19204091 [PubMed - as supplied by publisher]
Toxoplasma gondii glycosylphosphatidylinositols up-regulate MHC
Innate Immun. 2009 Feb;15(1):25-32.
Toxoplasma gondii glycosylphosphatidylinositols up-regulate major histocompatibility complex (MHC) molecule expression on primary murine macrophages
Debierre-Grockiego F, Molitor N, Schwarz RT, Lüder CG.
Institut für Virologie, AG Parasitologie, Philipps Universität Marburg, Marburg, Germany.
Toxoplasma gondii is an obligatory intracellular parasite able to block the IFN-gamma-induced up-regulation of major histocompatibility complex (MHC) class I and class II molecules. This facilitates parasite-mediated evasion of T-cell responses. Glycosylphosphatidylinositols (GPIs) are involved in the pathogenicity of protozoan parasites and we investigated if GPIs are responsible for inhibition of MHC expression on macrophages. In contrast to the blockade observed in cells infected with viable tachyzoites, T. gondii GPIs up-regulated MHC class I and class II molecules on the surface of both unstimulated and IFN-gamma-stimulated primary murine macrophages. This effect was correlated to the ability of GPIs to increase the antigen presentation to CD8(+) lymphocytes. T. gondii GPIs did not activate STAT1, one of the factors involved in the transcription of MHC class I and class II genes. However, the GPI-induced MHC class I up-regulation was abrogated by SN50, a specific NF-KB inhibitor. Up-regulation of surface MHC molecules by GPIs may lead to the elimination of non-infected cells of the host immune system, contributing to the immune escape strategy of T. gondii.
PMID: 19201822 [PubMed - in process]
Toxoplasma gondii glycosylphosphatidylinositols up-regulate major histocompatibility complex (MHC) molecule expression on primary murine macrophages
Debierre-Grockiego F, Molitor N, Schwarz RT, Lüder CG.
Institut für Virologie, AG Parasitologie, Philipps Universität Marburg, Marburg, Germany.
Toxoplasma gondii is an obligatory intracellular parasite able to block the IFN-gamma-induced up-regulation of major histocompatibility complex (MHC) class I and class II molecules. This facilitates parasite-mediated evasion of T-cell responses. Glycosylphosphatidylinositols (GPIs) are involved in the pathogenicity of protozoan parasites and we investigated if GPIs are responsible for inhibition of MHC expression on macrophages. In contrast to the blockade observed in cells infected with viable tachyzoites, T. gondii GPIs up-regulated MHC class I and class II molecules on the surface of both unstimulated and IFN-gamma-stimulated primary murine macrophages. This effect was correlated to the ability of GPIs to increase the antigen presentation to CD8(+) lymphocytes. T. gondii GPIs did not activate STAT1, one of the factors involved in the transcription of MHC class I and class II genes. However, the GPI-induced MHC class I up-regulation was abrogated by SN50, a specific NF-KB inhibitor. Up-regulation of surface MHC molecules by GPIs may lead to the elimination of non-infected cells of the host immune system, contributing to the immune escape strategy of T. gondii.
PMID: 19201822 [PubMed - in process]
Monday, February 09, 2009
Rab11A-controlled assembly of the inner membrane complex is required for completion of apicomplexan cytokinesis
PLoS Pathog. 2009 Jan;5(1):e1000270. Epub 2009 Jan 23
Rab11A-controlled assembly of the inner membrane complex is required for completion of apicomplexan cytokinesis
Agop-Nersesian C, Naissant B, Ben Rached F, Rauch M, Kretzschmar A, Thiberge S, Menard R, Ferguson DJ, Meissner M, Langsley G
Department of Parasitology, Hygieneinstitut, University Hospital Heidelberg, Heidelberg, Germany.
The final step during cell division is the separation of daughter cells, a process that requires the coordinated delivery and assembly of new membrane to the cleavage furrow. While most eukaryotic cells replicate by binary fission, replication of apicomplexan parasites involves the assembly of daughters (merozoites/tachyzoites) within the mother cell, using the so-called Inner Membrane Complex (IMC) as a scaffold. After de novo synthesis of the IMC and biogenesis or segregation of new organelles, daughters bud out of the mother cell to invade new host cells. Here, we demonstrate that the final step in parasite cell division involves delivery of new plasma membrane to the daughter cells, in a process requiring functional Rab11A. Importantly, Rab11A can be found in association with Myosin-Tail-Interacting-Protein (MTIP), also known as Myosin Light Chain 1 (MLC1), a member of a 4-protein motor complex called the glideosome that is known to be crucial for parasite invasion of host cells. Ablation of Rab11A function results in daughter parasites having an incompletely formed IMC that leads to a block at a late stage of cell division. A similar defect is observed upon inducible expression of a myosin A tail-only mutant. We propose a model where Rab11A-mediated vesicular traffic driven by an MTIP-Myosin motor is necessary for IMC maturation and to deliver new plasma membrane to daughter cells in order to complete cell division.
