Nat Struct Mol Biol. 2010 May;17(5):602-7. Epub 2010 May 2.
Toxoplasma gondii calcium-dependent protein kinase 1 is a target for selective kinase inhibitors
Ojo KK, Larson ET, Keyloun KR, Castaneda LJ, Derocher AE, Inampudi KK, Kim JE, Arakaki TL, Murphy RC, Zhang L, Napuli AJ, Maly DJ, Verlinde CL, Buckner FS, Parsons M, Hol WG, Merritt EA, Van Voorhis WC.
Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, Seattle, Washington, USA.
Abstract
New drugs are needed to treat toxoplasmosis. Toxoplasma gondii calcium-dependent protein kinases (TgCDPKs) are attractive targets because they are absent in mammals. We show that TgCDPK1 is inhibited by low nanomolar levels of bumped kinase inhibitors (BKIs), compounds inactive against mammalian kinases. Cocrystal structures of TgCDPK1 with BKIs confirm that the structural basis for selectivity is due to the unique glycine gatekeeper residue in the ATP-binding site. We show that BKIs interfere with an early step in T. gondii infection of human cells in culture. Furthermore, we show that TgCDPK1 is the in vivo target of BKIs because T. gondii expressing a glycine to methionine gatekeeper mutant enzyme show significantly decreased sensitivity to BKIs. Thus, design of selective TgCDPK1 inhibitors with low host toxicity may be achievable.
PMID: 20436472 [PubMed - indexed for MEDLINE]
Friday, May 28, 2010
Novel vacuoles in Toxoplasma
Mol Microbiol. 2010 May 25. [Epub ahead of print]
Novel vacuoles in Toxoplasma
van Dooren GG, Ralph SA.
Plant Cell Biology Research Centre, School of Botany, The University of Melbourne, Melbourne, Vic 3010, Australia.
PMID: 20500550 [PubMed - as supplied by publisher]
Novel vacuoles in Toxoplasma
van Dooren GG, Ralph SA.
Plant Cell Biology Research Centre, School of Botany, The University of Melbourne, Melbourne, Vic 3010, Australia.
PMID: 20500550 [PubMed - as supplied by publisher]
Wednesday, May 26, 2010
Protective Toxplasma gondii-specific T cell responses require the T cell-specific expression of protein kinase C-{theta}1
Infect Immun. 2010 May 24. [Epub ahead of print]
Protective Toxplasma gondii-specific T cell responses require the T cell-specific expression of protein kinase C-{theta}1
Nishanth G, Sakowicz-Burkiewicz M, Händel U, Kliche S, Wang X, Naumann M, Deckert M, Schlüter D.
Institut für Medizinische Mikrobiologie, OvG Universität Magdeburg, Magdeburg, Germany; Institut für Molekulare und Klinische Immunologie, OvG Universität Magdeburg, Magdeburg, Germany; Institut für Experimentelle Innere Medizin, OvG Universität Magdeburg, Magdeburg, Germany; Abteilung für Neuropathologie, Universitätsklinikum Köln, Köln, Germany.
Abstract
Protein kinase C (PKC)- is important for the activation of autoreactive T cells but is thought to be of minor importance for T cell responses in infectious diseases suggesting PKC- as target for the treatment of T cell-mediated autoimmune diseases. To explore the function of PKC- in a chronic persisting infection, in which T cells are crucial for pathogen control, we infected BALB/c PKC-(-/-) and PKC-(+/+) wildtype mice with Toxoplasma gondii. PKC-(-/-) mice succumbed to necrotizing Toxoplasma encephalitis due to an insufficient parasite control up to day 40, whereas wildtype mice survived. The number of T. gondii-specific CD4 and CD8 T cells was significantly reduced in PKC-(-/-) mice resulting in an impaired production of protective cytokines (interferon-gamma, tumor necrosis factor) and anti-parasitic effector molecules (inducible nitric oxide, IGTP) in spleen and brain. In addition, Th2 cells were reduced in infected PKC-(-/-) mice, paralleled by a diminished GATA3 expression of PKC-(-/-) CD4 T cells and a reduced T. gondii-specific IgG production in serum and cerebrospinal fluid. Western blot analysis of splenic CD4 and CD8 T cells revealed an impaired activation of the NF-kappaB, AP1, and MAPK pathway in T. gondii-infected PKC-(-/-) mice. Adoptive transfer of wildtype CD4 plus CD8 T cells significantly protected PKC-(-/-) mice from death by increasing numbers of interferon-gamma-producing T. gondii-specific CD4 and CD8 T cells illustrating a cell-autonomous, protective function of PKC- in T cells. These findings imply that PKC- inhibition drastically impairs T. gondii-specific T cells responses with fatal consequences for intracerebral parasite control and survival.
PMID: 20498263 [PubMed - as supplied by publisher]
Protective Toxplasma gondii-specific T cell responses require the T cell-specific expression of protein kinase C-{theta}1
Nishanth G, Sakowicz-Burkiewicz M, Händel U, Kliche S, Wang X, Naumann M, Deckert M, Schlüter D.
Institut für Medizinische Mikrobiologie, OvG Universität Magdeburg, Magdeburg, Germany; Institut für Molekulare und Klinische Immunologie, OvG Universität Magdeburg, Magdeburg, Germany; Institut für Experimentelle Innere Medizin, OvG Universität Magdeburg, Magdeburg, Germany; Abteilung für Neuropathologie, Universitätsklinikum Köln, Köln, Germany.
Abstract
Protein kinase C (PKC)- is important for the activation of autoreactive T cells but is thought to be of minor importance for T cell responses in infectious diseases suggesting PKC- as target for the treatment of T cell-mediated autoimmune diseases. To explore the function of PKC- in a chronic persisting infection, in which T cells are crucial for pathogen control, we infected BALB/c PKC-(-/-) and PKC-(+/+) wildtype mice with Toxoplasma gondii. PKC-(-/-) mice succumbed to necrotizing Toxoplasma encephalitis due to an insufficient parasite control up to day 40, whereas wildtype mice survived. The number of T. gondii-specific CD4 and CD8 T cells was significantly reduced in PKC-(-/-) mice resulting in an impaired production of protective cytokines (interferon-gamma, tumor necrosis factor) and anti-parasitic effector molecules (inducible nitric oxide, IGTP) in spleen and brain. In addition, Th2 cells were reduced in infected PKC-(-/-) mice, paralleled by a diminished GATA3 expression of PKC-(-/-) CD4 T cells and a reduced T. gondii-specific IgG production in serum and cerebrospinal fluid. Western blot analysis of splenic CD4 and CD8 T cells revealed an impaired activation of the NF-kappaB, AP1, and MAPK pathway in T. gondii-infected PKC-(-/-) mice. Adoptive transfer of wildtype CD4 plus CD8 T cells significantly protected PKC-(-/-) mice from death by increasing numbers of interferon-gamma-producing T. gondii-specific CD4 and CD8 T cells illustrating a cell-autonomous, protective function of PKC- in T cells. These findings imply that PKC- inhibition drastically impairs T. gondii-specific T cells responses with fatal consequences for intracerebral parasite control and survival.
PMID: 20498263 [PubMed - as supplied by publisher]
CD4 T-Cell Suppression by Cells from Toxoplasma gondii-Infected Retinas is Mediated by PD-L1
Infect Immun. 2010 May 24. [Epub ahead of print]
CD4 T-Cell Suppression by Cells from Toxoplasma gondii-Infected Retinas is Mediated by PD-L1
Charles E, Joshi S, Ash JD, Fox BA, Farris AD, Bzik DJ, Lang ML, Blader IJ.
Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104; Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104; Department of Microbiology and Immunology, Dartmouth Medical School, Lebanon, NH 03756; Arthritis and Immunology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104.
Abstract
In the inflamed retina, CD4(+) T-cells can cause retinal damage when they are not properly regulated. Since tissue expression of MHC Class II and co-stimulatory molecules is a key mechanism to regulate effector T-cells, we tested the hypothesis that upregulation of these proteins in the retina contributes to the regulation of CD4 T-cells. Here, we report that in retinas infected with the protozoan parasite Toxoplasma gondii MHC Class II is upregulated on infiltrating leukocytes as well as resident retinal cells including photoreceptors. Flow cytometric analysis indicated that B7 costimulatory family members (CD80, CD86, ICOS-L, and PD-L2) were not expressed on the Class II(+) cells. In contrast, PD-L1 (also named B7-H1 or CD274) was expressed on the majority of both hematopoietic and resident retinal Class II-expressing cells. Retinal cells from Toxoplasma-infected animals were able to suppress T-cell activation in a PD-L1-dependent manner. Finally, we demonstrate that MHC Class II and PD-L1 expression was critically dependent on IFNgamma expression. These data suggests that retinal MHC Class II and PD-L1 expression is a novel mechanism for the retina to protect itself from CD4 T-cell-mediated immune damage in ocular toxoplasmosis and other types of retinal immune responses.
PMID: 20498261 [PubMed - as supplied by publisher]
CD4 T-Cell Suppression by Cells from Toxoplasma gondii-Infected Retinas is Mediated by PD-L1
Charles E, Joshi S, Ash JD, Fox BA, Farris AD, Bzik DJ, Lang ML, Blader IJ.
Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104; Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104; Department of Microbiology and Immunology, Dartmouth Medical School, Lebanon, NH 03756; Arthritis and Immunology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104.
Abstract
In the inflamed retina, CD4(+) T-cells can cause retinal damage when they are not properly regulated. Since tissue expression of MHC Class II and co-stimulatory molecules is a key mechanism to regulate effector T-cells, we tested the hypothesis that upregulation of these proteins in the retina contributes to the regulation of CD4 T-cells. Here, we report that in retinas infected with the protozoan parasite Toxoplasma gondii MHC Class II is upregulated on infiltrating leukocytes as well as resident retinal cells including photoreceptors. Flow cytometric analysis indicated that B7 costimulatory family members (CD80, CD86, ICOS-L, and PD-L2) were not expressed on the Class II(+) cells. In contrast, PD-L1 (also named B7-H1 or CD274) was expressed on the majority of both hematopoietic and resident retinal Class II-expressing cells. Retinal cells from Toxoplasma-infected animals were able to suppress T-cell activation in a PD-L1-dependent manner. Finally, we demonstrate that MHC Class II and PD-L1 expression was critically dependent on IFNgamma expression. These data suggests that retinal MHC Class II and PD-L1 expression is a novel mechanism for the retina to protect itself from CD4 T-cell-mediated immune damage in ocular toxoplasmosis and other types of retinal immune responses.
PMID: 20498261 [PubMed - as supplied by publisher]
Regulation of CD8(+) T Cell Responses to Infection With Parasitic Protozoa
Exp Parasitol. 2010 May 19. [Epub ahead of print]
Regulation of CD8(+) T Cell Responses to Infection With Parasitic Protozoa
Jordan KA, Hunter CA.
