Immunol Rev. 2011 Mar;240(1):72-91. doi: 10.1111/j.1600-065X.2010.00990.x.
Invasion and intracellular survival by protozoan parasites
David Sibley L.
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, USA.
Abstract
Summary: Intracellular parasitism has arisen only a few times during the long ancestry of protozoan parasites including in diverse groups such as microsporidians, kinetoplastids, and apicomplexans. Strategies used to gain entry differ widely from injection (e.g. microsporidians), active penetration of the host cell (e.g. Toxoplasma), recruitment of lysosomes to a plasma membrane wound (e.g. Trypanosoma cruzi), to host cell-mediated phagocytosis (e.g. Leishmania). The resulting range of intracellular niches is equally diverse ranging from cytosolic (e.g. T. cruzi) to residing within a non-fusigenic vacuole (e.g. Toxoplasma, Encephalitozoon) or a modified phagolysosome (e.g. Leishmania). These lifestyle choices influence access to nutrients, interaction with host cell signaling pathways, and detection by pathogen recognition systems. As such, intracellular life requires a repertoire of adaptations to assure entry-exit from the cell, as well as to thwart innate immune mechanisms and prevent clearance. Elucidating these pathways at the cellular and molecular level may identify key steps that can be targeted to reduce parasite survival or augment immunologic responses and thereby prevent disease.
© 2011 John Wiley & Sons A/S.
PMID: 21349087 [PubMed - in process]
Saturday, February 26, 2011
Autophagy in immunity and cell-autonomous defense against intracellular microbes
Immunol Rev. 2011 Mar;240(1):92-104. doi: 10.1111/j.1600-065X.2010.00995.x.
Autophagy in immunity and cell-autonomous defense against intracellular microbes
Deretic V.
Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA.
Abstract
Summary: Autophagy was viewed until very recently primarily as a metabolic and intracellular biomass and organelle quality and quantity control pathway. It has now been recognized that autophagy represents a bona fide immunologic process with a wide array of roles in immunity. The immunologic functions of autophagy, as we understand them now, span both innate and adaptive immunity. They range from unique and sometimes highly specialized immunologic effectors and regulatory functions (referred to here as type I immunophagy) to generic homeostatic influence on immune cells (type II immunophagy), akin to the effects on survival and homeostasis of other cell types in the body. As a concept-building tool for understanding why and how autophagy is intertwined with immunity, it is useful to consider that the presently complex picture has emerged in increments, starting in part from the realization that autophagy acts as an evolutionarily ancient microbial clearance mechanism defending eukaryotic cells against intracellular pathogens. In this review, we build a stepwise model of how the core axis of autophagy as a cell-autonomous immune defense against microbes evolved into a complex but orderly web of intersections with innate and adaptive immunity processes. The connections between autophagy and conventional immunity systems include Toll-like receptors, Nod-like receptors, RIG-I-like receptors, damage-associated molecular patterns such as HMGB1, other known innate and adaptive immunity receptors and cytokines, sequestasome (p62)-like receptors that act as autophagy adapters, immunity-related GTPase IRGM, innate and adaptive functions of macrophages and dendritic cells, and differential effects on development and homeostasis of T- and B-lymphocyte subsets. The disease contexts covered here include tuberculosis, infections with human immunodeficiency virus and other viruses, Salmonella, Listeria, Shigella, Toxoplasma, and inflammatory disorders such as Crohn's disease and multiple sclerosis.
© 2011 John Wiley & Sons A/S.
PMID: 21349088 [PubMed - in process]
Autophagy in immunity and cell-autonomous defense against intracellular microbes
Deretic V.
Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM, USA.
Abstract
Summary: Autophagy was viewed until very recently primarily as a metabolic and intracellular biomass and organelle quality and quantity control pathway. It has now been recognized that autophagy represents a bona fide immunologic process with a wide array of roles in immunity. The immunologic functions of autophagy, as we understand them now, span both innate and adaptive immunity. They range from unique and sometimes highly specialized immunologic effectors and regulatory functions (referred to here as type I immunophagy) to generic homeostatic influence on immune cells (type II immunophagy), akin to the effects on survival and homeostasis of other cell types in the body. As a concept-building tool for understanding why and how autophagy is intertwined with immunity, it is useful to consider that the presently complex picture has emerged in increments, starting in part from the realization that autophagy acts as an evolutionarily ancient microbial clearance mechanism defending eukaryotic cells against intracellular pathogens. In this review, we build a stepwise model of how the core axis of autophagy as a cell-autonomous immune defense against microbes evolved into a complex but orderly web of intersections with innate and adaptive immunity processes. The connections between autophagy and conventional immunity systems include Toll-like receptors, Nod-like receptors, RIG-I-like receptors, damage-associated molecular patterns such as HMGB1, other known innate and adaptive immunity receptors and cytokines, sequestasome (p62)-like receptors that act as autophagy adapters, immunity-related GTPase IRGM, innate and adaptive functions of macrophages and dendritic cells, and differential effects on development and homeostasis of T- and B-lymphocyte subsets. The disease contexts covered here include tuberculosis, infections with human immunodeficiency virus and other viruses, Salmonella, Listeria, Shigella, Toxoplasma, and inflammatory disorders such as Crohn's disease and multiple sclerosis.
© 2011 John Wiley & Sons A/S.
PMID: 21349088 [PubMed - in process]
Immunology of Toxoplasma gondii
Immunol Rev. 2011 Mar;240(1):269-85. doi: 10.1111/j.1600-065X.2010.00992.x.
Immunology of Toxoplasma gondii
Munoz M, Liesenfeld O, Heimesaat MM.
Charite Medical School Berlin - Microbiology and Hygiene, Berlin, Germany.
Abstract
Summary: Toxoplasma gondii is an obligate intracellular parasite. Following oral infection the parasite crosses the intestinal epithelial barrier to disseminate throughout the body and establish latent infection in central nervous tissues. The clinical presentation ranges from asymptomatic to severe neurological disorders in immunocompromised individuals. Since the clinical presentation is diverse and depends, among other factors, on the immune status of the host, in the present review, we introduce parasitological, epidemiological, clinical, and molecular biological aspects of infection with T. gondii to set the stage for an in-depth discussion of host immune responses. Since immune responses in humans have not been investigated in detail the present review is exclusively referring to immune responses in experimental models of infection. Systemic and local immune responses in different models of infection are discussed, and a separate chapter introduces commonly used animal models of infection.
© 2011 John Wiley & Sons A/S.
PMID: 21349099 [PubMed - in process]
Immunology of Toxoplasma gondii
Munoz M, Liesenfeld O, Heimesaat MM.
