Science. 2010 Dec 23. [Epub ahead of print]
Intramembrane Cleavage of AMA1 Triggers Toxoplasma to Switch from an Invasive to a Replicative Mode
Santos JM, Ferguson DJ, Blackman MJ, Soldati-Favre D.
Department of Microbiology, Faculty of Medicine, University of Geneva, 1 rue-Michel Servet, 1211 Geneva 4, Switzerland.
Abstract
Apicomplexan parasites invade host cells and immediately initiate cell division. The extracellular parasite discharges transmembrane proteins onto its surface to mediate motility and invasion. These are shed by intramembrane cleavage, a process associated with invasion but otherwise poorly understood. Functional analysis of Toxoplasma rhomboid 4, a surface intramembrane protease, by conditional overexpression of a catalytically inactive form, produced a profound block in replication. This was completely rescued by expression of the cleaved cytoplasmic tail of Toxoplasma or Plasmodium apical membrane antigen 1 (AMA1). These results reveal an unexpected function for AMA1 in parasite replication, and suggest that invasion proteins help to promote parasite switch from an invasive to a replicative mode.
PMID: 21205639 [PubMed - as supplied by publisher]
Thursday, January 06, 2011
Full-parasites: database of full-length cDNAs of apicomplexa parasites, 2010 update
Nucleic Acids Res. 2011 Jan;39(Database issue):D625-31. Epub 2010 Nov 4.
Full-parasites: database of full-length cDNAs of apicomplexa parasites, 2010 update
Tuda J, Mongan AE, Tolba ME, Imada M, Yamagishi J, Xuan X, Wakaguri H, Sugano S, Sugimoto C, Suzuki Y.
Faculty of Medicine, Sam Ratulangi University, Kampus Unsrat, Bahu Manado 95115, Indonesia.
Abstract
Full-Parasites (http://fullmal.hgc.jp/) is a transcriptome database of apicomplexa parasites, which include Plasmodium and Toxoplasma species. The latest version of Full-Parasites contains a total of 105,786 EST sequences from 12 parasites, of which 5925 full-length cDNAs have been completely sequenced. Full-Parasites also contain more than 30 million transcription start sites (TSS) for Plasmodium falciparum (Pf) and Toxoplasma gondii (Tg), which were identified using our novel oligo-capping-based protocol. Various types of cDNA data resources were interconnected with our original database functionalities. Specifically, in this update, we have included two unique RNA-Seq data sets consisting of 730 million mapped RNA-Seq tags. One is a dataset of 16 time-lapse experiments of cultured bradyzoite differentiation for Tg. The other dataset includes 31 clinical samples of Pf. Parasite RNA was extracted together with host human RNA, and the extracted mixed RNA was used for RNA sequencing, with the expectation that gene expression information from the host and parasite would be simultaneously represented. By providing the largest unique full-length cDNA and dynamic transcriptome data, Full-Parasites is useful for understanding host-parasite interactions and will help to eventually elucidate how monophyletic organisms have evolved to become parasites by adopting complex life cycles.
PMID: 21051343 [PubMed - in process]
Full-parasites: database of full-length cDNAs of apicomplexa parasites, 2010 update
Tuda J, Mongan AE, Tolba ME, Imada M, Yamagishi J, Xuan X, Wakaguri H, Sugano S, Sugimoto C, Suzuki Y.
Faculty of Medicine, Sam Ratulangi University, Kampus Unsrat, Bahu Manado 95115, Indonesia.
Abstract
Full-Parasites (http://fullmal.hgc.jp/) is a transcriptome database of apicomplexa parasites, which include Plasmodium and Toxoplasma species. The latest version of Full-Parasites contains a total of 105,786 EST sequences from 12 parasites, of which 5925 full-length cDNAs have been completely sequenced. Full-Parasites also contain more than 30 million transcription start sites (TSS) for Plasmodium falciparum (Pf) and Toxoplasma gondii (Tg), which were identified using our novel oligo-capping-based protocol. Various types of cDNA data resources were interconnected with our original database functionalities. Specifically, in this update, we have included two unique RNA-Seq data sets consisting of 730 million mapped RNA-Seq tags. One is a dataset of 16 time-lapse experiments of cultured bradyzoite differentiation for Tg. The other dataset includes 31 clinical samples of Pf. Parasite RNA was extracted together with host human RNA, and the extracted mixed RNA was used for RNA sequencing, with the expectation that gene expression information from the host and parasite would be simultaneously represented. By providing the largest unique full-length cDNA and dynamic transcriptome data, Full-Parasites is useful for understanding host-parasite interactions and will help to eventually elucidate how monophyletic organisms have evolved to become parasites by adopting complex life cycles.
