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Louis M. Weiss - One of the best experts on this subject based on the ideXlab platform.
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toxoplasma gondii requires glycogen phosphorylase for balancing amylopectin storage and for efficient production of Brain Cysts
Mbio, 2017Co-Authors: Tatsuki Sugi, Tadakimi Tomita, Louis M. WeissAbstract:In immunocompromised hosts, latent infection with Toxoplasma gondii can reactivate from tissue Cysts, leading to encephalitis. A characteristic of T. gondii bradyzoites in tissue Cysts is the presence of amylopectin granules. The regulatory mechanisms and role of amylopectin accumulation in this organism are not fully understood. The T. gondii genome encodes a putative glycogen phosphorylase (TgGP), and mutants were constructed to manipulate the activity of TgGP and to evaluate the function of TgGP in amylopectin storage. Both a stop codon mutant (Pru/TgGPS25stop [expressing a Ser-to-stop codon change at position 25 in TgGP]) and a phosphorylation null mutant (Pru/TgGPS25A [expressing a Ser-to-Ala change at position 25 in TgGp]) mutated at Ser25 displayed amylopectin accumulation, while the phosphorylation-mimetic mutant (Pru/TgGPS25E [expressing a Ser-to-Glu change at position 25 in TgGp]) had minimal amylopectin accumulation under both tachyzoite and bradyzoite growth conditions. The expression of active TgGPS25S or TgGPS25E restored amylopectin catabolism in Pru/TgGPS25A To understand the relation between GP and calcium-dependent protein kinase 2 (CDPK2), which was recently reported to regulate amylopectin consumption, we knocked out CDPK2 in these mutants. PruΔcdpk2/TgGPS25E had minimal amylopectin accumulation, whereas the Δcdpk2 phenotype in the other GP mutants and parental lines displayed amylopectin accumulation. Both the inactive S25A and hyperactive S25E mutant produced Brain Cysts in infected mice, but the numbers of Cysts produced were significantly less than the number produced by the S25S wild-type GP parasite. Complementation that restored amylopectin regulation restored Brain Cyst production to the control levels seen in infected mice. These data suggest that T. gondii requires tight regulation of amylopectin expression for efficient production of Cysts and persistent infections and that GP phosphorylation is a regulatory mechanism involved in amylopectin storage and utilization.IMPORTANCEToxoplasma gondii is an obligate intracellular parasite that causes disease in immune-suppressed individuals, as well as a fetopathy in pregnant women who acquire infection for the first time during pregnancy. This parasite can differentiate between tachyzoites (seen in acute infection) and bradyzoites (seen in latent infection), and this differentiation is associated with disease relapse. A characteristic of bradyzoites is that they contain cytoplasmic amylopectin granules. The regulatory mechanisms and the roles of amylopectin granules during latent infection remain to be elucidated. We have identified a role of T. gondii glycogen phosphorylase (TgGP) in the regulation of starch digestion and a role of posttranslational modification of TgGP, i.e., phosphorylation of Ser25, in the regulation of amylopectin digestion. By manipulating TgGP activity in the parasite with genome editing, we found that the digestion and storage of amylopectin due to TgGP activity are both important for latency in the Brain.
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making home sweet and sturdy toxoplasma gondii ppgalnac ts glycosylate in hierarchical order and confer Cyst wall rigidity
Mbio, 2017Co-Authors: Tadakimi Tomita, Tatsuki Sugi, Rama R Yakubu, Louis M. WeissAbstract:ABSTRACT The protozoan intracellular parasite Toxoplasma gondii forms latent Cysts in the central nervous system (CNS) and persists for the lifetime of the host. This Cyst is cloaked with a glycosylated structure called the Cyst wall. Previously, we demonstrated that a mucin-like glycoprotein, CST1, localizes to the Cyst wall and confers structural rigidity on Brain Cysts in a mucin-like domain-dependent manner. The mucin-like domain of CST1 is composed of 20 units of threonine-rich tandem repeats that are O- GalNAc glycosylated. A family of enzymes termed polypeptide N -acetylgalactosaminyltransferases (ppGalNAc-Ts) initiates O -GalNAc glycosylation. To identify which isoforms of ppGalNAc-Ts are responsible for the glycosylation of the CST1 mucin-like domain and to evaluate the function of each ppGalNAc-T in the overall glycosylation of the Cyst wall, all five ppGalNAc-T isoforms were deleted individually from the T. gondii genome. The ppGalNAc-T2 and -T3 deletion mutants produced various glycosylation defects on the Cyst wall, implying that many Cyst wall glycoproteins are glycosylated by T2 and T3. Both T2 and T3 glycosylate the CST1 mucin-like domain, and this glycosylation is necessary for CST1 to confer structural rigidity on the Cyst wall. We established that T2 is required for the initial glycosylation of the mucin-like domain and that T3 is responsible for the sequential glycosylation on neighboring acceptor sites, demonstrating hierarchical glycosylation by two distinct initiating and filling-in ppGalNAc-Ts in an intact organism. IMPORTANCE Toxoplasma gondii is an obligate intracellular parasite that infects a third of the world’s population. It can cause severe congenital disease and devastating encephalitis in immunocompromised individuals. We identified two glycosyltransferases, ppGalNAc-T2 and -T3, which are responsible for glycosylating Cyst wall proteins in a hierarchical fashion. This glycosylation confers structural rigidity on the Brain Cyst. Our studies provide new insights into the mechanisms of O- GalNAc glycosylation in T. gondii .