Department of Pathobiology, University of Pennsylvania, 380 South University Ave, Philadelphia, PA 19104, USA.
Abstract
There are over 10,000 species of parasitic protozoa, a subset of which can cause considerable disease in humans. Here we examine in detail the complex immune response generated during infection with a subset of these parasites: Trypanosoma cruzi, Leishmania sp, Toxoplasma gondii, and Plasmodium sp. While these particular species perhaps represent the most studied parasites in terms of understanding how T cells function during infection, it is clear that the lessons learned from this body of work are also relevant to the other protozoa known to induce a CD8+ T cell response. This review will highlight some of the key studies that established that CD8+ T cells play a major role in protective immunity to protozoa, the factors that promote the generation as well as maintenance of the CD8+ T cell response during these infections, and draw attention to some of the gaps in our knowledge. Moreover, the development of new tools, including MHC Class I tetramer reagents and the use of TCR transgenic mice or genetically modified parasites, has provided a better appreciation of how parasite specific CD8+ T cell responses are initiated and new insights into their phenotypic plasticity. Copyright © 2010. Published by Elsevier Inc.
PMID: 20493842 [PubMed - as supplied by publisher]
Regulation of CD8(+) T Cell Responses to Infection With Parasitic Protozoa
Jordan KA, Hunter CA.
Department of Pathobiology, University of Pennsylvania, 380 South University Ave, Philadelphia, PA 19104, USA.
Abstract
There are over 10,000 species of parasitic protozoa, a subset of which can cause considerable disease in humans. Here we examine in detail the complex immune response generated during infection with a subset of these parasites: Trypanosoma cruzi, Leishmania sp, Toxoplasma gondii, and Plasmodium sp. While these particular species perhaps represent the most studied parasites in terms of understanding how T cells function during infection, it is clear that the lessons learned from this body of work are also relevant to the other protozoa known to induce a CD8+ T cell response. This review will highlight some of the key studies that established that CD8+ T cells play a major role in protective immunity to protozoa, the factors that promote the generation as well as maintenance of the CD8+ T cell response during these infections, and draw attention to some of the gaps in our knowledge. Moreover, the development of new tools, including MHC Class I tetramer reagents and the use of TCR transgenic mice or genetically modified parasites, has provided a better appreciation of how parasite specific CD8+ T cell responses are initiated and new insights into their phenotypic plasticity. Copyright © 2010. Published by Elsevier Inc.
PMID: 20493842 [PubMed - as supplied by publisher]
Sunday, May 23, 2010
A NEW ATYPICAL HIGHLY MOUSE VIRULENT TOXOPLASMA GONDII GENOTYPE ISOLATED FROM A WILD BLACK BEAR IN ALASKA
J Parasitol. 2010 Apr 5:1. [Epub ahead of print]
A NEW ATYPICAL HIGHLY MOUSE VIRULENT TOXOPLASMA GONDII GENOTYPE ISOLATED FROM A WILD BLACK BEAR IN ALASKA
Dubey JP, Chellaiah R, Ferreira L, Kwok O, Sinnett D, Majumdar D.
Abstract
Most strains of Toxoplasma gondii isolated in North America and Europe are grouped into three (Types I, II, III) genetic types and are considered clonal. Recent evidence suggests that illness due to toxoplasmosis in immunocompetent persons may be related to infection with atypical genotype; these strains are mouse virulent. In the present study, a new mouse virulent atypical T. gondii genotype was isolated from an asymptomatic black bear (Ursus americanus) from Alaska, USA. The bear had a titer of 1:1,600 in the modified agglutination test for T. gondii. Swiss Webster out-bred mice inoculated with bear heart homogenate died of acute toxoplasmosis, 12 days p.i. Cats fed tissues from chronically infected animals (30 day infection) shed oocysts but cats fed acutely infected mice ( 12 and 18 days p.i.) did not. The isolate (designated TgBbUS1) was mouse virulent, mice inoculated with 1 oocyst died of acute toxoplasmosis. The restricted fragment length polymorphism using 10 markers revealed that the strain possessed an atypical genotype; type I allele at loci SAG1, (5'-3')SAG2, SAG3, c22-8, c29-2, L358, and Apico, type II allele at locus alt.SAG2, and type III allele at loci BTUB, GRA6, and PK1. DNA sequencing at intron loci EF1, HP2 and UPRT1 revealed that the TgBbUS1 is a divergent T. gondii strain. These results indicate that mouse virulent atypical T. gondii genotypes are also circulating in wildlife in the US.
PMID: 20486739 [PubMed - as supplied by publisher]
A NEW ATYPICAL HIGHLY MOUSE VIRULENT TOXOPLASMA GONDII GENOTYPE ISOLATED FROM A WILD BLACK BEAR IN ALASKA
Dubey JP, Chellaiah R, Ferreira L, Kwok O, Sinnett D, Majumdar D.
Abstract
Most strains of Toxoplasma gondii isolated in North America and Europe are grouped into three (Types I, II, III) genetic types and are considered clonal. Recent evidence suggests that illness due to toxoplasmosis in immunocompetent persons may be related to infection with atypical genotype; these strains are mouse virulent. In the present study, a new mouse virulent atypical T. gondii genotype was isolated from an asymptomatic black bear (Ursus americanus) from Alaska, USA. The bear had a titer of 1:1,600 in the modified agglutination test for T. gondii. Swiss Webster out-bred mice inoculated with bear heart homogenate died of acute toxoplasmosis, 12 days p.i. Cats fed tissues from chronically infected animals (30 day infection) shed oocysts but cats fed acutely infected mice ( 12 and 18 days p.i.) did not. The isolate (designated TgBbUS1) was mouse virulent, mice inoculated with 1 oocyst died of acute toxoplasmosis. The restricted fragment length polymorphism using 10 markers revealed that the strain possessed an atypical genotype; type I allele at loci SAG1, (5'-3')SAG2, SAG3, c22-8, c29-2, L358, and Apico, type II allele at locus alt.SAG2, and type III allele at loci BTUB, GRA6, and PK1. DNA sequencing at intron loci EF1, HP2 and UPRT1 revealed that the TgBbUS1 is a divergent T. gondii strain. These results indicate that mouse virulent atypical T. gondii genotypes are also circulating in wildlife in the US.
PMID: 20486739 [PubMed - as supplied by publisher]
Novel roles for ATP-binding cassette G transporters in lipid redistribution in Toxoplasma
Mol Microbiol. 2010 May 12. [Epub ahead of print]
Novel roles for ATP-binding cassette G transporters in lipid redistribution in Toxoplasma
Ehrenman K, Sehgal A, Lige B, Stedman TT, Joiner KA, Coppens I.
Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD 21205, USA.
Abstract
Summary Toxoplasma is a protozoan parasite proficiently adapted to thrive in a parasitophorous vacuole (PV) formed in the cytoplasm of a large variety of mammalian cells. As an actively dividing organism, the parasite must adjust the lipid composition of its membranes to preserve organelle vitality and expand the size of the PV membrane to accommodate growing progeny. We showed that Toxoplasma takes up host lipids and can expel major lipids in an ATP-dependent process. In order to provide detailed mechanistic insights into lipid trafficking phenomena relevant to Toxoplasma biology, we characterized six parasite ATP-binding cassette (ABC) G family transporters and investigated their potential contribution to lipid homeostatic processes. All these transporters are expressed in the parasite and five of them are upregulated upon exposure to sterols. Four ABCG are localized to secretory organelles and the plasma membrane, and promote cholesterol and phospholipid efflux, reflecting the importance in exportation of large amounts of lipids into the PV. Interestingly, one ABCG that is associated with vesicles in the PV and the plasma membrane acts as a cholesterol importer. This last finding expands our current view on the role of some ABCG transporters in eukaryotic sterol influx.
PMID: 20487267 [PubMed - as supplied by publisher]
Novel roles for ATP-binding cassette G transporters in lipid redistribution in Toxoplasma
Ehrenman K, Sehgal A, Lige B, Stedman TT, Joiner KA, Coppens I.
Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD 21205, USA.
Abstract
Summary Toxoplasma is a protozoan parasite proficiently adapted to thrive in a parasitophorous vacuole (PV) formed in the cytoplasm of a large variety of mammalian cells. As an actively dividing organism, the parasite must adjust the lipid composition of its membranes to preserve organelle vitality and expand the size of the PV membrane to accommodate growing progeny. We showed that Toxoplasma takes up host lipids and can expel major lipids in an ATP-dependent process. In order to provide detailed mechanistic insights into lipid trafficking phenomena relevant to Toxoplasma biology, we characterized six parasite ATP-binding cassette (ABC) G family transporters and investigated their potential contribution to lipid homeostatic processes. All these transporters are expressed in the parasite and five of them are upregulated upon exposure to sterols. Four ABCG are localized to secretory organelles and the plasma membrane, and promote cholesterol and phospholipid efflux, reflecting the importance in exportation of large amounts of lipids into the PV. Interestingly, one ABCG that is associated with vesicles in the PV and the plasma membrane acts as a cholesterol importer. This last finding expands our current view on the role of some ABCG transporters in eukaryotic sterol influx.
PMID: 20487267 [PubMed - as supplied by publisher]
TLR9-Dependent Induction of Intestinal {alpha}-Defensins by Toxoplasma gondii
J Immunol. 2010 May 19. [Epub ahead of print]
TLR9-Dependent Induction of Intestinal {alpha}-Defensins by Toxoplasma gondii
Foureau DM, Mielcarz DW, Menard LC, Schulthess J, Werts C, Vasseur V, Ryffel B, Kasper LH, Buzoni-Gatel D.
Department of Medicine and Department of Microbiology and Immunology, Dartmouth Medical School, Lebanon, NH 03756;
Abstract
alpha-Defensins (or Cryptdins [Crps]) are a group of antimicrobial peptides produced as a component of Paneth cell (PC) secretory granules in the small intestine. In vivo ligation of TLR9 by synthetic agonists leads to PC degranulation, although the mechanism by which this occurs remains uncertain. In this report, we investigated TLR9-dependent mechanisms, triggered by the parasite Toxoplasma gondii, inducing Crp release in the lumen. Oral challenge of C57BL/6J (B6) wild-type (WT) mice with T. gondii induced TLR9 mRNA upregulation associated with a marked increase of type I IFN mRNA expression. PC secretory granules were released, and Crp-3/-5 mRNA expression by purified epithelial cells was increased following oral challenge of B6 WT mice. Although PCs failed to degranulate in infected B6 TLR9(-/-) mice, i.p. injection of mouse IFN-beta alone led to Crp-3/-5 mRNA upregulation in B6 WT and TLR9(-/-) mice. In addition, modulation of Crp mRNA expression in response to T. gondii infection was abrogated in B6 IFNAR(-/-) mice, which lack a functional type I IFN receptor. Taken together, these data demonstrate that T. gondii induces Crp-3/-5 production and release by PCs via a TLR9-dependent production of type I IFNs. Crps have a limited direct effect against T. gondii but may indirectly affect the early control of T. gondii invasiveness by promoting the initiation of a protective Th1 response against the parasite.