PMID: 19165333 [PubMed - in process]
Rab11A-controlled assembly of the inner membrane complex is required for completion of apicomplexan cytokinesis
Agop-Nersesian C, Naissant B, Ben Rached F, Rauch M, Kretzschmar A, Thiberge S, Menard R, Ferguson DJ, Meissner M, Langsley G
Department of Parasitology, Hygieneinstitut, University Hospital Heidelberg, Heidelberg, Germany.
The final step during cell division is the separation of daughter cells, a process that requires the coordinated delivery and assembly of new membrane to the cleavage furrow. While most eukaryotic cells replicate by binary fission, replication of apicomplexan parasites involves the assembly of daughters (merozoites/tachyzoites) within the mother cell, using the so-called Inner Membrane Complex (IMC) as a scaffold. After de novo synthesis of the IMC and biogenesis or segregation of new organelles, daughters bud out of the mother cell to invade new host cells. Here, we demonstrate that the final step in parasite cell division involves delivery of new plasma membrane to the daughter cells, in a process requiring functional Rab11A. Importantly, Rab11A can be found in association with Myosin-Tail-Interacting-Protein (MTIP), also known as Myosin Light Chain 1 (MLC1), a member of a 4-protein motor complex called the glideosome that is known to be crucial for parasite invasion of host cells. Ablation of Rab11A function results in daughter parasites having an incompletely formed IMC that leads to a block at a late stage of cell division. A similar defect is observed upon inducible expression of a myosin A tail-only mutant. We propose a model where Rab11A-mediated vesicular traffic driven by an MTIP-Myosin motor is necessary for IMC maturation and to deliver new plasma membrane to daughter cells in order to complete cell division.
PMID: 19165333 [PubMed - in process]
Sunday, February 08, 2009
Novel structural and regulatory features of rhoptry secretory kinases in Toxoplasma
EMBO J. 2009 Feb 5. [Epub ahead of print]
Novel structural and regulatory features of rhoptry secretory kinases in Toxoplasma gondii
Qiu W, Wernimont A, Tang K, Taylor S, Lunin V, Schapira M, Fentress S, Hui R, Sibley LD.
Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
Serine/threonine kinases secreted from rhoptry organelles constitute important virulence factors of Toxoplasma gondii. Rhoptry kinases are highly divergent and their structures and regulatory mechanism are hitherto unknown. Here, we report the X-ray crystal structures of two related pseudokinases named ROP2 and ROP8, which differ primarily in their substrate-binding site. ROP kinases contain a typical bilobate kinase fold and a novel N-terminal extension that both stabilizes the N-lobe and provides a unique means of regulation. Although ROP2 and ROP8 were catalytically inactive, they provided a template for homology modelling of the active kinase ROP18, a major virulence determinant of T. gondii. Autophosphorylation of key residues in the N-terminal extension resulted in ROP18 activation, which in turn phosphorylated ROP2 and ROP8. Mutagenesis and mass spectrometry experiments revealed that ROP18 was maximally activated when this phosphorylated N-terminus relieved autoinhibition resulting from extension of aliphatic side chains into the ATP-binding pocket. This novel means of regulation governs ROP kinases implicated in parasite virulence.
PMID: 19197235 [PubMed - as supplied by publisher]
Novel structural and regulatory features of rhoptry secretory kinases in Toxoplasma gondii
Qiu W, Wernimont A, Tang K, Taylor S, Lunin V, Schapira M, Fentress S, Hui R, Sibley LD.
Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
Serine/threonine kinases secreted from rhoptry organelles constitute important virulence factors of Toxoplasma gondii. Rhoptry kinases are highly divergent and their structures and regulatory mechanism are hitherto unknown. Here, we report the X-ray crystal structures of two related pseudokinases named ROP2 and ROP8, which differ primarily in their substrate-binding site. ROP kinases contain a typical bilobate kinase fold and a novel N-terminal extension that both stabilizes the N-lobe and provides a unique means of regulation. Although ROP2 and ROP8 were catalytically inactive, they provided a template for homology modelling of the active kinase ROP18, a major virulence determinant of T. gondii. Autophosphorylation of key residues in the N-terminal extension resulted in ROP18 activation, which in turn phosphorylated ROP2 and ROP8. Mutagenesis and mass spectrometry experiments revealed that ROP18 was maximally activated when this phosphorylated N-terminus relieved autoinhibition resulting from extension of aliphatic side chains into the ATP-binding pocket. This novel means of regulation governs ROP kinases implicated in parasite virulence.