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Toxoplasma gondii Requires Glycogen Phosphorylase for Balancing Amylopectin Storage and for Efficient Production of Brain Cysts
American Society for Microbiology, 2017Co-Authors: Tatsuki Sugi, Tadakimi Tomita, Louis M. Weiss, Jon P. BoyleAbstract:In immunocompromised hosts, latent infection with Toxoplasma gondii can reactivate from tissue Cysts, leading to encephalitis. A characteristic of T. gondii bradyzoites in tissue Cysts is the presence of amylopectin granules. The regulatory mechanisms and role of amylopectin accumulation in this organism are not fully understood. The T. gondii genome encodes a putative glycogen phosphorylase (TgGP), and mutants were constructed to manipulate the activity of TgGP and to evaluate the function of TgGP in amylopectin storage. Both a stop codon mutant (Pru/TgGPS25stop [expressing a Ser-to-stop codon change at position 25 in TgGP]) and a phosphorylation null mutant (Pru/TgGPS25A [expressing a Ser-to-Ala change at position 25 in TgGp]) mutated at Ser25 displayed amylopectin accumulation, while the phosphorylation-mimetic mutant (Pru/TgGPS25E [expressing a Ser-to-Glu change at position 25 in TgGp]) had minimal amylopectin accumulation under both tachyzoite and bradyzoite growth conditions. The expression of active TgGPS25S or TgGPS25E restored amylopectin catabolism in Pru/TgGPS25A. To understand the relation between GP and calcium-dependent protein kinase 2 (CDPK2), which was recently reported to regulate amylopectin consumption, we knocked out CDPK2 in these mutants. PruΔcdpk2/TgGPS25E had minimal amylopectin accumulation, whereas the Δcdpk2 phenotype in the other GP mutants and parental lines displayed amylopectin accumulation. Both the inactive S25A and hyperactive S25E mutant produced Brain Cysts in infected mice, but the numbers of Cysts produced were significantly less than the number produced by the S25S wild-type GP parasite. Complementation that restored amylopectin regulation restored Brain Cyst production to the control levels seen in infected mice. These data suggest that T. gondii requires tight regulation of amylopectin expression for efficient production of Cysts and persistent infections and that GP phosphorylation is a regulatory mechanism involved in amylopectin storage and utilization
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The Toxoplasma gondii Cyst Wall Protein CST1 Is Critical for Cyst Wall Integrity and Promotes Bradyzoite Persistence
PLoS Pathogens, 2013Co-Authors: Tadakimi Tomita, Yan Fen Ma, Ronald C. Taylor, Lye Meng Markillie, Barbara A. Fox, David J Bzik, Kami Kim, Louis M. WeissAbstract:Toxoplasma gondii infects up to one third of the world's population. A key to the success of T. gondii as a parasite is its ability to persist for the life of its host as bradyzoites within tissue Cysts. The glycosylated Cyst wall is the key structural feature that facilitates persistence and oral transmission of this parasite. Because most of the antibodies and reagents that recognize the Cyst wall recognize carbohydrates, identification of the components of the Cyst wall has been technically challenging. We have identified CST1 (TGME49_064660) as a 250 kDa SRS (SAG1 related sequence) domain protein with a large mucin-like domain. CST1 is responsible for the Dolichos biflorus Agglutinin (DBA) lectin binding characteristic of T. gondii Cysts. Deletion of CST1 results in reduced Cyst number and a fragile Brain Cyst phenotype characterized by a thinning and disruption of the underlying region of the Cyst wall. These defects are reversed by complementation of CST1. Additional complementation experiments demonstrate that the CST1-mucin domain is necessary for the formation of a normal Cyst wall structure, the ability of the Cyst to resist mechanical stress, and binding of DBA to the Cyst wall. RNA-seq transcriptome analysis demonstrated dysregulation of bradyzoite genes within the various cst1 mutants. These results indicate that CST1 functions as a key structural component that confers essential sturdiness to the T. gondii tissue Cyst critical for persistence of bradyzoite forms.
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Type II Toxoplasma gondii KU80 knockout strains enable functional analysis of genes required for Cyst development and latent infection.