Charite Medical School Berlin - Microbiology and Hygiene, Berlin, Germany.
Abstract
Summary: Toxoplasma gondii is an obligate intracellular parasite. Following oral infection the parasite crosses the intestinal epithelial barrier to disseminate throughout the body and establish latent infection in central nervous tissues. The clinical presentation ranges from asymptomatic to severe neurological disorders in immunocompromised individuals. Since the clinical presentation is diverse and depends, among other factors, on the immune status of the host, in the present review, we introduce parasitological, epidemiological, clinical, and molecular biological aspects of infection with T. gondii to set the stage for an in-depth discussion of host immune responses. Since immune responses in humans have not been investigated in detail the present review is exclusively referring to immune responses in experimental models of infection. Systemic and local immune responses in different models of infection are discussed, and a separate chapter introduces commonly used animal models of infection.
© 2011 John Wiley & Sons A/S.
PMID: 21349099 [PubMed - in process]
Evaluation of Five Antischizophrenic Agents Against Toxoplasma gondii in Human Cell Cultures
J Parasitol. 2011 Feb;97(1):148-51. Epub 2010 Sep 14.
Evaluation of Five Antischizophrenic Agents Against Toxoplasma gondii in Human Cell Cultures
Goodwin DG, Strobl JS, Lindsay DS.
a Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia 24061-0342.
Abstract
Abstract An increasing interest in the association of the presence of antibodies to Toxoplasma gondii and the development of schizophrenia in patients has been generated over the last several years. Some antischizophrenia agents have been shown to have activity against T. gondii in cell culture assays and to ameliorate behavioral changes associated with chronic T. gondii infection in rats. In the present study, we examined the effects of commonly used antipsychotic and mood stabilizing agents (haloperidol, clozapine, fluphenazine, trifluoperazine, and thioridazine) for activity against developing tachyzoites of the RH strain of T. gondii in human fibroblast cell cultures. Neither haloperidol nor clozapine had a measurable effect. Fluphenazine had an IC(50) of 1.7 µM, thioridazine had an IC(50) of 1.2 µM, and trifluoperazine had an IC(50) of 3.8 µM. Our study demonstrates that some agents used to treat schizophrenia have the ability to inhibit T. gondii proliferation in cell culture.
PMID: 21348624 [PubMed - in process]
Evaluation of Five Antischizophrenic Agents Against Toxoplasma gondii in Human Cell Cultures
Goodwin DG, Strobl JS, Lindsay DS.
a Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia 24061-0342.
Abstract
Abstract An increasing interest in the association of the presence of antibodies to Toxoplasma gondii and the development of schizophrenia in patients has been generated over the last several years. Some antischizophrenia agents have been shown to have activity against T. gondii in cell culture assays and to ameliorate behavioral changes associated with chronic T. gondii infection in rats. In the present study, we examined the effects of commonly used antipsychotic and mood stabilizing agents (haloperidol, clozapine, fluphenazine, trifluoperazine, and thioridazine) for activity against developing tachyzoites of the RH strain of T. gondii in human fibroblast cell cultures. Neither haloperidol nor clozapine had a measurable effect. Fluphenazine had an IC(50) of 1.7 µM, thioridazine had an IC(50) of 1.2 µM, and trifluoperazine had an IC(50) of 3.8 µM. Our study demonstrates that some agents used to treat schizophrenia have the ability to inhibit T. gondii proliferation in cell culture.
PMID: 21348624 [PubMed - in process]
The C-Terminus of Toxoplasma RON2 Provides the Crucial Link between AMA1 and the Host-Associated Invasion Complex
PLoS Pathog. 2011 Feb 10;7(2):e1001282.
The C-Terminus of Toxoplasma RON2 Provides the Crucial Link between AMA1 and the Host-Associated Invasion Complex
Tyler JS, Boothroyd JC.
Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, United States of America.
Abstract
Host cell invasion by apicomplexan parasites requires formation of the moving junction (MJ), a ring-like apposition between the parasite and host plasma membranes that the parasite migrates through during entry. The Toxoplasma MJ is a secreted complex including TgAMA1, a transmembrane protein on the parasite surface, and a complex of rhoptry neck proteins (TgRON2/4/5/8) described as host cell-associated. How these proteins connect the parasite and host cell has not previously been described. Here we show that TgRON2 localizes to the MJ and that two short segments flanking a hydrophobic stretch near its C-terminus (D3 and D4) independently associate with the ectodomain of TgAMA1. Pre-incubation of parasites with D3 (fused to glutathione S-transferase) dramatically reduces invasion but does not prevent injection of rhoptry bulb proteins. Hence, the entire C-terminal region of TgRON2 forms the crucial bridge between TgAMA1 and the rest of the MJ complex but this association is not required for rhoptry protein injection.
PMID: 21347354 [PubMed - in process]
The C-Terminus of Toxoplasma RON2 Provides the Crucial Link between AMA1 and the Host-Associated Invasion Complex
Tyler JS, Boothroyd JC.
Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, United States of America.
Abstract
Host cell invasion by apicomplexan parasites requires formation of the moving junction (MJ), a ring-like apposition between the parasite and host plasma membranes that the parasite migrates through during entry. The Toxoplasma MJ is a secreted complex including TgAMA1, a transmembrane protein on the parasite surface, and a complex of rhoptry neck proteins (TgRON2/4/5/8) described as host cell-associated. How these proteins connect the parasite and host cell has not previously been described. Here we show that TgRON2 localizes to the MJ and that two short segments flanking a hydrophobic stretch near its C-terminus (D3 and D4) independently associate with the ectodomain of TgAMA1. Pre-incubation of parasites with D3 (fused to glutathione S-transferase) dramatically reduces invasion but does not prevent injection of rhoptry bulb proteins. Hence, the entire C-terminal region of TgRON2 forms the crucial bridge between TgAMA1 and the rest of the MJ complex but this association is not required for rhoptry protein injection.
PMID: 21347354 [PubMed - in process]
Genetic mapping identifies novel highly protective antigens for an apicomplexan parasite
PLoS Pathog. 2011 Feb 10;7(2):e1001279.
Genetic mapping identifies novel highly protective antigens for an apicomplexan parasite
Blake DP, Billington KJ, Copestake SL, Oakes RD, Quail MA, Wan KL, Shirley MW, Smith AL.
Institute for Animal Health, Compton, Berkshire, United Kingdom.