PMID: 20488791 [PubMed - as supplied by publisher]
TLR9-Dependent Induction of Intestinal {alpha}-Defensins by Toxoplasma gondii
Foureau DM, Mielcarz DW, Menard LC, Schulthess J, Werts C, Vasseur V, Ryffel B, Kasper LH, Buzoni-Gatel D.
Department of Medicine and Department of Microbiology and Immunology, Dartmouth Medical School, Lebanon, NH 03756;
Abstract
alpha-Defensins (or Cryptdins [Crps]) are a group of antimicrobial peptides produced as a component of Paneth cell (PC) secretory granules in the small intestine. In vivo ligation of TLR9 by synthetic agonists leads to PC degranulation, although the mechanism by which this occurs remains uncertain. In this report, we investigated TLR9-dependent mechanisms, triggered by the parasite Toxoplasma gondii, inducing Crp release in the lumen. Oral challenge of C57BL/6J (B6) wild-type (WT) mice with T. gondii induced TLR9 mRNA upregulation associated with a marked increase of type I IFN mRNA expression. PC secretory granules were released, and Crp-3/-5 mRNA expression by purified epithelial cells was increased following oral challenge of B6 WT mice. Although PCs failed to degranulate in infected B6 TLR9(-/-) mice, i.p. injection of mouse IFN-beta alone led to Crp-3/-5 mRNA upregulation in B6 WT and TLR9(-/-) mice. In addition, modulation of Crp mRNA expression in response to T. gondii infection was abrogated in B6 IFNAR(-/-) mice, which lack a functional type I IFN receptor. Taken together, these data demonstrate that T. gondii induces Crp-3/-5 production and release by PCs via a TLR9-dependent production of type I IFNs. Crps have a limited direct effect against T. gondii but may indirectly affect the early control of T. gondii invasiveness by promoting the initiation of a protective Th1 response against the parasite.
PMID: 20488791 [PubMed - as supplied by publisher]
P2X7 Receptor-Mediated Killing of an Intracellular Parasite, Toxoplasma gondii, by Human and Murine Macrophages
J Immunol. 2010 May 19. [Epub ahead of print]
P2X7 Receptor-Mediated Killing of an Intracellular Parasite, Toxoplasma gondii, by Human and Murine Macrophages
Lees MP, Fuller SJ, McLeod R, Boulter NR, Miller CM, Zakrzewski AM, Mui EJ, Witola WH, Coyne JJ, Hargrave AC, Jamieson SE, Blackwell JM, Wiley JS, Smith NC.
Institute for the Biotechnology of Infectious Diseases, University of Technology, Sydney, Broadway;
Abstract
The P2X(7)R is highly expressed on the macrophage cell surface, and activation of infected cells by extracellular ATP has been shown to kill intracellular bacteria and parasites. Furthermore, single nucleotide polymorphisms that decrease receptor function reduce the ability of human macrophages to kill Mycobacterium tuberculosis and are associated with extrapulmonary tuberculosis. In this study, we show that macrophages from people with the 1513C (rs3751143, NM_002562.4:c.1487A>C) loss-of-function P2X(7)R single nucleotide polymorphism are less effective in killing intracellular Toxoplasma gondii after exposure to ATP compared with macrophages from people with the 1513A wild-type allele. Supporting a P2X(7)R-specific effect on T. gondii, macrophages from P2X(7)R knockout mice (P2X(7)R(-/-)) are unable to kill T. gondii as effectively as macrophages from wild-type mice. We show that P2X(7)R-mediated T. gondii killing occurs in parallel with host cell apoptosis and is independent of NO production.
PMID: 20488797 [PubMed - as supplied by publisher]
P2X7 Receptor-Mediated Killing of an Intracellular Parasite, Toxoplasma gondii, by Human and Murine Macrophages
Lees MP, Fuller SJ, McLeod R, Boulter NR, Miller CM, Zakrzewski AM, Mui EJ, Witola WH, Coyne JJ, Hargrave AC, Jamieson SE, Blackwell JM, Wiley JS, Smith NC.
Institute for the Biotechnology of Infectious Diseases, University of Technology, Sydney, Broadway;
Abstract
The P2X(7)R is highly expressed on the macrophage cell surface, and activation of infected cells by extracellular ATP has been shown to kill intracellular bacteria and parasites. Furthermore, single nucleotide polymorphisms that decrease receptor function reduce the ability of human macrophages to kill Mycobacterium tuberculosis and are associated with extrapulmonary tuberculosis. In this study, we show that macrophages from people with the 1513C (rs3751143, NM_002562.4:c.1487A>C) loss-of-function P2X(7)R single nucleotide polymorphism are less effective in killing intracellular Toxoplasma gondii after exposure to ATP compared with macrophages from people with the 1513A wild-type allele. Supporting a P2X(7)R-specific effect on T. gondii, macrophages from P2X(7)R knockout mice (P2X(7)R(-/-)) are unable to kill T. gondii as effectively as macrophages from wild-type mice. We show that P2X(7)R-mediated T. gondii killing occurs in parallel with host cell apoptosis and is independent of NO production.
PMID: 20488797 [PubMed - as supplied by publisher]
Saturday, May 22, 2010
Calcium-dependent protein kinase 1 is an essential regulator of exocytosis in Toxoplasma
Nature. 2010 May 20;465(7296):359-62.
Calcium-dependent protein kinase 1 is an essential regulator of exocytosis in Toxoplasma
Lourido S, Shuman J, Zhang C, Shokat KM, Hui R, Sibley LD.
Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Avenue, St Louis, Missouri 63110, USA.
Abstract
Calcium-regulated exocytosis is a ubiquitous process in eukaryotes, whereby secretory vesicles fuse with the plasma membrane and release their contents in response to an intracellular calcium surge. This process regulates various cellular functions such as plasma membrane repair in plants and animals, the discharge of defensive spikes in Paramecium, and the secretion of insulin from pancreatic cells, immune modulators from lymphocytes, and chemical transmitters from neurons. In animal cells, serine/threonine kinases including cAMP-dependent protein kinase, protein kinase C and calmodulin kinases have been implicated in calcium-signal transduction leading to regulated secretion. Although plants and protozoa also regulate secretion by means of intracellular calcium, the method by which these signals are relayed has not been explained. Here we show that the Toxoplasma gondii calcium-dependent protein kinase 1 (TgCDPK1) is an essential regulator of calcium-dependent exocytosis in this opportunistic human pathogen. Conditional suppression of TgCDPK1 revealed that it controls calcium-dependent secretion of specialized organelles called micronemes, resulting in a block of essential phenotypes including parasite motility, host-cell invasion, and egress. These phenotypes were recapitulated by using a chemical biology approach in which pyrazolopyrimidine-derived compounds specifically inhibited TgCDPK1 and disrupted the parasite's life cycle at stages dependent on microneme secretion. Inhibition was specific to TgCDPK1, because expression of a resistant mutant kinase reversed sensitivity to the inhibitor. TgCDPK1 is conserved among apicomplexans and belongs to a family of kinases shared with plants and ciliates, suggesting that related CDPKs may have a function in calcium-regulated secretion in other organisms. Because this kinase family is absent from mammalian hosts, it represents a validated target that may be exploitable for chemotherapy against T. gondii and related apicomplexans.
PMID: 20485436 [PubMed - in process]
Calcium-dependent protein kinase 1 is an essential regulator of exocytosis in Toxoplasma
Lourido S, Shuman J, Zhang C, Shokat KM, Hui R, Sibley LD.
Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Avenue, St Louis, Missouri 63110, USA.
Abstract
Calcium-regulated exocytosis is a ubiquitous process in eukaryotes, whereby secretory vesicles fuse with the plasma membrane and release their contents in response to an intracellular calcium surge. This process regulates various cellular functions such as plasma membrane repair in plants and animals, the discharge of defensive spikes in Paramecium, and the secretion of insulin from pancreatic cells, immune modulators from lymphocytes, and chemical transmitters from neurons. In animal cells, serine/threonine kinases including cAMP-dependent protein kinase, protein kinase C and calmodulin kinases have been implicated in calcium-signal transduction leading to regulated secretion. Although plants and protozoa also regulate secretion by means of intracellular calcium, the method by which these signals are relayed has not been explained. Here we show that the Toxoplasma gondii calcium-dependent protein kinase 1 (TgCDPK1) is an essential regulator of calcium-dependent exocytosis in this opportunistic human pathogen. Conditional suppression of TgCDPK1 revealed that it controls calcium-dependent secretion of specialized organelles called micronemes, resulting in a block of essential phenotypes including parasite motility, host-cell invasion, and egress. These phenotypes were recapitulated by using a chemical biology approach in which pyrazolopyrimidine-derived compounds specifically inhibited TgCDPK1 and disrupted the parasite's life cycle at stages dependent on microneme secretion. Inhibition was specific to TgCDPK1, because expression of a resistant mutant kinase reversed sensitivity to the inhibitor. TgCDPK1 is conserved among apicomplexans and belongs to a family of kinases shared with plants and ciliates, suggesting that related CDPKs may have a function in calcium-regulated secretion in other organisms. Because this kinase family is absent from mammalian hosts, it represents a validated target that may be exploitable for chemotherapy against T. gondii and related apicomplexans.
PMID: 20485436 [PubMed - in process]
Glycolipids are potential targets for protozoan parasite diseases
Trends Parasitol. 2010 May 17. [Epub ahead of print]
Glycolipids are potential targets for protozoan parasite diseases
Debierre-Grockiego F.
UMR Université-INRA 0483, UFR Sciences Pharmaceutiques, Immunologie Parasitaire, Vaccinologie et Biothérapies anti-infectieuses, 31 Avenue Monge, F-37200 Tours, France.
Abstract
Induction of sterilizing immunity by vaccination is extremely difficult because of the evasion mechanisms developed by parasites, and identification of new targets for therapy is therefore important. Glycosylphosphatidylinositols (GPIs) of parasites are glycolipids that participate in pathogenicity of parasitic diseases. Studies of Plasmodium falciparum and Trypanosoma brucei indicate that GPIs are good candidates for developing vaccines against malaria and sleeping sickness, respectively. By contrast, fatty acids isolated from P. falciparum and Toxoplasma gondii can inhibit the production of inflammatory cytokines induced by the GPIs in macrophages. GPIs are considered to be toxins that, if present in large amounts, induce irreversible damages to the host, and treatment with fatty acids could reduce this effect. Copyright © 2010 Elsevier Ltd. All rights reserved.
PMID: 20483663 [PubMed - as supplied by publisher]
Glycolipids are potential targets for protozoan parasite diseases
Debierre-Grockiego F.