Eukaryotic Cell, 2011Co-Authors: Barbara A. Fox, Tadakimi Tomita, Louis M. Weiss, Alejandra Falla, Leah M Rommereim, Jason P Gigley, Corinne Mercier, Marie-france Cesbron-delauw, David J BzikAbstract:Type II Toxoplasma gondii KU80 knockouts (Δku80) deficient in nonhomologous end joining were developed to delete the dominant pathway mediating random integration of targeting episomes. Gene targeting frequency in the type II Δku80 Δhxgprt strain measured at the orotate (OPRT) and the uracil (UPRT) phosphoribosyltransferase loci was highly efficient. To assess the potential of the type II Δku80 Δhxgprt strain to examine gene function affecting Cyst biology and latent stages of infection, we targeted the deletion of four parasite antigen genes (GRA4, GRA6, ROP7, and tgd057) that encode characterized CD8(+) T cell epitopes that elicit corresponding antigen-specific CD8(+) T cell populations associated with control of infection. Cyst development in these type II mutant strains was not found to be strictly dependent on antigen-specific CD8(+) T cell host responses. In contrast, a significant biological role was revealed for the dense granule proteins GRA4 and GRA6 in Cyst development since Brain tissue Cyst burdens were drastically reduced specifically in mutant strains with GRA4 and/or GRA6 deleted. Complementation of the Δgra4 and Δgra6 mutant strains using a functional allele of the deleted GRA coding region placed under the control of the endogenous UPRT locus was found to significantly restore Brain Cyst burdens. These results reveal that GRA proteins play a functional role in establishing Cyst burdens and latent infection. Collectively, our results suggest that a type II Δku80 Δhxgprt genetic background enables a higher-throughput functional analysis of the parasite genome to reveal fundamental aspects of parasite biology controlling virulence, pathogenesis, and transmission.
Tadakimi Tomita - One of the best experts on this subject based on the ideXlab platform.
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toxoplasma gondii requires glycogen phosphorylase for balancing amylopectin storage and for efficient production of Brain Cysts
Mbio, 2017Co-Authors: Tatsuki Sugi, Tadakimi Tomita, Louis M. WeissAbstract:In immunocompromised hosts, latent infection with Toxoplasma gondii can reactivate from tissue Cysts, leading to encephalitis. A characteristic of T. gondii bradyzoites in tissue Cysts is the presence of amylopectin granules. The regulatory mechanisms and role of amylopectin accumulation in this organism are not fully understood. The T. gondii genome encodes a putative glycogen phosphorylase (TgGP), and mutants were constructed to manipulate the activity of TgGP and to evaluate the function of TgGP in amylopectin storage. Both a stop codon mutant (Pru/TgGPS25stop [expressing a Ser-to-stop codon change at position 25 in TgGP]) and a phosphorylation null mutant (Pru/TgGPS25A [expressing a Ser-to-Ala change at position 25 in TgGp]) mutated at Ser25 displayed amylopectin accumulation, while the phosphorylation-mimetic mutant (Pru/TgGPS25E [expressing a Ser-to-Glu change at position 25 in TgGp]) had minimal amylopectin accumulation under both tachyzoite and bradyzoite growth conditions. The expression of active TgGPS25S or TgGPS25E restored amylopectin catabolism in Pru/TgGPS25A To understand the relation between GP and calcium-dependent protein kinase 2 (CDPK2), which was recently reported to regulate amylopectin consumption, we knocked out CDPK2 in these mutants. PruΔcdpk2/TgGPS25E had minimal amylopectin accumulation, whereas the Δcdpk2 phenotype in the other GP mutants and parental lines displayed amylopectin accumulation. Both the inactive S25A and hyperactive S25E mutant produced Brain Cysts in infected mice, but the numbers of Cysts produced were significantly less than the number produced by the S25S wild-type GP parasite. Complementation that restored amylopectin regulation restored Brain Cyst production to the control levels seen in infected mice. These data suggest that T. gondii requires tight regulation of amylopectin expression for efficient production of Cysts and persistent infections and that GP phosphorylation is a regulatory mechanism involved in amylopectin storage and utilization.IMPORTANCEToxoplasma gondii is an obligate intracellular parasite that causes disease in immune-suppressed individuals, as well as a fetopathy in pregnant women who acquire infection for the first time during pregnancy. This parasite can differentiate between tachyzoites (seen in acute infection) and bradyzoites (seen in latent infection), and this differentiation is associated with disease relapse. A characteristic of bradyzoites is that they contain cytoplasmic amylopectin granules. The regulatory mechanisms and the roles of amylopectin granules during latent infection remain to be elucidated. We have identified a role of T. gondii glycogen phosphorylase (TgGP) in the regulation of starch digestion and a role of posttranslational modification of TgGP, i.e., phosphorylation of Ser25, in the regulation of amylopectin digestion. By manipulating TgGP activity in the parasite with genome editing, we found that the digestion and storage of amylopectin due to TgGP activity are both important for latency in the Brain.