Abstract
Apicomplexan parasites are responsible for a myriad of diseases in humans and livestock; yet despite intensive effort, development of effective sub-unit vaccines remains a long-term goal. Antigenic complexity and our inability to identify protective antigens from the pool that induce response are serious challenges in the development of new vaccines. Using a combination of parasite genetics and selective barriers with population-based genetic fingerprinting, we have identified that immunity against the most important apicomplexan parasite of livestock (Eimeria spp.) was targeted against a few discrete regions of the genome. Herein we report the identification of six genomic regions and, within two of those loci, the identification of true protective antigens that confer immunity as sub-unit vaccines. The first of these is an Eimeria maxima homologue of apical membrane antigen-1 (AMA-1) and the second is a previously uncharacterised gene that we have termed 'immune mapped protein-1' (IMP-1). Significantly, homologues of the AMA-1 antigen are protective with a range of apicomplexan parasites including Plasmodium spp., which suggest that there may be some characteristic(s) of protective antigens shared across this diverse group of parasites. Interestingly, homologues of the IMP-1 antigen, which is protective against E. maxima infection, can be identified in Toxoplasma gondii and Neospora caninum. Overall, this study documents the discovery of novel protective antigens using a population-based genetic mapping approach allied with a protection-based screen of candidate genes. The identification of AMA-1 and IMP-1 represents a substantial step towards development of an effective anti-eimerian sub-unit vaccine and raises the possibility of identification of novel antigens for other apicomplexan parasites. Moreover, validation of the parasite genetics approach to identify effective antigens supports its adoption in other parasite systems where legitimate protective antigen identification is difficult.
PMID: 21347348 [PubMed - in process]
Genetic mapping identifies novel highly protective antigens for an apicomplexan parasite
Blake DP, Billington KJ, Copestake SL, Oakes RD, Quail MA, Wan KL, Shirley MW, Smith AL.
Institute for Animal Health, Compton, Berkshire, United Kingdom.
Abstract
Apicomplexan parasites are responsible for a myriad of diseases in humans and livestock; yet despite intensive effort, development of effective sub-unit vaccines remains a long-term goal. Antigenic complexity and our inability to identify protective antigens from the pool that induce response are serious challenges in the development of new vaccines. Using a combination of parasite genetics and selective barriers with population-based genetic fingerprinting, we have identified that immunity against the most important apicomplexan parasite of livestock (Eimeria spp.) was targeted against a few discrete regions of the genome. Herein we report the identification of six genomic regions and, within two of those loci, the identification of true protective antigens that confer immunity as sub-unit vaccines. The first of these is an Eimeria maxima homologue of apical membrane antigen-1 (AMA-1) and the second is a previously uncharacterised gene that we have termed 'immune mapped protein-1' (IMP-1). Significantly, homologues of the AMA-1 antigen are protective with a range of apicomplexan parasites including Plasmodium spp., which suggest that there may be some characteristic(s) of protective antigens shared across this diverse group of parasites. Interestingly, homologues of the IMP-1 antigen, which is protective against E. maxima infection, can be identified in Toxoplasma gondii and Neospora caninum. Overall, this study documents the discovery of novel protective antigens using a population-based genetic mapping approach allied with a protection-based screen of candidate genes. The identification of AMA-1 and IMP-1 represents a substantial step towards development of an effective anti-eimerian sub-unit vaccine and raises the possibility of identification of novel antigens for other apicomplexan parasites. Moreover, validation of the parasite genetics approach to identify effective antigens supports its adoption in other parasite systems where legitimate protective antigen identification is difficult.
PMID: 21347348 [PubMed - in process]
The RON2-AMA1 Interaction is a Critical Step in Moving Junction-Dependent Invasion by Apicomplexan Parasites
PLoS Pathog. 2011 Feb 10;7(2):e1001276.
The RON2-AMA1 Interaction is a Critical Step in Moving Junction-Dependent Invasion by Apicomplexan Parasites
Lamarque M, Besteiro S, Papoin J, Roques M, Vulliez-Le Normand B, Morlon-Guyot J, Dubremetz JF, Fauquenoy S, Tomavo S, Faber BW, Kocken CH, Thomas AW, Boulanger MJ, Bentley GA, Lebrun M.
UMR 5235 CNRS, Université de Montpellier 2, Montpellier, France.
Abstract
Obligate intracellular Apicomplexa parasites share a unique invasion mechanism involving a tight interaction between the host cell and the parasite surfaces called the moving junction (MJ). The MJ, which is the anchoring structure for the invasion process, is formed by secretion of a macromolecular complex (RON2/4/5/8), derived from secretory organelles called rhoptries, into the host cell membrane. AMA1, a protein secreted from micronemes and associated with the parasite surface during invasion, has been shown in vitro to bind the MJ complex through a direct association with RON2. Here we show that RON2 is inserted as an integral membrane protein in the host cell and, using several interaction assays with native or recombinant proteins, we define the region that binds AMA1. Our studies were performed both in Toxoplasma gondii and Plasmodium falciparum and although AMA1 and RON2 proteins have diverged between Apicomplexa species, we show an intra-species conservation of their interaction. More importantly, invasion inhibition assays using recombinant proteins demonstrate that the RON2-AMA1 interaction is crucial for both T. gondii and P. falciparum entry into their host cells. This work provides the first evidence that AMA1 uses the rhoptry neck protein RON2 as a receptor to promote invasion by Apicomplexa parasites.
PMID: 21347343 [PubMed - in process]
The RON2-AMA1 Interaction is a Critical Step in Moving Junction-Dependent Invasion by Apicomplexan Parasites
Lamarque M, Besteiro S, Papoin J, Roques M, Vulliez-Le Normand B, Morlon-Guyot J, Dubremetz JF, Fauquenoy S, Tomavo S, Faber BW, Kocken CH, Thomas AW, Boulanger MJ, Bentley GA, Lebrun M.
UMR 5235 CNRS, Université de Montpellier 2, Montpellier, France.
Abstract
Obligate intracellular Apicomplexa parasites share a unique invasion mechanism involving a tight interaction between the host cell and the parasite surfaces called the moving junction (MJ). The MJ, which is the anchoring structure for the invasion process, is formed by secretion of a macromolecular complex (RON2/4/5/8), derived from secretory organelles called rhoptries, into the host cell membrane. AMA1, a protein secreted from micronemes and associated with the parasite surface during invasion, has been shown in vitro to bind the MJ complex through a direct association with RON2. Here we show that RON2 is inserted as an integral membrane protein in the host cell and, using several interaction assays with native or recombinant proteins, we define the region that binds AMA1. Our studies were performed both in Toxoplasma gondii and Plasmodium falciparum and although AMA1 and RON2 proteins have diverged between Apicomplexa species, we show an intra-species conservation of their interaction. More importantly, invasion inhibition assays using recombinant proteins demonstrate that the RON2-AMA1 interaction is crucial for both T. gondii and P. falciparum entry into their host cells. This work provides the first evidence that AMA1 uses the rhoptry neck protein RON2 as a receptor to promote invasion by Apicomplexa parasites.