UMR Université-INRA 0483, UFR Sciences Pharmaceutiques, Immunologie Parasitaire, Vaccinologie et Biothérapies anti-infectieuses, 31 Avenue Monge, F-37200 Tours, France.
Abstract
Induction of sterilizing immunity by vaccination is extremely difficult because of the evasion mechanisms developed by parasites, and identification of new targets for therapy is therefore important. Glycosylphosphatidylinositols (GPIs) of parasites are glycolipids that participate in pathogenicity of parasitic diseases. Studies of Plasmodium falciparum and Trypanosoma brucei indicate that GPIs are good candidates for developing vaccines against malaria and sleeping sickness, respectively. By contrast, fatty acids isolated from P. falciparum and Toxoplasma gondii can inhibit the production of inflammatory cytokines induced by the GPIs in macrophages. GPIs are considered to be toxins that, if present in large amounts, induce irreversible damages to the host, and treatment with fatty acids could reduce this effect. Copyright © 2010 Elsevier Ltd. All rights reserved.
PMID: 20483663 [PubMed - as supplied by publisher]
Wednesday, May 19, 2010
A decade of epigenetic research in Toxoplasma gondii
Mol Biochem Parasitol. 2010 May 11. [Epub ahead of print]
A decade of epigenetic research in Toxoplasma gondii
Dixon SE, Stilger KL, Elias EV, Naguleswaran A, Sullivan WJ Jr.
Department of Pharmacology & Toxicology, Indiana University School of Medicine, Indianapolis, Indiana, 46202.
Abstract
In the past ten years, the field of parasitology has witnessed an explosion of studies investigating gene regulation. In this review, we will describe recent advances largely stemming from the study of Toxoplasma gondii, a significant opportunistic pathogen and useful model for other apicomplexan protozoa. Surprising findings have emerged, including the discovery of a wealth of epigenetic machinery in these primitive eukaryotes, unusual histone variants, and a battery of plant-like transcription factors. We will elaborate on how these unusual features impact parasite physiology and potential therapeutics as we summarize some of the key discoveries from the last decade. We will close by proposing a few questions to address in the next ten years. Copyright © 2010. Published by Elsevier B.V.
PMID: 20470832 [PubMed - as supplied by publisher]
A decade of epigenetic research in Toxoplasma gondii
Dixon SE, Stilger KL, Elias EV, Naguleswaran A, Sullivan WJ Jr.
Department of Pharmacology & Toxicology, Indiana University School of Medicine, Indianapolis, Indiana, 46202.
Abstract
In the past ten years, the field of parasitology has witnessed an explosion of studies investigating gene regulation. In this review, we will describe recent advances largely stemming from the study of Toxoplasma gondii, a significant opportunistic pathogen and useful model for other apicomplexan protozoa. Surprising findings have emerged, including the discovery of a wealth of epigenetic machinery in these primitive eukaryotes, unusual histone variants, and a battery of plant-like transcription factors. We will elaborate on how these unusual features impact parasite physiology and potential therapeutics as we summarize some of the key discoveries from the last decade. We will close by proposing a few questions to address in the next ten years. Copyright © 2010. Published by Elsevier B.V.
PMID: 20470832 [PubMed - as supplied by publisher]
In vitro differential phenotypic characteristics among type II Toxoplasma gondii strains from congenital toxoplasmosis in humans
J Parasitol. 2010 Feb 26:1. [Epub ahead of print]
In vitro differential phenotypic characteristics among type II Toxoplasma gondii strains from congenital toxoplasmosis in humans
Brenier-Pinchart MP, Bertini RL, Maubon D, Pelloux H.
Abstract
In France, more than 80% of human congenital toxoplasmosis is attributable to Toxoplasma strains belonging to type II lineage, while three main lineages have been described in Europe. Cell invasion, parasite replication and cyst formation of type II strains isolated from congenital infections were compared in a human cell model. The phenotype patterns of the four type II strains studied differed from each other: the two strains responsible for asymptomatic infection formed significantly fewer cysts than the two strains isolated from symptomatic toxoplasmosis. The capacity to form cysts was different among the type II strains studied and may be related to the clinical form.
PMID: 20476804 [PubMed - as supplied by publisher]
In vitro differential phenotypic characteristics among type II Toxoplasma gondii strains from congenital toxoplasmosis in humans
Brenier-Pinchart MP, Bertini RL, Maubon D, Pelloux H.
Abstract
In France, more than 80% of human congenital toxoplasmosis is attributable to Toxoplasma strains belonging to type II lineage, while three main lineages have been described in Europe. Cell invasion, parasite replication and cyst formation of type II strains isolated from congenital infections were compared in a human cell model. The phenotype patterns of the four type II strains studied differed from each other: the two strains responsible for asymptomatic infection formed significantly fewer cysts than the two strains isolated from symptomatic toxoplasmosis. The capacity to form cysts was different among the type II strains studied and may be related to the clinical form.
PMID: 20476804 [PubMed - as supplied by publisher]
Thursday, May 13, 2010
Metabolic pathways in the apicoplast of apicomplexa
Int Rev Cell Mol Biol. 2010;281:161-228.
Metabolic pathways in the apicoplast of apicomplexa
Seeber F, Soldati-Favre D.
Robert-Koch-Institut, Berlin, Germany.
Abstract
Intracellular parasites of the phylum Apicomplexa harbor a plastid-like organelle called apicoplast that is the most reduced organelle of this type known. Due to the medical importance of some members of Apicomplexa, a number of fully sequenced genomes are available that have allowed to assemble metabolic pathways also from the apicoplast and have revealed initial clues to its essential nature for parasite survival in the host. We provide a compilation of Internet resources useful to access, reconstruct, verify, or annotate metabolic pathways. Then we show detailed and updated metabolic maps and discuss the three major biosynthetic pathways leading to the generation of isoprenoids, fatty acids, and heme, and compare these routes in the different species. Moreover, several auxiliary pathways, like iron-sulfur cluster assembly, are covered and put into context with the major metabolic routes. Finally, we highlight some aspects that emerged from recent publications and were not discussed previously with regard to Apicomplexa. Copyright (c) 2010 Elsevier Inc. All rights reserved.
PMID: 20460186 [PubMed - in process]
Metabolic pathways in the apicoplast of apicomplexa
Seeber F, Soldati-Favre D.
Robert-Koch-Institut, Berlin, Germany.
Abstract
Intracellular parasites of the phylum Apicomplexa harbor a plastid-like organelle called apicoplast that is the most reduced organelle of this type known. Due to the medical importance of some members of Apicomplexa, a number of fully sequenced genomes are available that have allowed to assemble metabolic pathways also from the apicoplast and have revealed initial clues to its essential nature for parasite survival in the host. We provide a compilation of Internet resources useful to access, reconstruct, verify, or annotate metabolic pathways. Then we show detailed and updated metabolic maps and discuss the three major biosynthetic pathways leading to the generation of isoprenoids, fatty acids, and heme, and compare these routes in the different species. Moreover, several auxiliary pathways, like iron-sulfur cluster assembly, are covered and put into context with the major metabolic routes. Finally, we highlight some aspects that emerged from recent publications and were not discussed previously with regard to Apicomplexa. Copyright (c) 2010 Elsevier Inc. All rights reserved.
PMID: 20460186 [PubMed - in process]
Structure-activity relationships of carbocyclic 6-benzylthioinosine analogues as subversive substrates of Toxoplasma gondii adenosine kinase
Bioorg Med Chem. 2010 May 15;18(10):3403-12. Epub 2010 Apr 8.
Structure-activity relationships of carbocyclic 6-benzylthioinosine analogues as subversive substrates of Toxoplasma gondii adenosine kinase
Kim YA, Rawal RK, Yoo J, Sharon A, Jha AK, Chu CK, Rais RH, Al Safarjalani ON, Naguib FN, El Kouni MH.
Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, GA 30602, USA.
Abstract
Carbocyclic 6-benzylthioinosine analogues were synthesized and evaluated for their binding affinity against Toxoplasma gondii adenosine kinase [EC.2.7.1.20]. Various substituents on the aromatic ring of the 6-benzylthio group resulted in increased binding affinity to the enzyme as compared to the unsubstituted compound. Carbocyclic 6-(p-methylbenzylthio)inosine 9n exhibited the most potent binding affinity. Docking simulations were performed to position compound 9n into the T. gondii adenosine kinase active site to determine the probable binding mode. Experimental investigations and theoretical calculations further support that an oxygen atom of the sugar is not critical for the ligand-binding. In agreement with its binding affinity, carbocyclic 6-(p-methylbenzylthio)inosine 9n demonstrated significant anti-toxoplasma activity (IC(50)=11.9microM) in cell culture without any apparent host-toxicity. Copyright 2010 Elsevier Ltd. All rights reserved.
PMID: 20456959 [PubMed - in process]
Structure-activity relationships of carbocyclic 6-benzylthioinosine analogues as subversive substrates of Toxoplasma gondii adenosine kinase
Kim YA, Rawal RK, Yoo J, Sharon A, Jha AK, Chu CK, Rais RH, Al Safarjalani ON, Naguib FN, El Kouni MH.
Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, GA 30602, USA.
Abstract
Carbocyclic 6-benzylthioinosine analogues were synthesized and evaluated for their binding affinity against Toxoplasma gondii adenosine kinase [EC.2.7.1.20]. Various substituents on the aromatic ring of the 6-benzylthio group resulted in increased binding affinity to the enzyme as compared to the unsubstituted compound. Carbocyclic 6-(p-methylbenzylthio)inosine 9n exhibited the most potent binding affinity. Docking simulations were performed to position compound 9n into the T. gondii adenosine kinase active site to determine the probable binding mode. Experimental investigations and theoretical calculations further support that an oxygen atom of the sugar is not critical for the ligand-binding. In agreement with its binding affinity, carbocyclic 6-(p-methylbenzylthio)inosine 9n demonstrated significant anti-toxoplasma activity (IC(50)=11.9microM) in cell culture without any apparent host-toxicity. Copyright 2010 Elsevier Ltd. All rights reserved.
PMID: 20456959 [PubMed - in process]
Friday, May 07, 2010
Congenital toxoplasmosis: candidate host immune genes relevant for vertical transmission and pathogenesis
Genes Immun. 2010 May 6. [Epub ahead of print]
Congenital toxoplasmosis: candidate host immune genes relevant for vertical transmission and pathogenesis
Ortiz-Alegría LB, Caballero-Ortega H, Cañedo-Solares I, Rico-Torres CP, Sahagún-Ruiz A, Medina-Escutia ME, Correa D.
Laboratorio de Inmunología Experimental, Subdirección de Medicina Experimental, Instituto Nacional de Pediatría, SSA, México DF, Mexico.