PMID: 21051343 [PubMed - in process]
Wednesday, January 05, 2011
Strain-specific activation of the NF-{kappa}B pathway by GRA15, a novel Toxoplasma gondii dense granule protein
J Exp Med. 2011 Jan 3. [Epub ahead of print]
Strain-specific activation of the NF-{kappa}B pathway by GRA15, a novel Toxoplasma gondii dense granule protein
Rosowski EE, Lu D, Julien L, Rodda L, Gaiser RA, Jensen KD, Saeij JP.
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.
Abstract
NF-κB is an integral component of the immune response to Toxoplasma gondii. Although evidence exists that T. gondii can directly modulate the NF-κB pathway, the parasite-derived effectors involved are unknown. We determined that type II strains of T. gondii activate more NF-κB than type I or type III strains, and using forward genetics we found that this difference is a result of the polymorphic protein GRA15, a novel dense granule protein which T. gondii secretes into the host cell upon invasion. A GRA15-deficient type II strain has a severe defect in both NF-κB nuclear translocation and NF-κB-mediated transcription. Furthermore, human cells expressing type II GRA15 also activate NF-κB, demonstrating that GRA15 alone is sufficient for NF-κB activation. Along with the rhoptry protein ROP16, GRA15 is responsible for a large part of the strain differences in the induction of IL-12 secretion by infected mouse macrophages. In vivo bioluminescent imaging showed that a GRA15-deficient type II strain grows faster compared with wild-type, most likely through its reduced induction of IFN-γ. These results show for the first time that a dense granule protein can modulate host signaling pathways, and dense granule proteins can therefore join rhoptry proteins in T. gondii's host cell-modifying arsenal.
PMID: 21199955 [PubMed - as supplied by publisher]
Strain-specific activation of the NF-{kappa}B pathway by GRA15, a novel Toxoplasma gondii dense granule protein
Rosowski EE, Lu D, Julien L, Rodda L, Gaiser RA, Jensen KD, Saeij JP.
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.
Abstract
NF-κB is an integral component of the immune response to Toxoplasma gondii. Although evidence exists that T. gondii can directly modulate the NF-κB pathway, the parasite-derived effectors involved are unknown. We determined that type II strains of T. gondii activate more NF-κB than type I or type III strains, and using forward genetics we found that this difference is a result of the polymorphic protein GRA15, a novel dense granule protein which T. gondii secretes into the host cell upon invasion. A GRA15-deficient type II strain has a severe defect in both NF-κB nuclear translocation and NF-κB-mediated transcription. Furthermore, human cells expressing type II GRA15 also activate NF-κB, demonstrating that GRA15 alone is sufficient for NF-κB activation. Along with the rhoptry protein ROP16, GRA15 is responsible for a large part of the strain differences in the induction of IL-12 secretion by infected mouse macrophages. In vivo bioluminescent imaging showed that a GRA15-deficient type II strain grows faster compared with wild-type, most likely through its reduced induction of IFN-γ. These results show for the first time that a dense granule protein can modulate host signaling pathways, and dense granule proteins can therefore join rhoptry proteins in T. gondii's host cell-modifying arsenal.
PMID: 21199955 [PubMed - as supplied by publisher]
Phosphorylation of Mouse Immunity-Related GTPase (IRG) Resistance Proteins Is an Evasion Strategy for Virulent Toxoplasma gondii
PLoS Biol. 2010 Dec 21;8(12):e1000576.