PMID: 21347343 [PubMed - in process]
Actin Depolymerizing Factor controls actin turnover and gliding motility in Toxoplasma gondii
Mol Biol Cell. 2011 Feb 23. [Epub ahead of print]
Actin Depolymerizing Factor controls actin turnover and gliding motility in Toxoplasma gondii
Mehta S, Sibley LD.
Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110.
Abstract
Apicomplexan parasites rely on actin-based gliding motility to move across the substratum, cross biological barriers, and invade their host cells. Gliding motility depends on polymerization of parasite actin filaments, yet ∼98% of actin is non-filamentous in resting parasites. Previous studies suggest that the lack of actin filaments in the parasite is due to inherent instability, leaving uncertain the role for actin-binding proteins in controlling dynamics. We have previously shown that the single allele of Toxoplasma gondii ADF (TgADF) has strong actin monomer sequestering and weak filament severing activities in vitro. Here we used a conditional knockout strategy to investigate the role of TgADF in vivo. Suppression of TgADF led to accumulation of actin-rich filaments that were detected by immunofluorescence and EM. Parasites deficient in TgADF showed reduced speed of motility, increased aberrant patterns of motion, and inhibition of sustained helical gliding. Lack of TgADF also led to severe defects in entry and egress from host cells, thus blocking infection in vitro. These studies establish that the absence of stable actin structures in the parasite are not simply the result of intrinsic instability, but that TgADF is required for the rapid turnover of parasite actin filaments, gliding motility, and cell invasion.
PMID: 21346192 [PubMed - as supplied by publisher]
Actin Depolymerizing Factor controls actin turnover and gliding motility in Toxoplasma gondii
Mehta S, Sibley LD.
Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110.
Abstract
Apicomplexan parasites rely on actin-based gliding motility to move across the substratum, cross biological barriers, and invade their host cells. Gliding motility depends on polymerization of parasite actin filaments, yet ∼98% of actin is non-filamentous in resting parasites. Previous studies suggest that the lack of actin filaments in the parasite is due to inherent instability, leaving uncertain the role for actin-binding proteins in controlling dynamics. We have previously shown that the single allele of Toxoplasma gondii ADF (TgADF) has strong actin monomer sequestering and weak filament severing activities in vitro. Here we used a conditional knockout strategy to investigate the role of TgADF in vivo. Suppression of TgADF led to accumulation of actin-rich filaments that were detected by immunofluorescence and EM. Parasites deficient in TgADF showed reduced speed of motility, increased aberrant patterns of motion, and inhibition of sustained helical gliding. Lack of TgADF also led to severe defects in entry and egress from host cells, thus blocking infection in vitro. These studies establish that the absence of stable actin structures in the parasite are not simply the result of intrinsic instability, but that TgADF is required for the rapid turnover of parasite actin filaments, gliding motility, and cell invasion.
PMID: 21346192 [PubMed - as supplied by publisher]
Saturday, February 19, 2011
Toxoplasma gondii inhibits granzyme B-mediated apoptosis by the inhibition of granzyme B function in host cells
Int J Parasitol. 2011 Feb 14. [Epub ahead of print]
Toxoplasma gondii inhibits granzyme B-mediated apoptosis by the inhibition of granzyme B function in host cells
Yamada T, Tomita T, Weiss LM, Orlofsky A.
Department of Pathology Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York, 10461, USA.
Abstract
Host defense to the apicomplexan parasite Toxoplasma gondii is critically dependent on CD8(+) T cells, whose effector functions include the induction of apoptosis in target cells following the secretion of granzyme proteases. Here we demonstrate that T. gondii induces resistance of host cells to apoptosis induced by recombinant granzyme B. Granzyme B induction of caspase-independent cytochrome c release was blocked in T. gondii-infected cells. Prevention of apoptosis could not be attributed to altered expression of the Bcl-2 family of apoptotic regulatory proteins, but was instead associated with reduced granzyme B-mediated, caspase-independent cleavage of procaspase 3 to the p20 form in T. gondii-infected cells, as well as reduced granzyme B-mediated cleavage of the artificial granzyme B substrate, GranToxiLux. The reduction in granzyme B proteolytic function in T. gondii-infected cells could not be attributed to altered granzyme B uptake or reduced trafficking of granzyme B to the cytosol, implying a T. gondii-mediated inhibition of granzyme B activity. Apoptosis and GranToxiLux cleavage were similarly inhibited in T. gondii-infected cells exposed to the natural killer-like cell line YT-1. The endogenous granzyme B inhibitor PI-9 was not up-regulated in infected cells. We believe these findings represent the first demonstration of granzyme B inhibition by a cellular pathogen and indicate a new modality for host cell protection by T. gondii that may contribute to parasite immune evasion.
Copyright © 2011. Published by Elsevier Ltd.
PMID: 21329693 [PubMed - as supplied by publisher]
Toxoplasma gondii inhibits granzyme B-mediated apoptosis by the inhibition of granzyme B function in host cells
Yamada T, Tomita T, Weiss LM, Orlofsky A.
Department of Pathology Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York, 10461, USA.
Abstract
Host defense to the apicomplexan parasite Toxoplasma gondii is critically dependent on CD8(+) T cells, whose effector functions include the induction of apoptosis in target cells following the secretion of granzyme proteases. Here we demonstrate that T. gondii induces resistance of host cells to apoptosis induced by recombinant granzyme B. Granzyme B induction of caspase-independent cytochrome c release was blocked in T. gondii-infected cells. Prevention of apoptosis could not be attributed to altered expression of the Bcl-2 family of apoptotic regulatory proteins, but was instead associated with reduced granzyme B-mediated, caspase-independent cleavage of procaspase 3 to the p20 form in T. gondii-infected cells, as well as reduced granzyme B-mediated cleavage of the artificial granzyme B substrate, GranToxiLux. The reduction in granzyme B proteolytic function in T. gondii-infected cells could not be attributed to altered granzyme B uptake or reduced trafficking of granzyme B to the cytosol, implying a T. gondii-mediated inhibition of granzyme B activity. Apoptosis and GranToxiLux cleavage were similarly inhibited in T. gondii-infected cells exposed to the natural killer-like cell line YT-1. The endogenous granzyme B inhibitor PI-9 was not up-regulated in infected cells. We believe these findings represent the first demonstration of granzyme B inhibition by a cellular pathogen and indicate a new modality for host cell protection by T. gondii that may contribute to parasite immune evasion.
