Ann N Y Acad Sci. 2012 Jul;1258(1):135-42. doi: 10.1111/j.1749-6632.2012.06534.x.
The protozoan pathogen Toxoplasma gondii targets the paracellular pathway to invade the intestinal epithelium
Weight CM, Carding SR.
Institute of Food Research Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich, United Kingdom.
Toxoplasma gondii is a ubiquitous parasite found within all mammals and birds worldwide that can cause fatal infections in immunocompromised persons and fetuses. The parasite causes chronic infections by residing in long-living tissues of the muscle and brain. T. gondii infects the host through contaminated meat and water consumption with the gastrointestinal tract (GI tract) being the first point of contact with the host. The mechanisms by which the parasite invades the host through the GI tract are unknown, although it has been suggested that the paracellular pathway is important for parasite dissemination. Studies indicate that epithelial tight junction-associated proteins are affected by T. gondii, although which junctional proteins are affected and the nature of host protein-parasite interactions have not been established. We have uncovered evidence that T. gondii influences the cellular distribution of occludin to transmigrate the intestinal epithelium and suggest how candidate binding partners can be identified.
© 2012 New York Academy of Sciences.
PMID: 22731726 [PubMed - in process]
Wednesday, June 27, 2012
The GTPase activity of murine guanylate-binding protein 2 (mGBP2) controls the intracellular localization and recruitment to the parasitophorous vacuole of Toxoplasma gondii
J Biol Chem. 2012 Jun 22. [Epub ahead of print]
The GTPase activity of murine guanylate-binding protein 2 (mGBP2) controls the intracellular localization and recruitment to the parasitophorous vacuole of Toxoplasma gondii
Kravets E, Degrandi D, Weidtkamp-Peters S, Ries B, Konermann C, Felekyan S, Dargazanli JM, Praefcke GJ, Seidel CA, Schmitt L, Smits SH, Pfeffer K.
Heinrich-Heine University, Germany;
One of the most abundantly IFN-γ-induced protein families in different cell types are the 65-kDa guanylate-binding proteins (GBPs) which are recruited to the parasitophorous vacuole (PV) of the intracellular parasite T. gondii. Here, we elucidate the relationship between biochemistry and cellular host defense functions of mGBP2 in response to T. gondii. The wild type (WT) protein exhibits low affinities to guanine nucleotides, self-assembles upon guanosine 5' triphosphate (GTP) binding, forming tetramers in the activated state and stimulates the GTPase activity in a cooperative manner. The products of the two consecutive hydrolysis reactions are both GDP and GMP. The biochemical characterization of point mutants in the GTP-binding motifs of mGBP2 revealed amino acid residues that decrease the GTPase activity by orders of magnitude and strongly impair nucleotide binding and multimerization ability. Live cell imaging employing Multiparameter Fluorescence Image Spectroscopy (MFIS) shows that the inducible multimerization of mGBP2 is dependent on a functional GTPase-domain. The consistent results indicate that GTP-binding, self-assembly and stimulated hydrolysis activity are required for physiological localization of the protein in infected and uninfected cells. Ultimately, we show that the GTPase-domain regulates efficient recruitment to T. gondii in response to IFN-γ.
PMID: 22730319 [PubMed - as supplied by publisher]
The GTPase activity of murine guanylate-binding protein 2 (mGBP2) controls the intracellular localization and recruitment to the parasitophorous vacuole of Toxoplasma gondii
Kravets E, Degrandi D, Weidtkamp-Peters S, Ries B, Konermann C, Felekyan S, Dargazanli JM, Praefcke GJ, Seidel CA, Schmitt L, Smits SH, Pfeffer K.