Phosphorylation of Mouse Immunity-Related GTPase (IRG) Resistance Proteins Is an Evasion Strategy for Virulent Toxoplasma gondii
Steinfeldt T, Könen-Waisman S, Tong L, Pawlowski N, Lamkemeyer T, Sibley LD, Hunn JP, Howard JC.
Institute for Genetics, University of Cologne, Cologne, Germany.
Abstract
Virulence of complex pathogens in mammals is generally determined by multiple components of the pathogen interacting with the functional complexity and multiple layering of the mammalian immune system. It is most unusual for the resistance of a mammalian host to be overcome by the defeat of a single defence mechanism. In this study we uncover and analyse just such a case at the molecular level, involving the widespread intracellular protozoan pathogen Toxoplasma gondii and one of its most important natural hosts, the house mouse (Mus musculus). Natural polymorphism in virulence of Eurasian T. gondii strains for mice has been correlated in genetic screens with the expression of polymorphic rhoptry kinases (ROP kinases) secreted into the host cell during infection. We show that the molecular targets of the virulent allelic form of ROP18 kinase are members of a family of cellular GTPases, the interferon-inducible IRG (immunity-related GTPase) proteins, known from earlier work to be essential resistance factors in mice against avirulent strains of T. gondii. Virulent T. gondii strain ROP18 kinase phosphorylates several mouse IRG proteins. We show that the parasite kinase phosphorylates host Irga6 at two threonines in the nucleotide-binding domain, biochemically inactivating the GTPase and inhibiting its accumulation and action at the T. gondii parasitophorous vacuole membrane. Our analysis identifies the conformationally active switch I region of the GTP-binding site as an Achilles' heel of the IRG protein pathogen-resistance mechanism. The polymorphism of ROP18 in natural T. gondii populations indicates the existence of a dynamic, rapidly evolving ecological relationship between parasite virulence factors and host resistance factors. This system should be unusually fruitful for analysis at both ecological and molecular levels since both T. gondii and the mouse are widespread and abundant in the wild and are well-established model species with excellent analytical tools available.
PMID: 21203588 [PubMed - in process]
Phosphorylation of Mouse Immunity-Related GTPase (IRG) Resistance Proteins Is an Evasion Strategy for Virulent Toxoplasma gondii
Steinfeldt T, Könen-Waisman S, Tong L, Pawlowski N, Lamkemeyer T, Sibley LD, Hunn JP, Howard JC.
Institute for Genetics, University of Cologne, Cologne, Germany.
Abstract
Virulence of complex pathogens in mammals is generally determined by multiple components of the pathogen interacting with the functional complexity and multiple layering of the mammalian immune system. It is most unusual for the resistance of a mammalian host to be overcome by the defeat of a single defence mechanism. In this study we uncover and analyse just such a case at the molecular level, involving the widespread intracellular protozoan pathogen Toxoplasma gondii and one of its most important natural hosts, the house mouse (Mus musculus). Natural polymorphism in virulence of Eurasian T. gondii strains for mice has been correlated in genetic screens with the expression of polymorphic rhoptry kinases (ROP kinases) secreted into the host cell during infection. We show that the molecular targets of the virulent allelic form of ROP18 kinase are members of a family of cellular GTPases, the interferon-inducible IRG (immunity-related GTPase) proteins, known from earlier work to be essential resistance factors in mice against avirulent strains of T. gondii. Virulent T. gondii strain ROP18 kinase phosphorylates several mouse IRG proteins. We show that the parasite kinase phosphorylates host Irga6 at two threonines in the nucleotide-binding domain, biochemically inactivating the GTPase and inhibiting its accumulation and action at the T. gondii parasitophorous vacuole membrane. Our analysis identifies the conformationally active switch I region of the GTP-binding site as an Achilles' heel of the IRG protein pathogen-resistance mechanism. The polymorphism of ROP18 in natural T. gondii populations indicates the existence of a dynamic, rapidly evolving ecological relationship between parasite virulence factors and host resistance factors. This system should be unusually fruitful for analysis at both ecological and molecular levels since both T. gondii and the mouse are widespread and abundant in the wild and are well-established model species with excellent analytical tools available.