Copyright © 2011. Published by Elsevier Ltd.
PMID: 21329693 [PubMed - as supplied by publisher]
Wednesday, February 16, 2011
Toxoplasma gondii sequesters centromeres to a specific nuclear region throughout the cell cycle
Proc Natl Acad Sci U S A. 2011 Feb 14. [Epub ahead of print]
Toxoplasma gondii sequesters centromeres to a specific nuclear region throughout the cell cycle
Brooks CF, Francia ME, Gissot M, Croken MM, Kim K, Striepen B.
Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, GA 30602.
Abstract
Members of the eukaryotic phylum Apicomplexa are the cause of important human diseases including malaria, toxoplasmosis, and cryptosporidiosis. These obligate intracellular parasites produce new invasive stages through a complex budding process. The budding cycle is remarkably flexible and can produce varied numbers of progeny to adapt to different host-cell niches. How this complex process is coordinated remains poorly understood. Using Toxoplasma gondii as a genetic model, we show that a key element to this coordination is the centrocone, a unique elaboration of the nuclear envelope that houses the mitotic spindle. Exploiting transgenic parasite lines expressing epitope-tagged centromeric H3 variant CenH3, we identify the centromeres of T. gondii chromosomes by hybridization of chromatin immunoprecipitations to genome-wide microarrays (ChIP-chip). We demonstrate that centromere attachment to the centrocone persists throughout the parasite cell cycle and that centromeres localize to a single apical region within the nucleus. Centromere sequestration provides a mechanism for the organization of the Toxoplasma nucleus and the maintenance of genome integrity.
PMID: 21321216 [PubMed - as supplied by publisher]
Toxoplasma gondii sequesters centromeres to a specific nuclear region throughout the cell cycle
Brooks CF, Francia ME, Gissot M, Croken MM, Kim K, Striepen B.
Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, GA 30602.
Abstract
Members of the eukaryotic phylum Apicomplexa are the cause of important human diseases including malaria, toxoplasmosis, and cryptosporidiosis. These obligate intracellular parasites produce new invasive stages through a complex budding process. The budding cycle is remarkably flexible and can produce varied numbers of progeny to adapt to different host-cell niches. How this complex process is coordinated remains poorly understood. Using Toxoplasma gondii as a genetic model, we show that a key element to this coordination is the centrocone, a unique elaboration of the nuclear envelope that houses the mitotic spindle. Exploiting transgenic parasite lines expressing epitope-tagged centromeric H3 variant CenH3, we identify the centromeres of T. gondii chromosomes by hybridization of chromatin immunoprecipitations to genome-wide microarrays (ChIP-chip). We demonstrate that centromere attachment to the centrocone persists throughout the parasite cell cycle and that centromeres localize to a single apical region within the nucleus. Centromere sequestration provides a mechanism for the organization of the Toxoplasma nucleus and the maintenance of genome integrity.
PMID: 21321216 [PubMed - as supplied by publisher]
Induction of protective Th1 immune responses in mice by vaccination with recombinant Toxoplasma gondii nucleoside triphosphate hydrolase-II
Vaccine. 2011 Feb 9. [Epub ahead of print]
Induction of protective Th1 immune responses in mice by vaccination with recombinant Toxoplasma gondii nucleoside triphosphate hydrolase-II
Tan F, Hu X, Luo FJ, Pan CW, Chen XG.
Department of Parasitology, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, Guangdong 510515, PR China; Department of Parasitology, School of Basic Medical Sciences, Wenzhou Medical College, Wenzhou, Zhejiang 325035, PR China.
Abstract
The Toxoplasma gondii nucleoside triphosphate hydrolase (TgNTPase) has apyrase activity, degrading ATP to the di- and mono-phosphate forms and may be used by the parasite to salvage purines from the host cell for survival and replication. To study the immune-protective value of TgNTPase-II, BALB/c mice were immunized with a recombinant form of the antigen rTgNTPase-II combined with alum. All immunized mice produced specific anti-rTgNTPase-II immunoglobulins, with high IgG antibody titers and a mixed IgG1/IgG2a response, with predominance of IgG2a production. The cellular immune response was associated with the production of IFN-γ and IL-2 cytokines and the increase of the percentage of CD8+ T cells. Vaccinated mice displayed significant protection against acute infection with the virulent RH strain (P<0.05 in survival rate) and also chronic infection with PRU cyst (62.9% and 57.6% reduction in brain parasite load for rTgNTPase-II+alum and rTgNTPase-II alone vaccinated groups) compared to the non-vaccinated control group. In conclusion, rTgNTPase-II elicits a strong specific Th1 immune response providing partial protection against both T. gondii acute and chronic infection.
Copyright © 2011. Published by Elsevier Ltd.
PMID: 21315696 [PubMed - as supplied by publisher]
Induction of protective Th1 immune responses in mice by vaccination with recombinant Toxoplasma gondii nucleoside triphosphate hydrolase-II
Tan F, Hu X, Luo FJ, Pan CW, Chen XG.
Department of Parasitology, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, Guangdong 510515, PR China; Department of Parasitology, School of Basic Medical Sciences, Wenzhou Medical College, Wenzhou, Zhejiang 325035, PR China.
Abstract
The Toxoplasma gondii nucleoside triphosphate hydrolase (TgNTPase) has apyrase activity, degrading ATP to the di- and mono-phosphate forms and may be used by the parasite to salvage purines from the host cell for survival and replication. To study the immune-protective value of TgNTPase-II, BALB/c mice were immunized with a recombinant form of the antigen rTgNTPase-II combined with alum. All immunized mice produced specific anti-rTgNTPase-II immunoglobulins, with high IgG antibody titers and a mixed IgG1/IgG2a response, with predominance of IgG2a production. The cellular immune response was associated with the production of IFN-γ and IL-2 cytokines and the increase of the percentage of CD8+ T cells. Vaccinated mice displayed significant protection against acute infection with the virulent RH strain (P<0.05 in survival rate) and also chronic infection with PRU cyst (62.9% and 57.6% reduction in brain parasite load for rTgNTPase-II+alum and rTgNTPase-II alone vaccinated groups) compared to the non-vaccinated control group. In conclusion, rTgNTPase-II elicits a strong specific Th1 immune response providing partial protection against both T. gondii acute and chronic infection.
Copyright © 2011. Published by Elsevier Ltd.
PMID: 21315696 [PubMed - as supplied by publisher]
Genetic analyses of atypical Toxoplasma gondii strains reveal a fourth clonal lineage in North America
Int J Parasitol. 2011 Feb 11. [Epub ahead of print]
Genetic analyses of atypical Toxoplasma gondii strains reveal a fourth clonal lineage in North America
Khan A, Dubey JP, Su C, Ajioka JW, Rosenthal BM, Sibley LD.