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making home sweet and sturdy toxoplasma gondii ppgalnac ts glycosylate in hierarchical order and confer Cyst wall rigidity
Mbio, 2017Co-Authors: Tadakimi Tomita, Tatsuki Sugi, Rama R Yakubu, Louis M. WeissAbstract:ABSTRACT The protozoan intracellular parasite Toxoplasma gondii forms latent Cysts in the central nervous system (CNS) and persists for the lifetime of the host. This Cyst is cloaked with a glycosylated structure called the Cyst wall. Previously, we demonstrated that a mucin-like glycoprotein, CST1, localizes to the Cyst wall and confers structural rigidity on Brain Cysts in a mucin-like domain-dependent manner. The mucin-like domain of CST1 is composed of 20 units of threonine-rich tandem repeats that are O- GalNAc glycosylated. A family of enzymes termed polypeptide N -acetylgalactosaminyltransferases (ppGalNAc-Ts) initiates O -GalNAc glycosylation. To identify which isoforms of ppGalNAc-Ts are responsible for the glycosylation of the CST1 mucin-like domain and to evaluate the function of each ppGalNAc-T in the overall glycosylation of the Cyst wall, all five ppGalNAc-T isoforms were deleted individually from the T. gondii genome. The ppGalNAc-T2 and -T3 deletion mutants produced various glycosylation defects on the Cyst wall, implying that many Cyst wall glycoproteins are glycosylated by T2 and T3. Both T2 and T3 glycosylate the CST1 mucin-like domain, and this glycosylation is necessary for CST1 to confer structural rigidity on the Cyst wall. We established that T2 is required for the initial glycosylation of the mucin-like domain and that T3 is responsible for the sequential glycosylation on neighboring acceptor sites, demonstrating hierarchical glycosylation by two distinct initiating and filling-in ppGalNAc-Ts in an intact organism. IMPORTANCE Toxoplasma gondii is an obligate intracellular parasite that infects a third of the world’s population. It can cause severe congenital disease and devastating encephalitis in immunocompromised individuals. We identified two glycosyltransferases, ppGalNAc-T2 and -T3, which are responsible for glycosylating Cyst wall proteins in a hierarchical fashion. This glycosylation confers structural rigidity on the Brain Cyst. Our studies provide new insights into the mechanisms of O- GalNAc glycosylation in T. gondii .
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Toxoplasma gondii Requires Glycogen Phosphorylase for Balancing Amylopectin Storage and for Efficient Production of Brain Cysts
American Society for Microbiology, 2017Co-Authors: Tatsuki Sugi, Tadakimi Tomita, Louis M. Weiss, Jon P. BoyleAbstract:In immunocompromised hosts, latent infection with Toxoplasma gondii can reactivate from tissue Cysts, leading to encephalitis. A characteristic of T. gondii bradyzoites in tissue Cysts is the presence of amylopectin granules. The regulatory mechanisms and role of amylopectin accumulation in this organism are not fully understood. The T. gondii genome encodes a putative glycogen phosphorylase (TgGP), and mutants were constructed to manipulate the activity of TgGP and to evaluate the function of TgGP in amylopectin storage. Both a stop codon mutant (Pru/TgGPS25stop [expressing a Ser-to-stop codon change at position 25 in TgGP]) and a phosphorylation null mutant (Pru/TgGPS25A [expressing a Ser-to-Ala change at position 25 in TgGp]) mutated at Ser25 displayed amylopectin accumulation, while the phosphorylation-mimetic mutant (Pru/TgGPS25E [expressing a Ser-to-Glu change at position 25 in TgGp]) had minimal amylopectin accumulation under both tachyzoite and bradyzoite growth conditions. The expression of active TgGPS25S or TgGPS25E restored amylopectin catabolism in Pru/TgGPS25A. To understand the relation between GP and calcium-dependent protein kinase 2 (CDPK2), which was recently reported to regulate amylopectin consumption, we knocked out CDPK2 in these mutants. PruΔcdpk2/TgGPS25E had minimal amylopectin accumulation, whereas the Δcdpk2 phenotype in the other GP mutants and parental lines displayed amylopectin accumulation. Both the inactive S25A and hyperactive S25E mutant produced Brain Cysts in infected mice, but the numbers of Cysts produced were significantly less than the number produced by the S25S wild-type GP parasite. Complementation that restored amylopectin regulation restored Brain Cyst production to the control levels seen in infected mice. These data suggest that T. gondii requires tight regulation of amylopectin expression for efficient production of Cysts and persistent infections and that GP phosphorylation is a regulatory mechanism involved in amylopectin storage and utilization
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The Toxoplasma gondii Cyst Wall Protein CST1 Is Critical for Cyst Wall Integrity and Promotes Bradyzoite Persistence
PLoS Pathogens, 2013Co-Authors: Tadakimi Tomita, Yan Fen Ma, Ronald C. Taylor, Lye Meng Markillie, Barbara A. Fox, David J Bzik, Kami Kim, Louis M. WeissAbstract:Toxoplasma gondii infects up to one third of the world's population. A key to the success of T. gondii as a parasite is its ability to persist for the life of its host as bradyzoites within tissue Cysts. The glycosylated Cyst wall is the key structural feature that facilitates persistence and oral transmission of this parasite. Because most of the antibodies and reagents that recognize the Cyst wall recognize carbohydrates, identification of the components of the Cyst wall has been technically challenging. We have identified CST1 (TGME49_064660) as a 250 kDa SRS (SAG1 related sequence) domain protein with a large mucin-like domain. CST1 is responsible for the Dolichos biflorus Agglutinin (DBA) lectin binding characteristic of T. gondii Cysts. Deletion of CST1 results in reduced Cyst number and a fragile Brain Cyst phenotype characterized by a thinning and disruption of the underlying region of the Cyst wall. These defects are reversed by complementation of CST1. Additional complementation experiments demonstrate that the CST1-mucin domain is necessary for the formation of a normal Cyst wall structure, the ability of the Cyst to resist mechanical stress, and binding of DBA to the Cyst wall. RNA-seq transcriptome analysis demonstrated dysregulation of bradyzoite genes within the various cst1 mutants. These results indicate that CST1 functions as a key structural component that confers essential sturdiness to the T. gondii tissue Cyst critical for persistence of bradyzoite forms.