PMID: 21203588 [PubMed - in process]
TgVTC2 is Involved in Polyphosphate Accumulation in Toxoplasma gondii
Mol Biochem Parasitol. 2010 Dec 29. [Epub ahead of print]
TgVTC2 is Involved in Polyphosphate Accumulation in Toxoplasma gondii
Rooney PJ, Ayong L, Tobin CM, Moreno SN, Knoll LJ.
Department of Medical Microbiology and Immunology, University of Wisconsin School of Medicine and Public Health, Microbial Sciences Building Room 3345, 1550 Linden Drive, Madison, WI 53706, United States.
Abstract
Polyphosphate is found in every cell, having roles in diverse processes, including differentiation and response to stress. In this study, we characterize a Toxoplasma gondii mutant containing an insertion within the carboxy-terminal end of a homolog of Saccharomyces cerevisiae Vtc2p, a component of the polyphosphate synthetic machinery. Locus TgVTC2 encodes a 140kDa protein containing conserved SPX, VTC and transmembrane domains. TgVTC2 localizes in punctate spots within the cytoplasm that do not co-localize with known markers. The TgVTC2 mutant showed dramatically reduced polyphosphate accumulation, a defect restored by introduction of TgVTC2 to the mutant. Insertion within TgVTC2 resulted in increased transcript levels for two loci, including a putative FIKK kinase. These transcript levels were restored to wild-type levels upon complementation with the TgVTC2 locus. The TgVTC2 locus was refractory to knockout, and may be essential. Analysis of this TgVTC2 mutant will facilitate dissection of the T. gondii polyphosphate synthesis pathway.
Copyright © 2010. Published by Elsevier B.V.
PMID: 21195114 [PubMed - as supplied by publisher]
TgVTC2 is Involved in Polyphosphate Accumulation in Toxoplasma gondii
Rooney PJ, Ayong L, Tobin CM, Moreno SN, Knoll LJ.
Department of Medical Microbiology and Immunology, University of Wisconsin School of Medicine and Public Health, Microbial Sciences Building Room 3345, 1550 Linden Drive, Madison, WI 53706, United States.
Abstract
Polyphosphate is found in every cell, having roles in diverse processes, including differentiation and response to stress. In this study, we characterize a Toxoplasma gondii mutant containing an insertion within the carboxy-terminal end of a homolog of Saccharomyces cerevisiae Vtc2p, a component of the polyphosphate synthetic machinery. Locus TgVTC2 encodes a 140kDa protein containing conserved SPX, VTC and transmembrane domains. TgVTC2 localizes in punctate spots within the cytoplasm that do not co-localize with known markers. The TgVTC2 mutant showed dramatically reduced polyphosphate accumulation, a defect restored by introduction of TgVTC2 to the mutant. Insertion within TgVTC2 resulted in increased transcript levels for two loci, including a putative FIKK kinase. These transcript levels were restored to wild-type levels upon complementation with the TgVTC2 locus. The TgVTC2 locus was refractory to knockout, and may be essential. Analysis of this TgVTC2 mutant will facilitate dissection of the T. gondii polyphosphate synthesis pathway.
Copyright © 2010. Published by Elsevier B.V.
PMID: 21195114 [PubMed - as supplied by publisher]
Foodborne illness acquired in the United States-major pathogens
Emerg Infect Dis. 2011 Jan;17(1):7-15.
Foodborne illness acquired in the United States-major pathogens
Scallan E, Hoekstra RM, Angulo FJ, Tauxe RV, Widdowson MA, Roy SL, Jones JL, Griffin PM.
Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
Abstract
Estimates of foodborne illness can be used to direct food safety policy and interventions. We used data from active and passive surveillance and other sources to estimate that each year 31 major pathogens acquired in the United States caused 9.4 million episodes of foodborne illness (90% credible interval [CrI] 6.6-12.7 million), 55,961 hospitalizations (90% CrI 39,534-75,741), and 1,351 deaths (90% CrI 712-2,268). Most (58%) illnesses were caused by norovirus, followed by nontyphoidal Salmonella spp. (11%), Clostridium perfringens (10%), and Campylobacter spp. (9%). Leading causes of hospitalization were nontyphoidal Salmonella spp. (35%), norovirus (26%), Campylobacter spp. (15%), and Toxoplasma gondii (8%). Leading causes of death were nontyphoidal Salmonella spp. (28%), T. gondii (24%), Listeria monocytogenes (19%), and norovirus (11%). These estimates cannot be compared with prior (1999) estimates to assess trends because different methods were used. Additional data and more refined methods can improve future estimates.