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110, U.S.A.
Abstract
Toxoplasma gondii is a widespread parasite of animals that causes zoonotic infections in humans. Previous studies have revealed a strongly clonal population structure in North America and Europe, while strains from South America are genetically separate and more diverse. However, the composition within North America has been questioned by recent descriptions of genetically more variable strains from this region. Here, we examined an expanded set of isolates using sequenced-based phylogenetic and population analyses to re-evaluate the population structure of T. gondii in North America. Our findings reveal that isolates previously defined by atypical restriction fragment length polymorphism patterns fall into two discrete groups. In one case, these new isolates represent variants of an existing lineage, from which they differ only by minor mutational drift. However, in the second case, it is evident that these isolates define a completely new lineage that is common in North America. Support for this new lineage was based on phylogeny, principle components analysis, STRUCTURE analyses, and statistical analysis of gene flow between groups. This new group, referred to as haplogroup 12, contains divergent genotypes previously referred to as A and X, isolated from sea otters. Consistent with this, group 12 was found primarily in wild animals, as well as occasionally in humans. This new lineage also has a highly clonal population structure. Analysis of the inheritance of multilocus genotypes revealed that different strains within group 12 are the products of a single recombination event between type 2 and a unique parental lineage. Collectively, the archetypal type 2 has been associated with clonal expansion of a small number of lineages in the North, as a consequence of separate but infrequent genetic crosses with several different parental lines.
Copyright © 2011 Australian Society for Parasitology Inc. All rights reserved.
PMID: 21320505 [PubMed - as supplied by publisher]
Genetic analyses of atypical Toxoplasma gondii strains reveal a fourth clonal lineage in North America
Khan A, Dubey JP, Su C, Ajioka JW, Rosenthal BM, Sibley LD.
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110, U.S.A.
Abstract
Toxoplasma gondii is a widespread parasite of animals that causes zoonotic infections in humans. Previous studies have revealed a strongly clonal population structure in North America and Europe, while strains from South America are genetically separate and more diverse. However, the composition within North America has been questioned by recent descriptions of genetically more variable strains from this region. Here, we examined an expanded set of isolates using sequenced-based phylogenetic and population analyses to re-evaluate the population structure of T. gondii in North America. Our findings reveal that isolates previously defined by atypical restriction fragment length polymorphism patterns fall into two discrete groups. In one case, these new isolates represent variants of an existing lineage, from which they differ only by minor mutational drift. However, in the second case, it is evident that these isolates define a completely new lineage that is common in North America. Support for this new lineage was based on phylogeny, principle components analysis, STRUCTURE analyses, and statistical analysis of gene flow between groups. This new group, referred to as haplogroup 12, contains divergent genotypes previously referred to as A and X, isolated from sea otters. Consistent with this, group 12 was found primarily in wild animals, as well as occasionally in humans. This new lineage also has a highly clonal population structure. Analysis of the inheritance of multilocus genotypes revealed that different strains within group 12 are the products of a single recombination event between type 2 and a unique parental lineage. Collectively, the archetypal type 2 has been associated with clonal expansion of a small number of lineages in the North, as a consequence of separate but infrequent genetic crosses with several different parental lines.
Copyright © 2011 Australian Society for Parasitology Inc. All rights reserved.
PMID: 21320505 [PubMed - as supplied by publisher]
Sunday, February 06, 2011
Restoration of T Cell Responses to Toxoplasma gondii after Successful Combined Antiretroviral Therapy in Patients with AIDS with Previous Toxoplasmic
Clin Infect Dis. 2011 Mar;52(5):662-670.
Restoration of T Cell Responses to Toxoplasma gondii after Successful Combined Antiretroviral Therapy in Patients with AIDS with Previous Toxoplasmic Encephalitis
Lejeune M, Miró JM, De Lazzari E, García F, Claramonte X, Martínez E, Ribera E, Arrizabalaga J, Arribas JR, Domingo P, Ferrer E, Plana M, Valls ME, Podzamczer D, Pumarola T, Jacquet A, Mallolas J, Gatell JM, Gallart T; the Spanish Toxoplasma gondii Study Group.
Immunology Service, Hospital Clinic-Institut d'Investigacions Biomèdiques Augustí Pi i Suñer.
Abstract
Background. It is unknown whether a Toxoplasma gondii-specific T cell response is restored after successful combined antiretroviral therapy (cART) in patients with AIDS and current or previous toxoplasmic encephalitis (TE). Methods. We performed a multicenter cross-sectional study with 17 healthy T. gondii-positive human immunodeficiency virus (HIV)-1-uninfected individuals and 90 patients coinfected with HIV-1 and T. gondii distributed in 5 groups according to their CD4(+) T cell counts and T. gondii infection (with or without current or previous TE). We investigated the lymphocyte proliferative response (LPR) and interferon (IFN)-γ production in response to T. gondii soluble antigen extract (SATg) and as CD4(+) and CD8(+) T cell subsets. Results. SATg-specific LPR and IFN-γ production were not observed in many of the most immunosuppressed patients (CD4(+) T cell count, <200 cells/μL, with or without current or previous TE). By contrast, these responses occurred in a considerable percentage (LPR, 43%; IFN-γ production, 80%) of patients receiving successful cART (CD4(+) T cell count, >200 cells/μL) who presented with TE and had already stopped secondary TE prophylaxis. Similar results were found in immunocompetent asymptomatic patients who did not receive TE prophylaxis. The predictors of SATg-specific T cell responses and IFN-γ production were a cART-mediated increase in CD4(+) T cell count and LPR to phytohemagglutinin and viral suppression and a decrease in the activated (CD38(+)) CD8(+) T cell count, respectively. Conclusions. cART restores T. gondii-specific CD4 T cell responses in most patients with AIDS who had previous TE. Our data support the safety of withdrawing TE prophylaxis when the CD4(+) T cell count returns to levels >200 cells/μL.
PMID: 21292671 [PubMed - as supplied by publisher]
Restoration of T Cell Responses to Toxoplasma gondii after Successful Combined Antiretroviral Therapy in Patients with AIDS with Previous Toxoplasmic Encephalitis
Lejeune M, Miró JM, De Lazzari E, García F, Claramonte X, Martínez E, Ribera E, Arrizabalaga J, Arribas JR, Domingo P, Ferrer E, Plana M, Valls ME, Podzamczer D, Pumarola T, Jacquet A, Mallolas J, Gatell JM, Gallart T; the Spanish Toxoplasma gondii Study Group.