Abstract
Toxoplasma gondii infects a variety of vertebrate hosts, including humans. Transplacental passage of the parasite leads to congenital toxoplasmosis. A primary infection during the first weeks of gestation causes vertical transmission at low rate, although it causes major damage to the embryo. Transmission frequency increases to near 80% by the end of pregnancy, but the proportion of ill newborns is low. For transmission and pathogenesis, the parasite genetics is certainly important. Several host innate and adaptative immune response genes are induced during infection in adults, which control the rapidly replicating tachyzoite. The T helper 1 (Th1) response is protective, although it has to be modulated to avoid inflammatory damage. Paradoxical observations on this response pattern in congenital toxoplasmosis have been reported, as it may be protective or deleterious, inducing sterile abortion or favoring parasite transplacental passage. Regarding pregnancy, an early Th1 microenvironment is important for control of infectious diseases and successful implantation, although it has to be regulated to support trophoblast survival. Polymorphism of genes involved in these parallel phenomena, such as Toll-like receptors (TLRs), adhesins, cytokines, chemokines or their receptors, immunoglobulins or Fc receptors (FcRs), might be important in susceptibility for T. gondii vertical transmission, abortion or fetal pathology. In this study some examples are presented and discussed.Genes and Immunity advance online publication, 6 May 2010; doi:10.1038/gene.2010.21.
PMID: 20445562 [PubMed - as supplied by publisher]
Congenital toxoplasmosis: candidate host immune genes relevant for vertical transmission and pathogenesis
Ortiz-Alegría LB, Caballero-Ortega H, Cañedo-Solares I, Rico-Torres CP, Sahagún-Ruiz A, Medina-Escutia ME, Correa D.
Laboratorio de Inmunología Experimental, Subdirección de Medicina Experimental, Instituto Nacional de Pediatría, SSA, México DF, Mexico.
Abstract
Toxoplasma gondii infects a variety of vertebrate hosts, including humans. Transplacental passage of the parasite leads to congenital toxoplasmosis. A primary infection during the first weeks of gestation causes vertical transmission at low rate, although it causes major damage to the embryo. Transmission frequency increases to near 80% by the end of pregnancy, but the proportion of ill newborns is low. For transmission and pathogenesis, the parasite genetics is certainly important. Several host innate and adaptative immune response genes are induced during infection in adults, which control the rapidly replicating tachyzoite. The T helper 1 (Th1) response is protective, although it has to be modulated to avoid inflammatory damage. Paradoxical observations on this response pattern in congenital toxoplasmosis have been reported, as it may be protective or deleterious, inducing sterile abortion or favoring parasite transplacental passage. Regarding pregnancy, an early Th1 microenvironment is important for control of infectious diseases and successful implantation, although it has to be regulated to support trophoblast survival. Polymorphism of genes involved in these parallel phenomena, such as Toll-like receptors (TLRs), adhesins, cytokines, chemokines or their receptors, immunoglobulins or Fc receptors (FcRs), might be important in susceptibility for T. gondii vertical transmission, abortion or fetal pathology. In this study some examples are presented and discussed.Genes and Immunity advance online publication, 6 May 2010; doi:10.1038/gene.2010.21.
PMID: 20445562 [PubMed - as supplied by publisher]
Cathepsin L Occupies a Vacuolar Compartment and is a Protein Maturase within the Endo/Exocytic System of Toxoplasma gondii
Mol Microbiol. 2010 Apr 23. [Epub ahead of print]
Cathepsin L Occupies a Vacuolar Compartment and is a Protein Maturase within the Endo/Exocytic System of Toxoplasma gondii
Parussini F, Coppens I, Shah PP, Diamond SL, Carruthers VB.
Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, VT 05405 U.S.A..
Abstract
Abstract Regulated exocytosis allows the timely delivery of proteins and other macromolecules precisely when they are needed to fulfill their functions. The intracellular parasite Toxoplasma gondii has one of the most extensive regulated exocytic systems among all unicellular organisms, yet the basis of protein trafficking and proteolytic modification in this system is poorly understood. We demonstrate that a parasite cathepsin protease, TgCPL, occupies a newly recognized vacuolar compartment (VAC) that undergoes dynamic fragmentation during T. gondii replication. We also provide evidence that within the VAC or late endosome this protease mediates the proteolytic maturation of proproteins targeted to micronemes, regulated secretory organelles that deliver adhesive proteins to the parasite surface during cell invasion. Our findings suggest that processing of microneme precursors occurs within intermediate endocytic compartments within the exocytic system, indicating an extensive convergence of the endocytic and exocytic pathways in this human parasite.
PMID: 20444089 [PubMed - as supplied by publisher]
Cathepsin L Occupies a Vacuolar Compartment and is a Protein Maturase within the Endo/Exocytic System of Toxoplasma gondii
Parussini F, Coppens I, Shah PP, Diamond SL, Carruthers VB.
Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, VT 05405 U.S.A..
Abstract
Abstract Regulated exocytosis allows the timely delivery of proteins and other macromolecules precisely when they are needed to fulfill their functions. The intracellular parasite Toxoplasma gondii has one of the most extensive regulated exocytic systems among all unicellular organisms, yet the basis of protein trafficking and proteolytic modification in this system is poorly understood. We demonstrate that a parasite cathepsin protease, TgCPL, occupies a newly recognized vacuolar compartment (VAC) that undergoes dynamic fragmentation during T. gondii replication. We also provide evidence that within the VAC or late endosome this protease mediates the proteolytic maturation of proproteins targeted to micronemes, regulated secretory organelles that deliver adhesive proteins to the parasite surface during cell invasion. Our findings suggest that processing of microneme precursors occurs within intermediate endocytic compartments within the exocytic system, indicating an extensive convergence of the endocytic and exocytic pathways in this human parasite.
PMID: 20444089 [PubMed - as supplied by publisher]
Plastid associated porphobilinogen synthase from toxoplasma gondii: kinetic and structural properties validate therapeutic potential
J Biol Chem. 2010 May 4. [Epub ahead of print]
Plastid associated porphobilinogen synthase from toxoplasma gondii: kinetic and structural properties validate therapeutic potential
Shanmugam D, Wu B, Ramirez U, Jaffe EK, Roos DS.
University of Pennsylvania, United States;
Abstract
Apicomplexan parasites (including Plasmodium spp, and Toxoplasma gondii) employ a four carbon pathway for de novo heme biosynthesis, but this pathway is distinct from the animal/fungal C4 pathway in that it is distributed between three compartments: the mitochondrion, cytosol, and apicoplast -- a plastid acquired by secondary endosymbiosis of an alga. Parasite porphobilinogen synthase (PBGS) resides within the apicoplast, and phylogenetic analysis indicates a plant origin. The PBGS family exhibits a complex use of metal ions (Zn(2+)), Mg(2+)) and oligomeric states (dimers, hexamers, octamers). Recombinant T. gondii PBGS (TgPBGS) was purified as a stable ~320 kDa octamer, and low levels of dimmers -- but no hexamers -- were also observed. The enzyme displays a broad activity peak (pH 7 - 8.5), with a K(m) for aminolevulinic acid of ~150 muM and specific activity ~24 mumol porphobilinogen/mg protein/hr. Like the plant enzyme, TgPBGS responds to Mg(2+) but not Zn(2+), and shows two Mg(2+) affinities, interpreted as tight binding at both the active and allosteric sites. Unlike other Mg(2+)-binding PBGS, however, metal ions are not required for TgPBGS octamer stability. A mutant enzyme lacking the C-terminal 13 amino acids distinguishing parasite PBGS from plant and animal enzymes purified as a dimer, suggesting that the C-terminus is required for octamer stability. Parasite heme biosynthesis is inhibited (and parasites are killed) by succinylacetone, an active site-directed suicide substrate. The distinct phylogenetic, enzymatic and structural features of apicomplexan PBGS offers scope for developing selective inhibitors of the parasite enzyme based on its quarternary structure characteristics.
PMID: 20442414 [PubMed - as supplied by publisher]
Plastid associated porphobilinogen synthase from toxoplasma gondii: kinetic and structural properties validate therapeutic potential
Shanmugam D, Wu B, Ramirez U, Jaffe EK, Roos DS.
University of Pennsylvania, United States;
Abstract
Apicomplexan parasites (including Plasmodium spp, and Toxoplasma gondii) employ a four carbon pathway for de novo heme biosynthesis, but this pathway is distinct from the animal/fungal C4 pathway in that it is distributed between three compartments: the mitochondrion, cytosol, and apicoplast -- a plastid acquired by secondary endosymbiosis of an alga. Parasite porphobilinogen synthase (PBGS) resides within the apicoplast, and phylogenetic analysis indicates a plant origin. The PBGS family exhibits a complex use of metal ions (Zn(2+)), Mg(2+)) and oligomeric states (dimers, hexamers, octamers). Recombinant T. gondii PBGS (TgPBGS) was purified as a stable ~320 kDa octamer, and low levels of dimmers -- but no hexamers -- were also observed. The enzyme displays a broad activity peak (pH 7 - 8.5), with a K(m) for aminolevulinic acid of ~150 muM and specific activity ~24 mumol porphobilinogen/mg protein/hr. Like the plant enzyme, TgPBGS responds to Mg(2+) but not Zn(2+), and shows two Mg(2+) affinities, interpreted as tight binding at both the active and allosteric sites. Unlike other Mg(2+)-binding PBGS, however, metal ions are not required for TgPBGS octamer stability. A mutant enzyme lacking the C-terminal 13 amino acids distinguishing parasite PBGS from plant and animal enzymes purified as a dimer, suggesting that the C-terminus is required for octamer stability. Parasite heme biosynthesis is inhibited (and parasites are killed) by succinylacetone, an active site-directed suicide substrate. The distinct phylogenetic, enzymatic and structural features of apicomplexan PBGS offers scope for developing selective inhibitors of the parasite enzyme based on its quarternary structure characteristics.
PMID: 20442414 [PubMed - as supplied by publisher]
Action of a Pentacyclic Triterpenoid, Maslinic Acid, against Toxoplasma gondii
J Nat Prod. 2010 May 4. [Epub ahead of print]
Action of a Pentacyclic Triterpenoid, Maslinic Acid, against Toxoplasma gondii
De Pablos LM, González G, Rodrigues R, García Granados A, Parra A, Osuna A.
Biochemical and Molecular Parasitology Group, Biotechnology Institute, Campus de Fuentenueva, University of Granada, 18071, Granada, Spain, Laboratoire des Pathogenes Emergents (Fondation Merieux), Tour Cervi 20, Avenue Tony Garnier, 69007 Lyon, France, and Chemistry and Natural Product Biotechnology Group, Biotechnology Institute, Campus de Fuentenueva, University of Granada, 18071, Granada, Spain.