Heinrich-Heine University, Germany;
One of the most abundantly IFN-γ-induced protein families in different cell types are the 65-kDa guanylate-binding proteins (GBPs) which are recruited to the parasitophorous vacuole (PV) of the intracellular parasite T. gondii. Here, we elucidate the relationship between biochemistry and cellular host defense functions of mGBP2 in response to T. gondii. The wild type (WT) protein exhibits low affinities to guanine nucleotides, self-assembles upon guanosine 5' triphosphate (GTP) binding, forming tetramers in the activated state and stimulates the GTPase activity in a cooperative manner. The products of the two consecutive hydrolysis reactions are both GDP and GMP. The biochemical characterization of point mutants in the GTP-binding motifs of mGBP2 revealed amino acid residues that decrease the GTPase activity by orders of magnitude and strongly impair nucleotide binding and multimerization ability. Live cell imaging employing Multiparameter Fluorescence Image Spectroscopy (MFIS) shows that the inducible multimerization of mGBP2 is dependent on a functional GTPase-domain. The consistent results indicate that GTP-binding, self-assembly and stimulated hydrolysis activity are required for physiological localization of the protein in infected and uninfected cells. Ultimately, we show that the GTPase-domain regulates efficient recruitment to T. gondii in response to IFN-γ.
PMID: 22730319 [PubMed - as supplied by publisher]
Monday, June 25, 2012
Generalized Lévy walks and the role of chemokines in migration of effector CD8(+) T cells
Nature. 2012 May 27. doi: 10.1038/nature11098. [Epub ahead of print]
Generalized Lévy walks and the role of chemokines in migration of effector CD8(+) T cells
Harris TH, Banigan EJ, Christian DA, Konradt C, Tait Wojno ED, Norose K, Wilson EH, John B, Weninger W, Luster AD, Liu AJ, Hunter CA.
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, 380 South University Avenue, Philadelphia, Pennsylvania 19104, USA.
Chemokines have a central role in regulating processes essential to the immune function of T cells, such as their migration within lymphoid tissues and targeting of pathogens in sites of inflammation. Here we track T cells using multi-photon microscopy to demonstrate that the chemokine CXCL10 enhances the ability of CD8(+) T cells to control the pathogen Toxoplasma gondii in the brains of chronically infected mice. This chemokine boosts T-cell function in two different ways: it maintains the effector T-cell population in the brain and speeds up the average migration speed without changing the nature of the walk statistics. Notably, these statistics are not Brownian; rather, CD8(+) T-cell motility in the brain is well described by a generalized Lévy walk. According to our model, this unexpected feature enables T cells to find rare targets with more than an order of magnitude more efficiency than Brownian random walkers. Thus, CD8(+) T-cell behaviour is similar to Lévy strategies reported in organisms ranging from mussels to marine predators and monkeys, and CXCL10 aids T cells in shortening the average time taken to find rare targets.
PMID: 22722867 [PubMed - as supplied by publisher]
Generalized Lévy walks and the role of chemokines in migration of effector CD8(+) T cells
Harris TH, Banigan EJ, Christian DA, Konradt C, Tait Wojno ED, Norose K, Wilson EH, John B, Weninger W, Luster AD, Liu AJ, Hunter CA.
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, 380 South University Avenue, Philadelphia, Pennsylvania 19104, USA.
Chemokines have a central role in regulating processes essential to the immune function of T cells, such as their migration within lymphoid tissues and targeting of pathogens in sites of inflammation. Here we track T cells using multi-photon microscopy to demonstrate that the chemokine CXCL10 enhances the ability of CD8(+) T cells to control the pathogen Toxoplasma gondii in the brains of chronically infected mice. This chemokine boosts T-cell function in two different ways: it maintains the effector T-cell population in the brain and speeds up the average migration speed without changing the nature of the walk statistics. Notably, these statistics are not Brownian; rather, CD8(+) T-cell motility in the brain is well described by a generalized Lévy walk. According to our model, this unexpected feature enables T cells to find rare targets with more than an order of magnitude more efficiency than Brownian random walkers. Thus, CD8(+) T-cell behaviour is similar to Lévy strategies reported in organisms ranging from mussels to marine predators and monkeys, and CXCL10 aids T cells in shortening the average time taken to find rare targets.