Immunology Service, Hospital Clinic-Institut d'Investigacions Biomèdiques Augustí Pi i Suñer.
Abstract
Background. It is unknown whether a Toxoplasma gondii-specific T cell response is restored after successful combined antiretroviral therapy (cART) in patients with AIDS and current or previous toxoplasmic encephalitis (TE). Methods. We performed a multicenter cross-sectional study with 17 healthy T. gondii-positive human immunodeficiency virus (HIV)-1-uninfected individuals and 90 patients coinfected with HIV-1 and T. gondii distributed in 5 groups according to their CD4(+) T cell counts and T. gondii infection (with or without current or previous TE). We investigated the lymphocyte proliferative response (LPR) and interferon (IFN)-γ production in response to T. gondii soluble antigen extract (SATg) and as CD4(+) and CD8(+) T cell subsets. Results. SATg-specific LPR and IFN-γ production were not observed in many of the most immunosuppressed patients (CD4(+) T cell count, <200 cells/μL, with or without current or previous TE). By contrast, these responses occurred in a considerable percentage (LPR, 43%; IFN-γ production, 80%) of patients receiving successful cART (CD4(+) T cell count, >200 cells/μL) who presented with TE and had already stopped secondary TE prophylaxis. Similar results were found in immunocompetent asymptomatic patients who did not receive TE prophylaxis. The predictors of SATg-specific T cell responses and IFN-γ production were a cART-mediated increase in CD4(+) T cell count and LPR to phytohemagglutinin and viral suppression and a decrease in the activated (CD38(+)) CD8(+) T cell count, respectively. Conclusions. cART restores T. gondii-specific CD4 T cell responses in most patients with AIDS who had previous TE. Our data support the safety of withdrawing TE prophylaxis when the CD4(+) T cell count returns to levels >200 cells/μL.
PMID: 21292671 [PubMed - as supplied by publisher]
Thursday, February 03, 2011
Toxoplasma gondii: A bradyzoite-specific DnaK-tetratricopeptide repeat (DnaK-TPR) protein interacts with p23 co-chaperone protein
Exp Parasitol. 2011 Jan 28. [Epub ahead of print]
Toxoplasma gondii: A bradyzoite-specific DnaK-tetratricopeptide repeat (DnaK-TPR) protein interacts with p23 co-chaperone protein
Ueno A, Dautu G, Haga K, Munyaka B, Carmen G, Kobayashi Y, Igarashi M.
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, 2-13 Inada-cho, Obihiro, Hokkaido, 080-8555, Japan; Department of Pathobiological Science, School of Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, Japan.
Abstract
The DnaK-tetratricopeptide repeat (DnaK-TPR) gene (ToxoDB ID, TGME49_002020) is expressed predominantly at the bradyzoite stage. DnaK-TPR protein has a heat shock protein (DnaK) and tetratricopeptide repeat (TPR) domains with amino acid sequence similarity to the counterparts of other organisms (40.2 to 43.7% to DnaK domain and 41.1 to 66.0% to TPR domain). These findings allowed us to infer that DnaK-TPR protein is important in the tachyzoite-to-bradyzoite development or maintenance of cyst structure although the function of this gene is still unknown. An immunofluorescence assay (IFA) revealed that DnaK-TPR protein was expressed in T. gondii-encysted and in vitro-induced bradyzoites and distributed in the whole part of parasite cells. We conducted yeast two-hybrid screening to identify proteins interacting with DnaK-TPR protein, and demonstrated that DnaK-TPR protein interacts with p23 co-chaperone protein (Tgp23). It was expected that DnaK-TPR protein would have a function as a molecular chaperon in bradyzoite cells associated with Tgp23. Possible mechanisms for this gene are discussed.
Copyright © 2011. Published by Elsevier Inc.
PMID: 21281637 [PubMed - as supplied by publisher]
Toxoplasma gondii: A bradyzoite-specific DnaK-tetratricopeptide repeat (DnaK-TPR) protein interacts with p23 co-chaperone protein
Ueno A, Dautu G, Haga K, Munyaka B, Carmen G, Kobayashi Y, Igarashi M.
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, 2-13 Inada-cho, Obihiro, Hokkaido, 080-8555, Japan; Department of Pathobiological Science, School of Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido, Japan.
Abstract
The DnaK-tetratricopeptide repeat (DnaK-TPR) gene (ToxoDB ID, TGME49_002020) is expressed predominantly at the bradyzoite stage. DnaK-TPR protein has a heat shock protein (DnaK) and tetratricopeptide repeat (TPR) domains with amino acid sequence similarity to the counterparts of other organisms (40.2 to 43.7% to DnaK domain and 41.1 to 66.0% to TPR domain). These findings allowed us to infer that DnaK-TPR protein is important in the tachyzoite-to-bradyzoite development or maintenance of cyst structure although the function of this gene is still unknown. An immunofluorescence assay (IFA) revealed that DnaK-TPR protein was expressed in T. gondii-encysted and in vitro-induced bradyzoites and distributed in the whole part of parasite cells. We conducted yeast two-hybrid screening to identify proteins interacting with DnaK-TPR protein, and demonstrated that DnaK-TPR protein interacts with p23 co-chaperone protein (Tgp23). It was expected that DnaK-TPR protein would have a function as a molecular chaperon in bradyzoite cells associated with Tgp23. Possible mechanisms for this gene are discussed.
Copyright © 2011. Published by Elsevier Inc.
PMID: 21281637 [PubMed - as supplied by publisher]
Wednesday, February 02, 2011
The activation mechanism of Irga6, an interferon-inducible GTPase contributing to mouse resistance against Toxoplasma gondii
BMC Biol. 2011 Jan 28;9(1):7. [Epub ahead of print]
The activation mechanism of Irga6, an interferon-inducible GTPase contributing to mouse resistance against Toxoplasma gondii
Pawlowski N, Khaminets A, Hunn JP, Papic N, Schmidt A, Uthaiah RC, Lange R, Vopper G, Martens S, Wolf E, Howard JC.
Abstract
ABSTRACT:
BACKGROUND: The interferon-inducible immunity-related GTPases (IRG proteins/p47 GTPases) are a distinctive family of GTPases that function as powerful cell-autonomous resistance factors. The IRG protein, Irga6 (IIGP1), participates in the disruption of the vacuolar membrane surrounding the intracellular parasite, Toxoplasma gondii, through which it communicates with its cellular hosts. Some aspects of the protein's behaviour have suggested a dynamin-like molecular mode of action, in that the energy released by GTP hydrolysis is transduced into mechanical work that results in deformation and ultimately rupture of the vacuolar membrane.