Abstract
The action of maslinic acid (2alpha,3beta-dihydroxyolean-12-en-28-oic acid) (1), a pentacyclic derivative present in the pressed fruits of the olive (Olea europaea), has been studied against the tachyzoites of Toxoplasma gondii. The capability of tachyzoites to infect Vero cells treated with 1 was affected. The LD(50) values were 58.2 muM for the isolated tachyzoites and 236 muM for the noninfected Vero cells. Zymograms of the T. gondii proteases incubated with 1 showed a dosage-dependent inhibition of some of the proteases. The parasites treated with 1 showed gliding motility and ultrastructural alterations. The present findings suggest that protease activity of the parasite required for cell invasion is the action target for maslinic acid (1).
PMID: 20441162 [PubMed - as supplied by publisher]
Action of a Pentacyclic Triterpenoid, Maslinic Acid, against Toxoplasma gondii
De Pablos LM, González G, Rodrigues R, García Granados A, Parra A, Osuna A.
Biochemical and Molecular Parasitology Group, Biotechnology Institute, Campus de Fuentenueva, University of Granada, 18071, Granada, Spain, Laboratoire des Pathogenes Emergents (Fondation Merieux), Tour Cervi 20, Avenue Tony Garnier, 69007 Lyon, France, and Chemistry and Natural Product Biotechnology Group, Biotechnology Institute, Campus de Fuentenueva, University of Granada, 18071, Granada, Spain.
Abstract
The action of maslinic acid (2alpha,3beta-dihydroxyolean-12-en-28-oic acid) (1), a pentacyclic derivative present in the pressed fruits of the olive (Olea europaea), has been studied against the tachyzoites of Toxoplasma gondii. The capability of tachyzoites to infect Vero cells treated with 1 was affected. The LD(50) values were 58.2 muM for the isolated tachyzoites and 236 muM for the noninfected Vero cells. Zymograms of the T. gondii proteases incubated with 1 showed a dosage-dependent inhibition of some of the proteases. The parasites treated with 1 showed gliding motility and ultrastructural alterations. The present findings suggest that protease activity of the parasite required for cell invasion is the action target for maslinic acid (1).
PMID: 20441162 [PubMed - as supplied by publisher]
Wednesday, May 05, 2010
Evaluation of the antigenic value of recombinant Toxoplasma gondii HSP20 to detect specific immunoglobulin G antibodies in Toxoplasma infected humans
Exp Parasitol. 2010 Apr 27. [Epub ahead of print]
Evaluation of the antigenic value of recombinant Toxoplasma gondii HSP20 to detect specific immunoglobulin G antibodies in Toxoplasma infected humans
Cóceres VM, Becher ML, De Napoli MG, Corvi MM, Clemente M, Angel SO.
Laboratorio de Parasitología Molecular, Instituto de Investigaciones Biotecnológicas-Instituto Tecnologico Chascomús (IIB-INTECH), UNSAM/CONICET, Chascomús (7130), Argentina.
Abstract
Recombinant Toxoplasma gondii small heat shock protein HSP20, surface antigen SAG1 and dense granule GRA7 were analyzed by IgG-ELISA with serum samples of Toxoplasma infected humans grouped as I (IgG+, IgM+), II (IgG+, IgM-) and III (IgG-, IgM-). rHSP20 reacted against 80% and 62.5% of serum samples from groups I and II, respectively. rSAG1 was recognized by 85% of the samples from group I and 70.8% from group II, whereas rGRA7 was recognized by 85% and 66.6% of the serum samples from groups I and II respectively. When a combination of two or three recombinant antigens was used, the sensitivity values improved to 85-95% for group I and 87.5-91.7% for group II. All combinations tested produced similar reactivity profiles. None of the recombinant proteins reacted against group III serum samples. In conclusion, we demonstrated that T. gondii HSP20 elicits an important B-cell response during human infection, and could be suitable for the development of serodiagnosis tools. Copyright © 2010. Published by Elsevier Inc.
PMID: 20433835 [PubMed - as supplied by publisher]
Evaluation of the antigenic value of recombinant Toxoplasma gondii HSP20 to detect specific immunoglobulin G antibodies in Toxoplasma infected humans
Cóceres VM, Becher ML, De Napoli MG, Corvi MM, Clemente M, Angel SO.
Laboratorio de Parasitología Molecular, Instituto de Investigaciones Biotecnológicas-Instituto Tecnologico Chascomús (IIB-INTECH), UNSAM/CONICET, Chascomús (7130), Argentina.
Abstract
Recombinant Toxoplasma gondii small heat shock protein HSP20, surface antigen SAG1 and dense granule GRA7 were analyzed by IgG-ELISA with serum samples of Toxoplasma infected humans grouped as I (IgG+, IgM+), II (IgG+, IgM-) and III (IgG-, IgM-). rHSP20 reacted against 80% and 62.5% of serum samples from groups I and II, respectively. rSAG1 was recognized by 85% of the samples from group I and 70.8% from group II, whereas rGRA7 was recognized by 85% and 66.6% of the serum samples from groups I and II respectively. When a combination of two or three recombinant antigens was used, the sensitivity values improved to 85-95% for group I and 87.5-91.7% for group II. All combinations tested produced similar reactivity profiles. None of the recombinant proteins reacted against group III serum samples. In conclusion, we demonstrated that T. gondii HSP20 elicits an important B-cell response during human infection, and could be suitable for the development of serodiagnosis tools. Copyright © 2010. Published by Elsevier Inc.
PMID: 20433835 [PubMed - as supplied by publisher]
Toxoplasma gondii deoxyribose phosphate aldolase-like protein (TgDPA) interacts with actin depolymerizing factor (TgADF)
Mol Biochem Parasitol. 2010 Apr 27. [Epub ahead of print]
Toxoplasma gondii deoxyribose phosphate aldolase-like protein (TgDPA) interacts with actin depolymerizing factor (TgADF) to enhance the actin filament dynamics in the bradyzoite stage
Ueno A, Dautu G, Saiki E, Haga K, Igarashi M.
National Research Center for Protozoan Diseases (NRCPD), Obihiro University of Agriculture and Veterinary Medicine, 2-13 Inada-cho, Obihiro, Hokkaido, 080-8555, Japan.
Abstract
The Toxoplasma gondii deoxyribose phosphate aldolase-like (TgDPA) gene is expressed predominantly in bradyzoites. This finding allowed us to infer that TgDPA is important in the tachyzoite-to-bradyzoite development or maintenance of cyst structure although the function of this gene is still unknown. We conducted yeast two-hybrid screening to identify proteins interacting with TgDPA, and the actin depolymerizing factor (TgADF) gene was obtained. Co-immunoprecipitation and a GST pull-down assay demonstrated that TgDPA interacts with TgADF. To reveal the significance of the protein-protein interaction between TgDPA and TgADF, actin polymerization and disassembly kinetics were examined. Addition of GST-TgDPA to TgADF lowered the extent of actin polymerization and enhanced the filamentous actin disassembly. These results demonstrated that this is the novel protein-protein interaction in T. gondii, and that TgDPA can enhance the activity of TgADF. This phenomenon might play an important role in T. gondii bradyzoites by affecting the actin turnover. Copyright © 2010. Published by Elsevier B.V.
PMID: 20433874 [PubMed - as supplied by publisher]
Toxoplasma gondii deoxyribose phosphate aldolase-like protein (TgDPA) interacts with actin depolymerizing factor (TgADF) to enhance the actin filament dynamics in the bradyzoite stage
Ueno A, Dautu G, Saiki E, Haga K, Igarashi M.
National Research Center for Protozoan Diseases (NRCPD), Obihiro University of Agriculture and Veterinary Medicine, 2-13 Inada-cho, Obihiro, Hokkaido, 080-8555, Japan.
Abstract
The Toxoplasma gondii deoxyribose phosphate aldolase-like (TgDPA) gene is expressed predominantly in bradyzoites. This finding allowed us to infer that TgDPA is important in the tachyzoite-to-bradyzoite development or maintenance of cyst structure although the function of this gene is still unknown. We conducted yeast two-hybrid screening to identify proteins interacting with TgDPA, and the actin depolymerizing factor (TgADF) gene was obtained. Co-immunoprecipitation and a GST pull-down assay demonstrated that TgDPA interacts with TgADF. To reveal the significance of the protein-protein interaction between TgDPA and TgADF, actin polymerization and disassembly kinetics were examined. Addition of GST-TgDPA to TgADF lowered the extent of actin polymerization and enhanced the filamentous actin disassembly. These results demonstrated that this is the novel protein-protein interaction in T. gondii, and that TgDPA can enhance the activity of TgADF. This phenomenon might play an important role in T. gondii bradyzoites by affecting the actin turnover. Copyright © 2010. Published by Elsevier B.V.
PMID: 20433874 [PubMed - as supplied by publisher]
Host Cell Invasion by Toxoplasma gondii is Temporally Regulated by the Host Microtubule Cytoskeleton
Eukaryot Cell. 2010 Apr 30. [Epub ahead of print]
Host Cell Invasion by Toxoplasma gondii is Temporally Regulated by the Host Microtubule Cytoskeleton
Sweeney KR, Morrissette NS, Lachapelle S, Blader IJ.
Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104; Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697.
Abstract
Toxoplasma gondii is an obligate intracellular protozoan parasite that invades and replicates within most nucleated cells of warm-blooded animals. The basis for this wide host cell tropism is unknown but could be because parasites invade host cells using distinct pathways and/or repertoires of host factors. Using synchronized parasite invasion assays, we find that host microtubule disruption significantly reduces parasite invasion into host cells early after stimulating parasite invasion but not at later time points. Host microtubules are specifically associated with the moving junction, which is the site of contact between the host cell and the invading parasite. Host microtubules are specifically associated with the moving junction of those parasites invading early after stimulating invasion but not with those invading later. Disruption of host microtubules has no effect on parasite contact, attachment, motility, or rate of penetration. Rather host microtubules hasten the time before parasites commence invasion. This effect on parasite invasion is distinct from the role that host microtubules play in bacterial and viral infections where they function to traffic the pathogen or pathogen-derived material from the host cell's periphery to its interior. These data indicate that the host microtubule cytoskeleton is a structure used by Toxoplasma to rapidly infect its host cell and highlight a novel function for host microtubules in microbial pathogenesis.
PMID: 20435700 [PubMed - as supplied by publisher]
Host Cell Invasion by Toxoplasma gondii is Temporally Regulated by the Host Microtubule Cytoskeleton
Sweeney KR, Morrissette NS, Lachapelle S, Blader IJ.
Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104; Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697.