PMID: 22722867 [PubMed - as supplied by publisher]
Wednesday, June 06, 2012
Toxoplasma ISP4 is a central IMC sub-compartment protein whose localization depends on palmitoylation but not myristoylation
Mol Biochem Parasitol. 2012 May 30. [Epub ahead of print]
Toxoplasma ISP4 is a central IMC sub-compartment protein whose localization depends on palmitoylation but not myristoylation
Fung C, Beck JR, Robertson SD, Gubbels MJ, Bradley PJ.
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles CA 90095-1489 USA.
Apicomplexan parasites utilize a peripheral membrane system called the inner membrane complex (IMC) to facilitate host cell invasion and parasite replication. We recently identified a novel family of Toxoplasma IMC Sub-compartment Proteins (ISP1/2/3) that localize to sub-domains of the IMC using a targeting mechanism that is dependent on coordinated myristoylation and palmitoylation of a series of residues in the N-terminus of the protein. While the precise functions of the ISPs are unknown, deletion of ISP2 results in replication defects, suggesting that this family of proteins plays a role in daughter cell formation. Here we have characterized a fourth ISP family member (ISP4) and discovered that this protein localizes to the central IMC sub-compartment, similar to ISP2. Like ISP1/3, ISP4 is dispensable for the tachyzoite lytic cycle as the disruption of ISP4 does not produce any gross replication or growth defects. Surprisingly, targeting of ISP4 to the IMC membranes is dependent on residues predicted for palmitoylation but not myristoylation, setting its trafficking apart from the other ISP proteins and demonstrating distinct mechanisms of protein localization to the IMC membranes, even within a family of highly-related proteins.
Copyright © 2012. Published by Elsevier B.V.
PMID: 22659420
Toxoplasma ISP4 is a central IMC sub-compartment protein whose localization depends on palmitoylation but not myristoylation
Fung C, Beck JR, Robertson SD, Gubbels MJ, Bradley PJ.
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles CA 90095-1489 USA.
Apicomplexan parasites utilize a peripheral membrane system called the inner membrane complex (IMC) to facilitate host cell invasion and parasite replication. We recently identified a novel family of Toxoplasma IMC Sub-compartment Proteins (ISP1/2/3) that localize to sub-domains of the IMC using a targeting mechanism that is dependent on coordinated myristoylation and palmitoylation of a series of residues in the N-terminus of the protein. While the precise functions of the ISPs are unknown, deletion of ISP2 results in replication defects, suggesting that this family of proteins plays a role in daughter cell formation. Here we have characterized a fourth ISP family member (ISP4) and discovered that this protein localizes to the central IMC sub-compartment, similar to ISP2. Like ISP1/3, ISP4 is dispensable for the tachyzoite lytic cycle as the disruption of ISP4 does not produce any gross replication or growth defects. Surprisingly, targeting of ISP4 to the IMC membranes is dependent on residues predicted for palmitoylation but not myristoylation, setting its trafficking apart from the other ISP proteins and demonstrating distinct mechanisms of protein localization to the IMC membranes, even within a family of highly-related proteins.
Copyright © 2012. Published by Elsevier B.V.
PMID: 22659420
Saturday, June 02, 2012
Toxoplasma Skp1 is Modified by a Cytoplasmic Prolyl 4-Hydroxylase Associated with Oxygen Sensing in the Social Amoeba Dictyostelium
J Biol Chem. 2012 May 30. [Epub ahead of print]
Toxoplasma Skp1 is Modified by a Cytoplasmic Prolyl 4-Hydroxylase Associated with Oxygen Sensing in the Social Amoeba Dictyostelium
Xu Y, Brown KM, Wang ZA, van der Wel H, Teygong C, Zhang D, Blader IJ, West CM.
OUHSC, United States.