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Type II Toxoplasma gondii KU80 knockout strains enable functional analysis of genes required for Cyst development and latent infection.
Eukaryotic Cell, 2011Co-Authors: Barbara A. Fox, Tadakimi Tomita, Louis M. Weiss, Alejandra Falla, Leah M Rommereim, Jason P Gigley, Corinne Mercier, Marie-france Cesbron-delauw, David J BzikAbstract:Type II Toxoplasma gondii KU80 knockouts (Δku80) deficient in nonhomologous end joining were developed to delete the dominant pathway mediating random integration of targeting episomes. Gene targeting frequency in the type II Δku80 Δhxgprt strain measured at the orotate (OPRT) and the uracil (UPRT) phosphoribosyltransferase loci was highly efficient. To assess the potential of the type II Δku80 Δhxgprt strain to examine gene function affecting Cyst biology and latent stages of infection, we targeted the deletion of four parasite antigen genes (GRA4, GRA6, ROP7, and tgd057) that encode characterized CD8(+) T cell epitopes that elicit corresponding antigen-specific CD8(+) T cell populations associated with control of infection. Cyst development in these type II mutant strains was not found to be strictly dependent on antigen-specific CD8(+) T cell host responses. In contrast, a significant biological role was revealed for the dense granule proteins GRA4 and GRA6 in Cyst development since Brain tissue Cyst burdens were drastically reduced specifically in mutant strains with GRA4 and/or GRA6 deleted. Complementation of the Δgra4 and Δgra6 mutant strains using a functional allele of the deleted GRA coding region placed under the control of the endogenous UPRT locus was found to significantly restore Brain Cyst burdens. These results reveal that GRA proteins play a functional role in establishing Cyst burdens and latent infection. Collectively, our results suggest that a type II Δku80 Δhxgprt genetic background enables a higher-throughput functional analysis of the parasite genome to reveal fundamental aspects of parasite biology controlling virulence, pathogenesis, and transmission.
David J Bzik - One of the best experts on this subject based on the ideXlab platform.
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nuclear glycolytic enzyme enolase of toxoplasma gondii functions as a transcriptional regulator
PLOS ONE, 2014Co-Authors: Thomas Mouveaux, Barbara A. Fox, David J Bzik, Gabrielle Oria, Elisabeth Werkmeister, Christian Slomianny, Stanislas TomavoAbstract:Apicomplexan parasites including Toxoplasma gondii have complex life cycles within different hosts and their infectivity relies on their capacity to regulate gene expression. However, little is known about the nuclear factors that regulate gene expression in these pathogens. Here, we report that T. gondii enolase TgENO2 is targeted to the nucleus of actively replicating parasites, where it specifically binds to nuclear chromatin in vivo. Using a ChIP-Seq technique, we provide evidence for TgENO2 enrichment at the 5′ untranslated gene regions containing the putative promoters of 241 nuclear genes. Ectopic expression of HA-tagged TgENO1 or TgENO2 led to changes in transcript levels of numerous gene targets. Targeted disruption of TgENO1 gene results in a decrease in Brain Cyst burden of chronically infected mice and in changes in transcript levels of several nuclear genes. Complementation of this knockout mutant with ectopic TgENO1-HA fully restored normal transcript levels. Our findings reveal that enolase functions extend beyond glycolytic activity and include a direct role in coordinating gene regulation in T. gondii.
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The Toxoplasma gondii Cyst Wall Protein CST1 Is Critical for Cyst Wall Integrity and Promotes Bradyzoite Persistence
PLoS Pathogens, 2013Co-Authors: Tadakimi Tomita, Yan Fen Ma, Ronald C. Taylor, Lye Meng Markillie, Barbara A. Fox, David J Bzik, Kami Kim, Louis M. WeissAbstract:Toxoplasma gondii infects up to one third of the world's population. A key to the success of T. gondii as a parasite is its ability to persist for the life of its host as bradyzoites within tissue Cysts. The glycosylated Cyst wall is the key structural feature that facilitates persistence and oral transmission of this parasite. Because most of the antibodies and reagents that recognize the Cyst wall recognize carbohydrates, identification of the components of the Cyst wall has been technically challenging. We have identified CST1 (TGME49_064660) as a 250 kDa SRS (SAG1 related sequence) domain protein with a large mucin-like domain. CST1 is responsible for the Dolichos biflorus Agglutinin (DBA) lectin binding characteristic of T. gondii Cysts. Deletion of CST1 results in reduced Cyst number and a fragile Brain Cyst phenotype characterized by a thinning and disruption of the underlying region of the Cyst wall. These defects are reversed by complementation of CST1. Additional complementation experiments demonstrate that the CST1-mucin domain is necessary for the formation of a normal Cyst wall structure, the ability of the Cyst to resist mechanical stress, and binding of DBA to the Cyst wall. RNA-seq transcriptome analysis demonstrated dysregulation of bradyzoite genes within the various cst1 mutants. These results indicate that CST1 functions as a key structural component that confers essential sturdiness to the T. gondii tissue Cyst critical for persistence of bradyzoite forms.