RESULTS: Irga6 forms GTP-dependent oligomers in vitro and thereby activates hydrolysis of the GTP substrate. In this study we define the catalytic G-domain interface by mutagenesis and present a structural model, of how GTP hydrolysis is activated in Irga6 complexes, based on the substrate-twinning reaction mechanism of the signal recognition particle (SRP) and its receptor (SRalpha). In conformity with this model, we show that the bound nucleotide is part of the catalytic interface and that the 3'hydroxyl of the GTP ribose bound to each subunit is essential for trans-activation of hydrolysis of the GTP bound to the other subunit. We show that both positive and negative regulatory interactions between IRG proteins occur via the catalytic interface. Furthermore, mutations that disrupt the catalytic interface also prevent Irga6 from accumulating on the parasitophorous vacuole membrane of T. gondii, showing that GTP-dependent Irga6 activation is an essential component of the resistance mechanism.
CONCLUSIONS: The catalytic interface of Irga6 defined in the present experiments can probably be used as a paradigm for the nucleotide-dependent interactions of all members of the large family of IRG GTPases, both activating and regulatory. Understanding the activation mechanism of Irga6 will help to explain the mechanism by which IRG proteins exercise their resistance function. We find no support from sequence or G-domain structure for the idea that IRG proteins and the SRP GTPases have a common phylogenetic origin. It therefore seems probable, if surprising, that the substrate-assisted catalytic mechanism has been independently evolved in the two protein families.
PMID: 21276251 [PubMed - as supplied by publisher]
The activation mechanism of Irga6, an interferon-inducible GTPase contributing to mouse resistance against Toxoplasma gondii
Pawlowski N, Khaminets A, Hunn JP, Papic N, Schmidt A, Uthaiah RC, Lange R, Vopper G, Martens S, Wolf E, Howard JC.
Abstract
ABSTRACT:
BACKGROUND: The interferon-inducible immunity-related GTPases (IRG proteins/p47 GTPases) are a distinctive family of GTPases that function as powerful cell-autonomous resistance factors. The IRG protein, Irga6 (IIGP1), participates in the disruption of the vacuolar membrane surrounding the intracellular parasite, Toxoplasma gondii, through which it communicates with its cellular hosts. Some aspects of the protein's behaviour have suggested a dynamin-like molecular mode of action, in that the energy released by GTP hydrolysis is transduced into mechanical work that results in deformation and ultimately rupture of the vacuolar membrane.
RESULTS: Irga6 forms GTP-dependent oligomers in vitro and thereby activates hydrolysis of the GTP substrate. In this study we define the catalytic G-domain interface by mutagenesis and present a structural model, of how GTP hydrolysis is activated in Irga6 complexes, based on the substrate-twinning reaction mechanism of the signal recognition particle (SRP) and its receptor (SRalpha). In conformity with this model, we show that the bound nucleotide is part of the catalytic interface and that the 3'hydroxyl of the GTP ribose bound to each subunit is essential for trans-activation of hydrolysis of the GTP bound to the other subunit. We show that both positive and negative regulatory interactions between IRG proteins occur via the catalytic interface. Furthermore, mutations that disrupt the catalytic interface also prevent Irga6 from accumulating on the parasitophorous vacuole membrane of T. gondii, showing that GTP-dependent Irga6 activation is an essential component of the resistance mechanism.
CONCLUSIONS: The catalytic interface of Irga6 defined in the present experiments can probably be used as a paradigm for the nucleotide-dependent interactions of all members of the large family of IRG GTPases, both activating and regulatory. Understanding the activation mechanism of Irga6 will help to explain the mechanism by which IRG proteins exercise their resistance function. We find no support from sequence or G-domain structure for the idea that IRG proteins and the SRP GTPases have a common phylogenetic origin. It therefore seems probable, if surprising, that the substrate-assisted catalytic mechanism has been independently evolved in the two protein families.
PMID: 21276251 [PubMed - as supplied by publisher]
Toxoplasma gondii toxolysin 4 is an extensively processed putative metalloproteinase secreted from micronemes
Mol Biochem Parasitol. 2011 Jan 25. [Epub ahead of print]
Toxoplasma gondii toxolysin 4 is an extensively processed putative metalloproteinase secreted from micronemes
Laliberté J, Carruthers VB.
Department of Microbiology and Immunology, University of Michigan School of Medicine, 1150W. Medical Center Dr., Ann Arbor, MI 48109 USA.
Abstract
Proteases play central roles in cell invasion by Toxoplasma gondii and other apicomplexan parasites. Herein we report the cloning and characterization of a novel secretory putative metalloproteinase, Toxolysin 4 (TLN4). T. gondii tachyzoites store TLN4 in the micronemes and secrete it in response to elevated calcium, suggesting a possible role in cell invasion. TLN4 is initially synthesized as a large (∼260kDa) precursor, which is extensively processed into multiple proteolytic fragments within the parasite secretory system. At least some of these proteolytic fragments remain associated in a large molecular complex. Whereas precomplementation with the TLN4 cDNA allowed disruption of the TLN4 gene, multiple attempts to directly knockout TLN4 without precomplementation failed. TLN4 knockout parasites were detected by PCR in transfected populations but were lost from the cultures during drug selection and growth suggesting that TLN4 contributes to parasite fitness.
Copyright © 2011. Published by Elsevier B.V.
PMID: 21277910 [PubMed - as supplied by publisher]
Toxoplasma gondii toxolysin 4 is an extensively processed putative metalloproteinase secreted from micronemes
Laliberté J, Carruthers VB.
Department of Microbiology and Immunology, University of Michigan School of Medicine, 1150W. Medical Center Dr., Ann Arbor, MI 48109 USA.
Abstract
Proteases play central roles in cell invasion by Toxoplasma gondii and other apicomplexan parasites. Herein we report the cloning and characterization of a novel secretory putative metalloproteinase, Toxolysin 4 (TLN4). T. gondii tachyzoites store TLN4 in the micronemes and secrete it in response to elevated calcium, suggesting a possible role in cell invasion. TLN4 is initially synthesized as a large (∼260kDa) precursor, which is extensively processed into multiple proteolytic fragments within the parasite secretory system. At least some of these proteolytic fragments remain associated in a large molecular complex. Whereas precomplementation with the TLN4 cDNA allowed disruption of the TLN4 gene, multiple attempts to directly knockout TLN4 without precomplementation failed. TLN4 knockout parasites were detected by PCR in transfected populations but were lost from the cultures during drug selection and growth suggesting that TLN4 contributes to parasite fitness.
Copyright © 2011. Published by Elsevier B.V.
PMID: 21277910 [PubMed - as supplied by publisher]
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