PMID: 21192848 [PubMed - in process]
Foodborne illness acquired in the United States-major pathogens
Scallan E, Hoekstra RM, Angulo FJ, Tauxe RV, Widdowson MA, Roy SL, Jones JL, Griffin PM.
Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
Abstract
Estimates of foodborne illness can be used to direct food safety policy and interventions. We used data from active and passive surveillance and other sources to estimate that each year 31 major pathogens acquired in the United States caused 9.4 million episodes of foodborne illness (90% credible interval [CrI] 6.6-12.7 million), 55,961 hospitalizations (90% CrI 39,534-75,741), and 1,351 deaths (90% CrI 712-2,268). Most (58%) illnesses were caused by norovirus, followed by nontyphoidal Salmonella spp. (11%), Clostridium perfringens (10%), and Campylobacter spp. (9%). Leading causes of hospitalization were nontyphoidal Salmonella spp. (35%), norovirus (26%), Campylobacter spp. (15%), and Toxoplasma gondii (8%). Leading causes of death were nontyphoidal Salmonella spp. (28%), T. gondii (24%), Listeria monocytogenes (19%), and norovirus (11%). These estimates cannot be compared with prior (1999) estimates to assess trends because different methods were used. Additional data and more refined methods can improve future estimates.
PMID: 21192848 [PubMed - in process]
Self-Mating in the Definitive Host Potentiates Clonal Outbreaks of the Apicomplexan Parasites Sarcocystis neurona and Toxoplasma gondii
PLoS Genet. 2010 Dec 23;6(12):e1001261.
Self-Mating in the Definitive Host Potentiates Clonal Outbreaks of the Apicomplexan Parasites Sarcocystis neurona and Toxoplasma gondii
Wendte JM, Miller MA, Lambourn DM, Magargal SL, Jessup DA, Grigg ME.
Molecular Parasitology Unit, Laboratory of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
Abstract
Tissue-encysting coccidia, including Toxoplasma gondii and Sarcocystis neurona, are heterogamous parasites with sexual and asexual life stages in definitive and intermediate hosts, respectively. During its sexual life stage, T. gondii reproduces either by genetic out-crossing or via clonal amplification of a single strain through self-mating. Out-crossing has been experimentally verified as a potent mechanism capable of producing offspring possessing a range of adaptive and virulence potentials. In contrast, selfing and other life history traits, such as asexual expansion of tissue-cysts by oral transmission among intermediate hosts, have been proposed to explain the genetic basis for the clonal population structure of T. gondii. In this study, we investigated the contributing roles self-mating and sexual recombination play in nature to maintain clonal population structures and produce or expand parasite clones capable of causing disease epidemics for two tissue encysting parasites. We applied high-resolution genotyping against strains isolated from a T. gondii waterborne outbreak that caused symptomatic disease in 155 immune-competent people in Brazil and a S. neurona outbreak that resulted in a mass mortality event in Southern sea otters. In both cases, a single, genetically distinct clone was found infecting outbreak-exposed individuals. Furthermore, the T. gondii outbreak clone was one of several apparently recombinant progeny recovered from the local environment. Since oocysts or sporocysts were the infectious form implicated in each outbreak, the expansion of the epidemic clone can be explained by self-mating. The results also show that out-crossing preceded selfing to produce the virulent T. gondii clone. For the tissue encysting coccidia, self-mating exists as a key adaptation potentiating the epidemic expansion and transmission of newly emerged parasite clones that can profoundly shape parasite population genetic structures or cause devastating disease outbreaks.