Abstract
Toxoplasma gondii is an obligate intracellular protozoan parasite that invades and replicates within most nucleated cells of warm-blooded animals. The basis for this wide host cell tropism is unknown but could be because parasites invade host cells using distinct pathways and/or repertoires of host factors. Using synchronized parasite invasion assays, we find that host microtubule disruption significantly reduces parasite invasion into host cells early after stimulating parasite invasion but not at later time points. Host microtubules are specifically associated with the moving junction, which is the site of contact between the host cell and the invading parasite. Host microtubules are specifically associated with the moving junction of those parasites invading early after stimulating invasion but not with those invading later. Disruption of host microtubules has no effect on parasite contact, attachment, motility, or rate of penetration. Rather host microtubules hasten the time before parasites commence invasion. This effect on parasite invasion is distinct from the role that host microtubules play in bacterial and viral infections where they function to traffic the pathogen or pathogen-derived material from the host cell's periphery to its interior. These data indicate that the host microtubule cytoskeleton is a structure used by Toxoplasma to rapidly infect its host cell and highlight a novel function for host microtubules in microbial pathogenesis.
PMID: 20435700 [PubMed - as supplied by publisher]
Toxoplasma gondii: the severity of toxoplasmic encephalitis in C57BL/6 mice is associated with increased ALCAM and VCAM-1 expression
Exp Parasitol. 2010 Apr 28. [Epub ahead of print]
Toxoplasma gondii: the severity of toxoplasmic encephalitis in C57BL/6 mice is associated with increased ALCAM and VCAM-1 expression in the central nervous system and higher blood-brain barrier permeability
Silva NM, Manzan RM, Carneiro WP, Milanezi CM, Silva JS, Ferro EA, Mineo JR.
Laboratory of Immunopathology and Institute of Biomedical Sciences, Federal University of Uberlândia, Uberlândia, MG, Brazil 38400-902.
Abstract
In order to investigate the differential ALCAM, ICAM-1 and VCAM-1 adhesion molecules mRNA expression and the blood-brain barrier (BBB) permeability in C57BL/6 and BALB/c mice in Toxoplasma gondii infection, animals were infected with ME-49 strain. It was observed higher ALCAM on day 9 and VCAM-1 expression on days 9 and 14 of infection in the central nervous system (CNS) of C57BL/6 compared to BALB/c mice. The expression of ICAM-1 was high and similar in the CNS of both lineages of infected mice. In addition, C57BL/6 presented higher BBB permeability and higher IFN-gamma and iNOS expression in the CNS compared to BALB/c mice. The CNS of C57BL/6 mice presented elevated tissue pathology and parasitism. In conclusion, our data suggest that the higher adhesion molecules expression and higher BBB permeability contributed to the major inflammatory cell infiltration into the CNS of C57BL/6 mice that was not efficient to control the parasite. Copyright © 2010. Published by Elsevier Inc.
PMID: 20434443 [PubMed - as supplied by publisher]
Toxoplasma gondii: the severity of toxoplasmic encephalitis in C57BL/6 mice is associated with increased ALCAM and VCAM-1 expression in the central nervous system and higher blood-brain barrier permeability
Silva NM, Manzan RM, Carneiro WP, Milanezi CM, Silva JS, Ferro EA, Mineo JR.
Laboratory of Immunopathology and Institute of Biomedical Sciences, Federal University of Uberlândia, Uberlândia, MG, Brazil 38400-902.
Abstract
In order to investigate the differential ALCAM, ICAM-1 and VCAM-1 adhesion molecules mRNA expression and the blood-brain barrier (BBB) permeability in C57BL/6 and BALB/c mice in Toxoplasma gondii infection, animals were infected with ME-49 strain. It was observed higher ALCAM on day 9 and VCAM-1 expression on days 9 and 14 of infection in the central nervous system (CNS) of C57BL/6 compared to BALB/c mice. The expression of ICAM-1 was high and similar in the CNS of both lineages of infected mice. In addition, C57BL/6 presented higher BBB permeability and higher IFN-gamma and iNOS expression in the CNS compared to BALB/c mice. The CNS of C57BL/6 mice presented elevated tissue pathology and parasitism. In conclusion, our data suggest that the higher adhesion molecules expression and higher BBB permeability contributed to the major inflammatory cell infiltration into the CNS of C57BL/6 mice that was not efficient to control the parasite. Copyright © 2010. Published by Elsevier Inc.
PMID: 20434443 [PubMed - as supplied by publisher]
Toxoplasma gondii calcium-dependent protein kinase 1 is a target for selective kinase inhibitors
Nat Struct Mol Biol. 2010 May 2. [Epub ahead of print]
Toxoplasma gondii calcium-dependent protein kinase 1 is a target for selective kinase inhibitors
Ojo KK, Larson ET, Keyloun KR, Castaneda LJ, Derocher AE, Inampudi KK, Kim JE, Arakaki TL, Murphy RC, Zhang L, Napuli AJ, Maly DJ, Verlinde CL, Buckner FS, Parsons M, Hol WG, Merritt EA, Van Voorhis WC.
Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, Seattle, Washington, USA.
Abstract
New drugs are needed to treat toxoplasmosis. Toxoplasma gondii calcium-dependent protein kinases (TgCDPKs) are attractive targets because they are absent in mammals. We show that TgCDPK1 is inhibited by low nanomolar levels of bumped kinase inhibitors (BKIs), compounds inactive against mammalian kinases. Cocrystal structures of TgCDPK1 with BKIs confirm that the structural basis for selectivity is due to the unique glycine gatekeeper residue in the ATP-binding site. We show that BKIs interfere with an early step in T. gondii infection of human cells in culture. Furthermore, we show that TgCDPK1 is the in vivo target of BKIs because T. gondii expressing a glycine to methionine gatekeeper mutant enzyme show significantly decreased sensitivity to BKIs. Thus, design of selective TgCDPK1 inhibitors with low host toxicity may be achievable.
PMID: 20436472 [PubMed - as supplied by publisher]
Toxoplasma gondii calcium-dependent protein kinase 1 is a target for selective kinase inhibitors
Ojo KK, Larson ET, Keyloun KR, Castaneda LJ, Derocher AE, Inampudi KK, Kim JE, Arakaki TL, Murphy RC, Zhang L, Napuli AJ, Maly DJ, Verlinde CL, Buckner FS, Parsons M, Hol WG, Merritt EA, Van Voorhis WC.
Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, Seattle, Washington, USA.
Abstract
New drugs are needed to treat toxoplasmosis. Toxoplasma gondii calcium-dependent protein kinases (TgCDPKs) are attractive targets because they are absent in mammals. We show that TgCDPK1 is inhibited by low nanomolar levels of bumped kinase inhibitors (BKIs), compounds inactive against mammalian kinases. Cocrystal structures of TgCDPK1 with BKIs confirm that the structural basis for selectivity is due to the unique glycine gatekeeper residue in the ATP-binding site. We show that BKIs interfere with an early step in T. gondii infection of human cells in culture. Furthermore, we show that TgCDPK1 is the in vivo target of BKIs because T. gondii expressing a glycine to methionine gatekeeper mutant enzyme show significantly decreased sensitivity to BKIs. Thus, design of selective TgCDPK1 inhibitors with low host toxicity may be achievable.
PMID: 20436472 [PubMed - as supplied by publisher]
Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium
Nat Struct Mol Biol. 2010 May 2. [Epub ahead of print]
Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium
Wernimont AK, Artz JD, Finerty P Jr, Lin YH, Amani M, Allali-Hassani A, Senisterra G, Vedadi M, Tempel W, Mackenzie F, Chau I, Lourido S, Sibley LD, Hui R.
Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
Abstract
Calcium-dependent protein kinases (CDPKs) have pivotal roles in the calcium-signaling pathway in plants, ciliates and apicomplexan parasites and comprise a calmodulin-dependent kinase (CaMK)-like kinase domain regulated by a calcium-binding domain in the C terminus. To understand this intramolecular mechanism of activation, we solved the structures of the autoinhibited (apo) and activated (calcium-bound) conformations of CDPKs from the apicomplexan parasites Toxoplasma gondii and Cryptosporidium parvum. In the apo form, the C-terminal CDPK activation domain (CAD) resembles a calmodulin protein with an unexpected long helix in the N terminus that inhibits the kinase domain in the same manner as CaMKII. Calcium binding triggers the reorganization of the CAD into a highly intricate fold, leading to its relocation around the base of the kinase domain to a site remote from the substrate binding site. This large conformational change constitutes a distinct mechanism in calcium signal-transduction pathways.
PMID: 20436473 [PubMed - as supplied by publisher]
Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium
Wernimont AK, Artz JD, Finerty P Jr, Lin YH, Amani M, Allali-Hassani A, Senisterra G, Vedadi M, Tempel W, Mackenzie F, Chau I, Lourido S, Sibley LD, Hui R.
Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
Abstract
Calcium-dependent protein kinases (CDPKs) have pivotal roles in the calcium-signaling pathway in plants, ciliates and apicomplexan parasites and comprise a calmodulin-dependent kinase (CaMK)-like kinase domain regulated by a calcium-binding domain in the C terminus. To understand this intramolecular mechanism of activation, we solved the structures of the autoinhibited (apo) and activated (calcium-bound) conformations of CDPKs from the apicomplexan parasites Toxoplasma gondii and Cryptosporidium parvum. In the apo form, the C-terminal CDPK activation domain (CAD) resembles a calmodulin protein with an unexpected long helix in the N terminus that inhibits the kinase domain in the same manner as CaMKII. Calcium binding triggers the reorganization of the CAD into a highly intricate fold, leading to its relocation around the base of the kinase domain to a site remote from the substrate binding site. This large conformational change constitutes a distinct mechanism in calcium signal-transduction pathways.
PMID: 20436473 [PubMed - as supplied by publisher]
Toxoplasmosis in allo-SCT patients: risk factors and outcomes at a transplantation center with a low incidence
Bone Marrow Transplant. 2010 May 3. [Epub ahead of print]
Toxoplasmosis in allo-SCT patients: risk factors and outcomes at a transplantation center with a low incidence
Mulanovich VE, Ahmed SI, Oztürk T, Khokhar FA, Kontoyiannis DP, de Lima M.
Department of Infectious Diseases, Infection Control and Employee Health, MD Anderson Cancer Center, Houston, TX, USA.
Abstract
Toxoplasmosis in allo-SCT patients is rare in the United States but has a mortality of 60-90%. In this retrospective study, we identified patients with definite and probable toxoplasmosis after allo-SCT at our institution from 1994 to 2009 using ICD-9 codes and the pathology database. Of 3626 patients who underwent allogeneic SCT, we identified 8 with definite toxoplasmosis and 1 with probable toxoplasmosis, an incidence of 0.25%. International patients had a significantly higher incidence of toxoplasmosis than did US patients (1.6 versus 0.15% (P=0.002)). Three patients presented with toxoplasmosis <30 days after transplantation and six developed toxoplasmosis within 100 days of starting high-dose corticosteroids. Two patients were diagnosed postmortem. Six of the remaining seven patients died despite >/=2 weeks of therapy. Co-morbidities, particularly infections, were the primary cause of death in one case and a contributing factor in the remaining six cases. On the basis of these results, we conclude that all allo-SCT patients from countries with high Toxoplasma seropositivity and seropositive patients from the United States should undergo serial PCR screening during the first month after transplantation and during corticosteroid use. All patients who test positive should undergo preemptive therapy.Bone Marrow Transplantation advance online publication, 3 May 2010; doi:10.1038/bmt.2010.102.