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Type II Toxoplasma gondii KU80 knockout strains enable functional analysis of genes required for Cyst development and latent infection.
Eukaryotic Cell, 2011Co-Authors: Barbara A. Fox, Tadakimi Tomita, Louis M. Weiss, Alejandra Falla, Leah M Rommereim, Jason P Gigley, Corinne Mercier, Marie-france Cesbron-delauw, David J BzikAbstract:Type II Toxoplasma gondii KU80 knockouts (Δku80) deficient in nonhomologous end joining were developed to delete the dominant pathway mediating random integration of targeting episomes. Gene targeting frequency in the type II Δku80 Δhxgprt strain measured at the orotate (OPRT) and the uracil (UPRT) phosphoribosyltransferase loci was highly efficient. To assess the potential of the type II Δku80 Δhxgprt strain to examine gene function affecting Cyst biology and latent stages of infection, we targeted the deletion of four parasite antigen genes (GRA4, GRA6, ROP7, and tgd057) that encode characterized CD8(+) T cell epitopes that elicit corresponding antigen-specific CD8(+) T cell populations associated with control of infection. Cyst development in these type II mutant strains was not found to be strictly dependent on antigen-specific CD8(+) T cell host responses. In contrast, a significant biological role was revealed for the dense granule proteins GRA4 and GRA6 in Cyst development since Brain tissue Cyst burdens were drastically reduced specifically in mutant strains with GRA4 and/or GRA6 deleted. Complementation of the Δgra4 and Δgra6 mutant strains using a functional allele of the deleted GRA coding region placed under the control of the endogenous UPRT locus was found to significantly restore Brain Cyst burdens. These results reveal that GRA proteins play a functional role in establishing Cyst burdens and latent infection. Collectively, our results suggest that a type II Δku80 Δhxgprt genetic background enables a higher-throughput functional analysis of the parasite genome to reveal fundamental aspects of parasite biology controlling virulence, pathogenesis, and transmission.
Barbara A. Fox - One of the best experts on this subject based on the ideXlab platform.
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nuclear glycolytic enzyme enolase of toxoplasma gondii functions as a transcriptional regulator
PLOS ONE, 2014Co-Authors: Thomas Mouveaux, Barbara A. Fox, David J Bzik, Gabrielle Oria, Elisabeth Werkmeister, Christian Slomianny, Stanislas TomavoAbstract:Apicomplexan parasites including Toxoplasma gondii have complex life cycles within different hosts and their infectivity relies on their capacity to regulate gene expression. However, little is known about the nuclear factors that regulate gene expression in these pathogens. Here, we report that T. gondii enolase TgENO2 is targeted to the nucleus of actively replicating parasites, where it specifically binds to nuclear chromatin in vivo. Using a ChIP-Seq technique, we provide evidence for TgENO2 enrichment at the 5′ untranslated gene regions containing the putative promoters of 241 nuclear genes. Ectopic expression of HA-tagged TgENO1 or TgENO2 led to changes in transcript levels of numerous gene targets. Targeted disruption of TgENO1 gene results in a decrease in Brain Cyst burden of chronically infected mice and in changes in transcript levels of several nuclear genes. Complementation of this knockout mutant with ectopic TgENO1-HA fully restored normal transcript levels. Our findings reveal that enolase functions extend beyond glycolytic activity and include a direct role in coordinating gene regulation in T. gondii.
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The Toxoplasma gondii Cyst Wall Protein CST1 Is Critical for Cyst Wall Integrity and Promotes Bradyzoite Persistence
PLoS Pathogens, 2013Co-Authors: Tadakimi Tomita, Yan Fen Ma, Ronald C. Taylor, Lye Meng Markillie, Barbara A. Fox, David J Bzik, Kami Kim, Louis M. WeissAbstract:Toxoplasma gondii infects up to one third of the world's population. A key to the success of T. gondii as a parasite is its ability to persist for the life of its host as bradyzoites within tissue Cysts. The glycosylated Cyst wall is the key structural feature that facilitates persistence and oral transmission of this parasite. Because most of the antibodies and reagents that recognize the Cyst wall recognize carbohydrates, identification of the components of the Cyst wall has been technically challenging. We have identified CST1 (TGME49_064660) as a 250 kDa SRS (SAG1 related sequence) domain protein with a large mucin-like domain. CST1 is responsible for the Dolichos biflorus Agglutinin (DBA) lectin binding characteristic of T. gondii Cysts. Deletion of CST1 results in reduced Cyst number and a fragile Brain Cyst phenotype characterized by a thinning and disruption of the underlying region of the Cyst wall. These defects are reversed by complementation of CST1. Additional complementation experiments demonstrate that the CST1-mucin domain is necessary for the formation of a normal Cyst wall structure, the ability of the Cyst to resist mechanical stress, and binding of DBA to the Cyst wall. RNA-seq transcriptome analysis demonstrated dysregulation of bradyzoite genes within the various cst1 mutants. These results indicate that CST1 functions as a key structural component that confers essential sturdiness to the T. gondii tissue Cyst critical for persistence of bradyzoite forms.