In diverse types of organisms, cellular hypoxic responses are mediated by prolyl 4-hydroxylases that use O2 and α-ketoglutarate as substrates to hydroxylate conserved proline residues in target proteins. Whereas in metazoans, these enzymes control the stability of the HIFα family of transcription factor subunits, the Dictyostelium enzyme (DdPhyA) contributes to O2 regulation of development by a divergent mechanism involving hydroxylation and subsequent glycosylation of DdSkp1, an adaptor subunit in E3SCFubiquitin ligases. Sequences related to DdPhyA, DdSkp1, and the glycosyltransferases that cap Skp1-hydroxyproline occur also in the genomes of Toxoplasma and other protists, suggesting that this O2 sensing mechanism may be widespread. Here we show by disruption of the TgphyA locus that this enzyme is required for Skp1 glycosylation in Toxoplasma and that disrupted parasites grow slowly at physiological O2 levels. Conservation of cellular function was tested by expression of TgPhyA in DdphyA-null cells. Simple gene replacement did not rescue Skp1 glycosylation, whereas overexpression not only corrected Skp1 modification but also restored the O2 requirement to a level comparable to that of overexpressed DdPhyA. Bacterially expressed TgPhyA protein can prolyl hydroxylate both Toxoplasma and Dictyostelium Skp1s. Kinetic analyses showed that TgPhyA has similar properties to DdPhyA, including a superimposable dependence on the concentration of its co-substrate α-ketoglutarate. Remarkably, however, TgPhyA had a significantly higher apparent affinity for O2. The findings suggest that Skp1 hydroxylation by PhyA is a conserved process among protists and that this biochemical pathway may indirectly sense O2 by detecting the levels of O2-regulated metabolites such as α-ketoglutarate.
PMID: 22648409 [PubMed - as supplied by publisher]
Toxoplasma Skp1 is Modified by a Cytoplasmic Prolyl 4-Hydroxylase Associated with Oxygen Sensing in the Social Amoeba Dictyostelium
Xu Y, Brown KM, Wang ZA, van der Wel H, Teygong C, Zhang D, Blader IJ, West CM.
OUHSC, United States.
In diverse types of organisms, cellular hypoxic responses are mediated by prolyl 4-hydroxylases that use O2 and α-ketoglutarate as substrates to hydroxylate conserved proline residues in target proteins. Whereas in metazoans, these enzymes control the stability of the HIFα family of transcription factor subunits, the Dictyostelium enzyme (DdPhyA) contributes to O2 regulation of development by a divergent mechanism involving hydroxylation and subsequent glycosylation of DdSkp1, an adaptor subunit in E3SCFubiquitin ligases. Sequences related to DdPhyA, DdSkp1, and the glycosyltransferases that cap Skp1-hydroxyproline occur also in the genomes of Toxoplasma and other protists, suggesting that this O2 sensing mechanism may be widespread. Here we show by disruption of the TgphyA locus that this enzyme is required for Skp1 glycosylation in Toxoplasma and that disrupted parasites grow slowly at physiological O2 levels. Conservation of cellular function was tested by expression of TgPhyA in DdphyA-null cells. Simple gene replacement did not rescue Skp1 glycosylation, whereas overexpression not only corrected Skp1 modification but also restored the O2 requirement to a level comparable to that of overexpressed DdPhyA. Bacterially expressed TgPhyA protein can prolyl hydroxylate both Toxoplasma and Dictyostelium Skp1s. Kinetic analyses showed that TgPhyA has similar properties to DdPhyA, including a superimposable dependence on the concentration of its co-substrate α-ketoglutarate. Remarkably, however, TgPhyA had a significantly higher apparent affinity for O2. The findings suggest that Skp1 hydroxylation by PhyA is a conserved process among protists and that this biochemical pathway may indirectly sense O2 by detecting the levels of O2-regulated metabolites such as α-ketoglutarate.
PMID: 22648409 [PubMed - as supplied by publisher]
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