PMID: 20436521 [PubMed - as supplied by publisher]
Toxoplasmosis in allo-SCT patients: risk factors and outcomes at a transplantation center with a low incidence
Mulanovich VE, Ahmed SI, Oztürk T, Khokhar FA, Kontoyiannis DP, de Lima M.
Department of Infectious Diseases, Infection Control and Employee Health, MD Anderson Cancer Center, Houston, TX, USA.
Abstract
Toxoplasmosis in allo-SCT patients is rare in the United States but has a mortality of 60-90%. In this retrospective study, we identified patients with definite and probable toxoplasmosis after allo-SCT at our institution from 1994 to 2009 using ICD-9 codes and the pathology database. Of 3626 patients who underwent allogeneic SCT, we identified 8 with definite toxoplasmosis and 1 with probable toxoplasmosis, an incidence of 0.25%. International patients had a significantly higher incidence of toxoplasmosis than did US patients (1.6 versus 0.15% (P=0.002)). Three patients presented with toxoplasmosis <30 days after transplantation and six developed toxoplasmosis within 100 days of starting high-dose corticosteroids. Two patients were diagnosed postmortem. Six of the remaining seven patients died despite >/=2 weeks of therapy. Co-morbidities, particularly infections, were the primary cause of death in one case and a contributing factor in the remaining six cases. On the basis of these results, we conclude that all allo-SCT patients from countries with high Toxoplasma seropositivity and seropositive patients from the United States should undergo serial PCR screening during the first month after transplantation and during corticosteroid use. All patients who test positive should undergo preemptive therapy.Bone Marrow Transplantation advance online publication, 3 May 2010; doi:10.1038/bmt.2010.102.
PMID: 20436521 [PubMed - as supplied by publisher]
Does atovaquone prolong the disease-free interval of toxoplasmic retinochoroiditis?
Graefes Arch Clin Exp Ophthalmol. 2010 May 2. [Epub ahead of print]
Does atovaquone prolong the disease-free interval of toxoplasmic retinochoroiditis?
Winterhalter S, Severing K, Stammen J, Maier AK, Godehardt E, Joussen AM.
Department of Ophthalmology, University Hospital Düsseldorf, Düsseldorf, Germany, billewin@gmx.de.
Abstract
BACKGROUND: To evaluate the efficacy of suppressing a recurrence of Toxoplasma retinochoroiditis after treatment with atovaquone. METHODS: Retrospective, nonrandomized, clinical trial. Forty-one immunocompetent patients were treated for Toxoplasma retinochoroiditis with atovaquone between 1999 and 2006. The diagnosis was based on clinical signs alone. Atovaquone was given 750 mg two to three times daily together with oral steroids. Lesion location, time interval until recurrence, visual function, and adverse events were recorded. RESULTS: Forty-two eyes of 41 patients were treated with atovaquone for Toxoplasma retinochoroiditis. Side-effects were usually mild and only one patient stopped therapy with atovaquone because of nausea. Reactivation of retinochoroiditis occurred in 18 patients (44%) during a time interval of 3-70 months. CONCLUSIONS: The therapy of Toxoplasma retinochoroiditis with atovaquone is well tolerated. Our data suggests that therapy with atovaquone has the potential to prolong the time to recurrence of Toxoplasma retinochoroiditis. A prospective randomized comparative long-term clinical trial would be necessary to confirm our data.
PMID: 20437247 [PubMed - as supplied by publisher]
Does atovaquone prolong the disease-free interval of toxoplasmic retinochoroiditis?
Winterhalter S, Severing K, Stammen J, Maier AK, Godehardt E, Joussen AM.
Department of Ophthalmology, University Hospital Düsseldorf, Düsseldorf, Germany, billewin@gmx.de.
Abstract
BACKGROUND: To evaluate the efficacy of suppressing a recurrence of Toxoplasma retinochoroiditis after treatment with atovaquone. METHODS: Retrospective, nonrandomized, clinical trial. Forty-one immunocompetent patients were treated for Toxoplasma retinochoroiditis with atovaquone between 1999 and 2006. The diagnosis was based on clinical signs alone. Atovaquone was given 750 mg two to three times daily together with oral steroids. Lesion location, time interval until recurrence, visual function, and adverse events were recorded. RESULTS: Forty-two eyes of 41 patients were treated with atovaquone for Toxoplasma retinochoroiditis. Side-effects were usually mild and only one patient stopped therapy with atovaquone because of nausea. Reactivation of retinochoroiditis occurred in 18 patients (44%) during a time interval of 3-70 months. CONCLUSIONS: The therapy of Toxoplasma retinochoroiditis with atovaquone is well tolerated. Our data suggests that therapy with atovaquone has the potential to prolong the time to recurrence of Toxoplasma retinochoroiditis. A prospective randomized comparative long-term clinical trial would be necessary to confirm our data.
PMID: 20437247 [PubMed - as supplied by publisher]
Monday, May 03, 2010
Sialic acids: Key determinants for invasion by the Apicomplexa
Int J Parasitol. 2010 Apr 26. [Epub ahead of print]
Sialic acids: Key determinants for invasion by the Apicomplexa
Friedrich N, Matthews S, Soldati-Favre D.
Department of Microbiology and Molecular Medicine, CMU, University of Geneva, 1 Rue Michel-Servet, CH-1211 Geneva 4, Switzerland.
Abstract
Sialic acids are ubiquitously found on the surface of all vertebrate cells at the extremities of glycan chains and widely exploited by viruses and bacteria to enter host cells. Carbohydrate-bearing receptors are equally important for host cell invasion by the obligate intracellular protozoan parasites of the phylum Apicomplexa. Host cell entry is an active process relying crucially on proteins that engage with receptors on the host cell surface and promote adhesion and internalization. Assembly into complexes, proteolytic processing, and oligomerization are important requirements for the functionality of these adhesins. The combination of adhesive proteins with varying stringency in specificity confers some flexibility to the parasite in face of receptor heterogeneity and immune pressure. Sialic acids are now recognized to critically contribute to selective host cell recognition by various species of the phylum. Copyright © 2010. Published by Elsevier Ltd.
PMID: 20430033 [PubMed - as supplied by publisher]
Sialic acids: Key determinants for invasion by the Apicomplexa
Friedrich N, Matthews S, Soldati-Favre D.
Department of Microbiology and Molecular Medicine, CMU, University of Geneva, 1 Rue Michel-Servet, CH-1211 Geneva 4, Switzerland.
Abstract
Sialic acids are ubiquitously found on the surface of all vertebrate cells at the extremities of glycan chains and widely exploited by viruses and bacteria to enter host cells. Carbohydrate-bearing receptors are equally important for host cell invasion by the obligate intracellular protozoan parasites of the phylum Apicomplexa. Host cell entry is an active process relying crucially on proteins that engage with receptors on the host cell surface and promote adhesion and internalization. Assembly into complexes, proteolytic processing, and oligomerization are important requirements for the functionality of these adhesins. The combination of adhesive proteins with varying stringency in specificity confers some flexibility to the parasite in face of receptor heterogeneity and immune pressure. Sialic acids are now recognized to critically contribute to selective host cell recognition by various species of the phylum. Copyright © 2010. Published by Elsevier Ltd.
PMID: 20430033 [PubMed - as supplied by publisher]
Comparative Analysis of Stage Specific Gene Regulation of Apicomplexan parasites: Plasmodium falciparum and Toxoplasma
Infect Disord Drug Targets. 2010 Apr 29. [Epub ahead of print]
Comparative Analysis of Stage Specific Gene Regulation of Apicomplexan parasites: Plasmodium falciparum and Toxoplasma gondii
Gopalakrishnan AM, López-Estraño C.
Department of Biology, Life Sciences Bldg. Room 409B. The University of Memphis. 3774 Walker Ave. Memphis, Tennessee 38152. USA. cestrano@memphis.edu.
Abstract
Apicomplexans comprise some of the most life threatening parasites infecting human and livestock and includes Plasmodium and Toxoplasma, the causative agents of malaria and toxoplasmosis respectively, in humans as well as Neospora caninum (abortion in livestock, neosporosis in dogs), Cryptosporidium (Diarrheal cryptosporidiosis and opportunistic infections in AIDS patients) and Eimeria (poultry coccidiosis). These parasites are characterized by a complex life cycle usually alternating between sexual and asexual cycles in different hosts. The need to adapt to different host environments demands a tight regulation of gene expression during parasite development. Therefore, the understanding of parasite biology will facilitate the control of the infection and the disease. In this review we emphasize the progress made so far in gene regulation in two medically important parasites, namely Plasmodium falciparum and Toxoplasma gondii, as well as other less known apicomplexan. The genome of both Plasmodium and Toxoplasma has been sequenced and since then there has been a significant progress in understanding the molecular mechanisms that control stage specific gene expression in the two parasites. In addition, the information gained in each of the parasite can be used in studying mechanisms that are still elusive in the other apicomplexans that are not readily available. Additionally, they can serve as model system for other disease causing Apicomplexan parasites.
PMID: 20429866 [PubMed - as supplied by publisher]
Comparative Analysis of Stage Specific Gene Regulation of Apicomplexan parasites: Plasmodium falciparum and Toxoplasma gondii
Gopalakrishnan AM, López-Estraño C.
Department of Biology, Life Sciences Bldg. Room 409B. The University of Memphis. 3774 Walker Ave. Memphis, Tennessee 38152. USA. cestrano@memphis.edu.
Abstract
Apicomplexans comprise some of the most life threatening parasites infecting human and livestock and includes Plasmodium and Toxoplasma, the causative agents of malaria and toxoplasmosis respectively, in humans as well as Neospora caninum (abortion in livestock, neosporosis in dogs), Cryptosporidium (Diarrheal cryptosporidiosis and opportunistic infections in AIDS patients) and Eimeria (poultry coccidiosis). These parasites are characterized by a complex life cycle usually alternating between sexual and asexual cycles in different hosts. The need to adapt to different host environments demands a tight regulation of gene expression during parasite development. Therefore, the understanding of parasite biology will facilitate the control of the infection and the disease. In this review we emphasize the progress made so far in gene regulation in two medically important parasites, namely Plasmodium falciparum and Toxoplasma gondii, as well as other less known apicomplexan. The genome of both Plasmodium and Toxoplasma has been sequenced and since then there has been a significant progress in understanding the molecular mechanisms that control stage specific gene expression in the two parasites. In addition, the information gained in each of the parasite can be used in studying mechanisms that are still elusive in the other apicomplexans that are not readily available. Additionally, they can serve as model system for other disease causing Apicomplexan parasites.
PMID: 20429866 [PubMed - as supplied by publisher]
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