PMID: 19197235 [PubMed - as supplied by publisher]
Disruption of the Toxoplasma gondii parasitophorous vacuole by IFNgamma-inducible immunity-related GTPases (IRG proteins) triggers necrotic cell death
PLoS Pathog. 2009 Feb;5(2):e1000288. Epub 2009 Feb 6
Disruption of the Toxoplasma gondii parasitophorous vacuole by IFNgamma-inducible immunity-related GTPases (IRG proteins) triggers necrotic cell death
Zhao YO, Khaminets A, Hunn JP, Howard JC.
Institute for Genetics, University of Cologne, Cologne, Germany.
Toxoplasma gondii is a natural intracellular protozoal pathogen of mice and other small mammals. After infection, the parasite replicates freely in many cell types (tachyzoite stage) before undergoing a phase transition and encysting in brain and muscle (bradyzoite stage). In the mouse, early immune resistance to the tachyzoite stage is mediated by the family of interferon-inducible immunity-related GTPases (IRG proteins), but little is known of the nature of this resistance. We reported earlier that IRG proteins accumulate on intracellular vacuoles containing the pathogen, and that the vacuolar membrane subsequently ruptures. In this report, live-cell imaging microscopy has been used to follow this process and its consequences in real time. We show that the rupture of the vacuole is inevitably followed by death of the intracellular parasite, shown by its permeability to cytosolic protein markers. Death of the parasite is followed by the death of the infected cell. The death of the cell has features of pyronecrosis, including membrane permeabilisation and release of the inflammatory protein, HMGB1, but caspase-1 cleavage is not detected. This sequence of events occurs on a large scale only following infection of IFNgamma-induced cells with an avirulent strain of T. gondii, and is reduced by expression of a dominant negative mutant IRG protein. Cells infected by virulent strains rarely undergo necrosis. We did not find autophagy to play any role in the key steps leading to the death of the parasite. We conclude that IRG proteins resist infection by avirulent T. gondii by a novel mechanism involving disruption of the vacuolar membrane, which in turn ultimately leads to the necrotic death of the infected cell.
Publication Types:
Research Support, Non-U.S. Gov't
PMID: 19197351 [PubMed - in process]
Disruption of the Toxoplasma gondii parasitophorous vacuole by IFNgamma-inducible immunity-related GTPases (IRG proteins) triggers necrotic cell death
Zhao YO, Khaminets A, Hunn JP, Howard JC.
Institute for Genetics, University of Cologne, Cologne, Germany.
Toxoplasma gondii is a natural intracellular protozoal pathogen of mice and other small mammals. After infection, the parasite replicates freely in many cell types (tachyzoite stage) before undergoing a phase transition and encysting in brain and muscle (bradyzoite stage). In the mouse, early immune resistance to the tachyzoite stage is mediated by the family of interferon-inducible immunity-related GTPases (IRG proteins), but little is known of the nature of this resistance. We reported earlier that IRG proteins accumulate on intracellular vacuoles containing the pathogen, and that the vacuolar membrane subsequently ruptures. In this report, live-cell imaging microscopy has been used to follow this process and its consequences in real time. We show that the rupture of the vacuole is inevitably followed by death of the intracellular parasite, shown by its permeability to cytosolic protein markers. Death of the parasite is followed by the death of the infected cell. The death of the cell has features of pyronecrosis, including membrane permeabilisation and release of the inflammatory protein, HMGB1, but caspase-1 cleavage is not detected. This sequence of events occurs on a large scale only following infection of IFNgamma-induced cells with an avirulent strain of T. gondii, and is reduced by expression of a dominant negative mutant IRG protein. Cells infected by virulent strains rarely undergo necrosis. We did not find autophagy to play any role in the key steps leading to the death of the parasite. We conclude that IRG proteins resist infection by avirulent T. gondii by a novel mechanism involving disruption of the vacuolar membrane, which in turn ultimately leads to the necrotic death of the infected cell.
Publication Types:
Research Support, Non-U.S. Gov't
PMID: 19197351 [PubMed - in process]
Vaccination concepts against Toxoplasma gondii
Expert Rev Vaccines. 2009 Feb;8(2):215-25
Vaccination concepts against Toxoplasma gondii
Garcia JL.