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Type II Toxoplasma gondii KU80 knockout strains enable functional analysis of genes required for Cyst development and latent infection.
Eukaryotic Cell, 2011Co-Authors: Barbara A. Fox, Tadakimi Tomita, Louis M. Weiss, Alejandra Falla, Leah M Rommereim, Jason P Gigley, Corinne Mercier, Marie-france Cesbron-delauw, David J BzikAbstract:Type II Toxoplasma gondii KU80 knockouts (Δku80) deficient in nonhomologous end joining were developed to delete the dominant pathway mediating random integration of targeting episomes. Gene targeting frequency in the type II Δku80 Δhxgprt strain measured at the orotate (OPRT) and the uracil (UPRT) phosphoribosyltransferase loci was highly efficient. To assess the potential of the type II Δku80 Δhxgprt strain to examine gene function affecting Cyst biology and latent stages of infection, we targeted the deletion of four parasite antigen genes (GRA4, GRA6, ROP7, and tgd057) that encode characterized CD8(+) T cell epitopes that elicit corresponding antigen-specific CD8(+) T cell populations associated with control of infection. Cyst development in these type II mutant strains was not found to be strictly dependent on antigen-specific CD8(+) T cell host responses. In contrast, a significant biological role was revealed for the dense granule proteins GRA4 and GRA6 in Cyst development since Brain tissue Cyst burdens were drastically reduced specifically in mutant strains with GRA4 and/or GRA6 deleted. Complementation of the Δgra4 and Δgra6 mutant strains using a functional allele of the deleted GRA coding region placed under the control of the endogenous UPRT locus was found to significantly restore Brain Cyst burdens. These results reveal that GRA proteins play a functional role in establishing Cyst burdens and latent infection. Collectively, our results suggest that a type II Δku80 Δhxgprt genetic background enables a higher-throughput functional analysis of the parasite genome to reveal fundamental aspects of parasite biology controlling virulence, pathogenesis, and transmission.
Tatsuki Sugi - One of the best experts on this subject based on the ideXlab platform.
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toxoplasma gondii requires glycogen phosphorylase for balancing amylopectin storage and for efficient production of Brain Cysts
Mbio, 2017Co-Authors: Tatsuki Sugi, Tadakimi Tomita, Louis M. WeissAbstract:In immunocompromised hosts, latent infection with Toxoplasma gondii can reactivate from tissue Cysts, leading to encephalitis. A characteristic of T. gondii bradyzoites in tissue Cysts is the presence of amylopectin granules. The regulatory mechanisms and role of amylopectin accumulation in this organism are not fully understood. The T. gondii genome encodes a putative glycogen phosphorylase (TgGP), and mutants were constructed to manipulate the activity of TgGP and to evaluate the function of TgGP in amylopectin storage. Both a stop codon mutant (Pru/TgGPS25stop [expressing a Ser-to-stop codon change at position 25 in TgGP]) and a phosphorylation null mutant (Pru/TgGPS25A [expressing a Ser-to-Ala change at position 25 in TgGp]) mutated at Ser25 displayed amylopectin accumulation, while the phosphorylation-mimetic mutant (Pru/TgGPS25E [expressing a Ser-to-Glu change at position 25 in TgGp]) had minimal amylopectin accumulation under both tachyzoite and bradyzoite growth conditions. The expression of active TgGPS25S or TgGPS25E restored amylopectin catabolism in Pru/TgGPS25A To understand the relation between GP and calcium-dependent protein kinase 2 (CDPK2), which was recently reported to regulate amylopectin consumption, we knocked out CDPK2 in these mutants. PruΔcdpk2/TgGPS25E had minimal amylopectin accumulation, whereas the Δcdpk2 phenotype in the other GP mutants and parental lines displayed amylopectin accumulation. Both the inactive S25A and hyperactive S25E mutant produced Brain Cysts in infected mice, but the numbers of Cysts produced were significantly less than the number produced by the S25S wild-type GP parasite. Complementation that restored amylopectin regulation restored Brain Cyst production to the control levels seen in infected mice. These data suggest that T. gondii requires tight regulation of amylopectin expression for efficient production of Cysts and persistent infections and that GP phosphorylation is a regulatory mechanism involved in amylopectin storage and utilization.IMPORTANCEToxoplasma gondii is an obligate intracellular parasite that causes disease in immune-suppressed individuals, as well as a fetopathy in pregnant women who acquire infection for the first time during pregnancy. This parasite can differentiate between tachyzoites (seen in acute infection) and bradyzoites (seen in latent infection), and this differentiation is associated with disease relapse. A characteristic of bradyzoites is that they contain cytoplasmic amylopectin granules. The regulatory mechanisms and the roles of amylopectin granules during latent infection remain to be elucidated. We have identified a role of T. gondii glycogen phosphorylase (TgGP) in the regulation of starch digestion and a role of posttranslational modification of TgGP, i.e., phosphorylation of Ser25, in the regulation of amylopectin digestion. By manipulating TgGP activity in the parasite with genome editing, we found that the digestion and storage of amylopectin due to TgGP activity are both important for latency in the Brain.