Department of Preventive Veterinary Medicine, Londrina State University, Campus Universitário, Rodovia Celso Garcia Cid, Pr 445 Km 380, Cx. Postal 6001, Londrina, PR 86051-990, Brazil. jlgarcia@uel.br
Toxoplasma gondii is a parasite that infects animals and humans worldwide. Despite the current knowledge of immunology, pathology and genetics related to the parasite, a safe vaccine for prevention of the infection in both humans and animals does not exist. Here, we review some aspects concerning vaccination against T. gondii.
PMID: 19196201 [PubMed - in process]
Vaccination concepts against Toxoplasma gondii
Garcia JL.
Department of Preventive Veterinary Medicine, Londrina State University, Campus Universitário, Rodovia Celso Garcia Cid, Pr 445 Km 380, Cx. Postal 6001, Londrina, PR 86051-990, Brazil. jlgarcia@uel.br
Toxoplasma gondii is a parasite that infects animals and humans worldwide. Despite the current knowledge of immunology, pathology and genetics related to the parasite, a safe vaccine for prevention of the infection in both humans and animals does not exist. Here, we review some aspects concerning vaccination against T. gondii.
PMID: 19196201 [PubMed - in process]
Wednesday, February 04, 2009
Exosomes are an effective vaccine against congenital toxoplasmosis in mice
Vaccine. 2009 Jan 29. [Epub ahead of print]
Exosomes are an effective vaccine against congenital toxoplasmosis in mice
Beauvillain C, Juste MO, Dion S, Pierre J, Dimier-Poisson I.
Université François-Rabelais de Tours, INRA, 31, Avenue Monge, 37200 Tours, France; UMR 0483 Université-INRA d'Immunologie Parasitaire et Vaccinologie, Bio-Thérapies Anti-Infectieuses, IFR des Agents Transmissibles et Infectiologie, UFR des Sciences Pharmaceutiques, 31, Avenue Monge, 37200 Tours, France.
Toxoplasmosis is a serious disease in humans and may cause abortion or congenital infection if a woman is exposed to the disease for the first time during pregnancy. Infection before pregnancy normally results in immunity protecting the foetus, suggesting that it may be possible to block vertical transmission of the parasite by appropriate vaccination before pregnancy. We found that the vaccination of CBA/J mice, before pregnancy, with exosomes secreted by SRDCs pulsed in vitro with Toxoplasma gondii-derived antigens (TAg) induced a protective response in the pups. Indeed, vaccination resulted in the presence of significantly fewer cysts in pup brains. This protection was associated with strong humoral responses in the serum in vivo. We also observed cellular responses in vivo, with cell proliferation associated with the production of cytokines by the splenocytes. Thus, exosomes are nucleic acid-free vesicles able to induce immune responses correlated with protection against T. gondii infection in a congenital model. They are therefore a potentially useful tool for vaccination against infectious disease.
PMID: 19186199 [PubMed - as supplied by publisher]
Exosomes are an effective vaccine against congenital toxoplasmosis in mice
Beauvillain C, Juste MO, Dion S, Pierre J, Dimier-Poisson I.
Université François-Rabelais de Tours, INRA, 31, Avenue Monge, 37200 Tours, France; UMR 0483 Université-INRA d'Immunologie Parasitaire et Vaccinologie, Bio-Thérapies Anti-Infectieuses, IFR des Agents Transmissibles et Infectiologie, UFR des Sciences Pharmaceutiques, 31, Avenue Monge, 37200 Tours, France.
Toxoplasmosis is a serious disease in humans and may cause abortion or congenital infection if a woman is exposed to the disease for the first time during pregnancy. Infection before pregnancy normally results in immunity protecting the foetus, suggesting that it may be possible to block vertical transmission of the parasite by appropriate vaccination before pregnancy. We found that the vaccination of CBA/J mice, before pregnancy, with exosomes secreted by SRDCs pulsed in vitro with Toxoplasma gondii-derived antigens (TAg) induced a protective response in the pups. Indeed, vaccination resulted in the presence of significantly fewer cysts in pup brains. This protection was associated with strong humoral responses in the serum in vivo. We also observed cellular responses in vivo, with cell proliferation associated with the production of cytokines by the splenocytes. Thus, exosomes are nucleic acid-free vesicles able to induce immune responses correlated with protection against T. gondii infection in a congenital model. They are therefore a potentially useful tool for vaccination against infectious disease.
PMID: 19186199 [PubMed - as supplied by publisher]
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