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making home sweet and sturdy toxoplasma gondii ppgalnac ts glycosylate in hierarchical order and confer Cyst wall rigidity
Mbio, 2017Co-Authors: Tadakimi Tomita, Tatsuki Sugi, Rama R Yakubu, Louis M. WeissAbstract:ABSTRACT The protozoan intracellular parasite Toxoplasma gondii forms latent Cysts in the central nervous system (CNS) and persists for the lifetime of the host. This Cyst is cloaked with a glycosylated structure called the Cyst wall. Previously, we demonstrated that a mucin-like glycoprotein, CST1, localizes to the Cyst wall and confers structural rigidity on Brain Cysts in a mucin-like domain-dependent manner. The mucin-like domain of CST1 is composed of 20 units of threonine-rich tandem repeats that are O- GalNAc glycosylated. A family of enzymes termed polypeptide N -acetylgalactosaminyltransferases (ppGalNAc-Ts) initiates O -GalNAc glycosylation. To identify which isoforms of ppGalNAc-Ts are responsible for the glycosylation of the CST1 mucin-like domain and to evaluate the function of each ppGalNAc-T in the overall glycosylation of the Cyst wall, all five ppGalNAc-T isoforms were deleted individually from the T. gondii genome. The ppGalNAc-T2 and -T3 deletion mutants produced various glycosylation defects on the Cyst wall, implying that many Cyst wall glycoproteins are glycosylated by T2 and T3. Both T2 and T3 glycosylate the CST1 mucin-like domain, and this glycosylation is necessary for CST1 to confer structural rigidity on the Cyst wall. We established that T2 is required for the initial glycosylation of the mucin-like domain and that T3 is responsible for the sequential glycosylation on neighboring acceptor sites, demonstrating hierarchical glycosylation by two distinct initiating and filling-in ppGalNAc-Ts in an intact organism. IMPORTANCE Toxoplasma gondii is an obligate intracellular parasite that infects a third of the world’s population. It can cause severe congenital disease and devastating encephalitis in immunocompromised individuals. We identified two glycosyltransferases, ppGalNAc-T2 and -T3, which are responsible for glycosylating Cyst wall proteins in a hierarchical fashion. This glycosylation confers structural rigidity on the Brain Cyst. Our studies provide new insights into the mechanisms of O- GalNAc glycosylation in T. gondii .
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Toxoplasma gondii Requires Glycogen Phosphorylase for Balancing Amylopectin Storage and for Efficient Production of Brain Cysts
American Society for Microbiology, 2017Co-Authors: Tatsuki Sugi, Tadakimi Tomita, Louis M. Weiss, Jon P. BoyleAbstract:In immunocompromised hosts, latent infection with Toxoplasma gondii can reactivate from tissue Cysts, leading to encephalitis. A characteristic of T. gondii bradyzoites in tissue Cysts is the presence of amylopectin granules. The regulatory mechanisms and role of amylopectin accumulation in this organism are not fully understood. The T. gondii genome encodes a putative glycogen phosphorylase (TgGP), and mutants were constructed to manipulate the activity of TgGP and to evaluate the function of TgGP in amylopectin storage. Both a stop codon mutant (Pru/TgGPS25stop [expressing a Ser-to-stop codon change at position 25 in TgGP]) and a phosphorylation null mutant (Pru/TgGPS25A [expressing a Ser-to-Ala change at position 25 in TgGp]) mutated at Ser25 displayed amylopectin accumulation, while the phosphorylation-mimetic mutant (Pru/TgGPS25E [expressing a Ser-to-Glu change at position 25 in TgGp]) had minimal amylopectin accumulation under both tachyzoite and bradyzoite growth conditions. The expression of active TgGPS25S or TgGPS25E restored amylopectin catabolism in Pru/TgGPS25A. To understand the relation between GP and calcium-dependent protein kinase 2 (CDPK2), which was recently reported to regulate amylopectin consumption, we knocked out CDPK2 in these mutants. PruΔcdpk2/TgGPS25E had minimal amylopectin accumulation, whereas the Δcdpk2 phenotype in the other GP mutants and parental lines displayed amylopectin accumulation. Both the inactive S25A and hyperactive S25E mutant produced Brain Cysts in infected mice, but the numbers of Cysts produced were significantly less than the number produced by the S25S wild-type GP parasite. Complementation that restored amylopectin regulation restored Brain Cyst production to the control levels seen in infected mice. These data suggest that T. gondii requires tight regulation of amylopectin expression for efficient production of Cysts and persistent infections and that GP phosphorylation is a regulatory mechanism involved in amylopectin storage and utilization