PMID: 21203443 [PubMed - in process]
Self-Mating in the Definitive Host Potentiates Clonal Outbreaks of the Apicomplexan Parasites Sarcocystis neurona and Toxoplasma gondii
Wendte JM, Miller MA, Lambourn DM, Magargal SL, Jessup DA, Grigg ME.
Molecular Parasitology Unit, Laboratory of Parasitic Diseases, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
Abstract
Tissue-encysting coccidia, including Toxoplasma gondii and Sarcocystis neurona, are heterogamous parasites with sexual and asexual life stages in definitive and intermediate hosts, respectively. During its sexual life stage, T. gondii reproduces either by genetic out-crossing or via clonal amplification of a single strain through self-mating. Out-crossing has been experimentally verified as a potent mechanism capable of producing offspring possessing a range of adaptive and virulence potentials. In contrast, selfing and other life history traits, such as asexual expansion of tissue-cysts by oral transmission among intermediate hosts, have been proposed to explain the genetic basis for the clonal population structure of T. gondii. In this study, we investigated the contributing roles self-mating and sexual recombination play in nature to maintain clonal population structures and produce or expand parasite clones capable of causing disease epidemics for two tissue encysting parasites. We applied high-resolution genotyping against strains isolated from a T. gondii waterborne outbreak that caused symptomatic disease in 155 immune-competent people in Brazil and a S. neurona outbreak that resulted in a mass mortality event in Southern sea otters. In both cases, a single, genetically distinct clone was found infecting outbreak-exposed individuals. Furthermore, the T. gondii outbreak clone was one of several apparently recombinant progeny recovered from the local environment. Since oocysts or sporocysts were the infectious form implicated in each outbreak, the expansion of the epidemic clone can be explained by self-mating. The results also show that out-crossing preceded selfing to produce the virulent T. gondii clone. For the tissue encysting coccidia, self-mating exists as a key adaptation potentiating the epidemic expansion and transmission of newly emerged parasite clones that can profoundly shape parasite population genetic structures or cause devastating disease outbreaks.
PMID: 21203443 [PubMed - in process]
Wednesday, December 29, 2010
Host Cholesterol Synthesis Contributes to Growth of Intracellular Toxoplasma gondii in Macrophages
J Vet Med Sci. 2010 Dec 24. [Epub ahead of print]
Host Cholesterol Synthesis Contributes to Growth of Intracellular Toxoplasma gondii in Macrophages
Nishikawa Y, Ibrahim HM, Kameyama K, Shiga I, Hiasa J, Xuan X.
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine.
Abstract
The intracellular protozoan Toxoplasma gondii lacks the ability to synthesize sterol and scavenges cholesterol from the low-density lipoprotein receptor (LDLR) pathway of its host to facilitate replication. Sterol biosynthesis inhibitors, however, have a demonstrated anti-Toxoplasma effect. In this study, we examined the host mevalonate pathway as a novel source of cholesterol for T. gondii and its effects on parasite growth in macrophages. Parasite growth did not significantly change in the absence of LDLR or when LDL was exogenously supplemented. Lovastatin and compactin, both inhibitors of hydroxymethylglutaryl-CoA (HMG-CoA) reductase in the mevalonate pathway, significantly inhibited T. gondii growth in both wild-type and LDLR-knockout macrophages. Parasite growth was also suppressed by squalestatin, an inhibitor of squalene synthase, despite mevalonate producing isoprenoid intermediates in host cells. The present study demonstrates that lovastatin, compactin and squalestatin have anti-Toxoplasma activities and that the host cholesterol synthesis may contribute to parasite growth in macrophages.
PMID: 21187676
Host Cholesterol Synthesis Contributes to Growth of Intracellular Toxoplasma gondii in Macrophages
Nishikawa Y, Ibrahim HM, Kameyama K, Shiga I, Hiasa J, Xuan X.
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine.
Abstract
The intracellular protozoan Toxoplasma gondii lacks the ability to synthesize sterol and scavenges cholesterol from the low-density lipoprotein receptor (LDLR) pathway of its host to facilitate replication. Sterol biosynthesis inhibitors, however, have a demonstrated anti-Toxoplasma effect. In this study, we examined the host mevalonate pathway as a novel source of cholesterol for T. gondii and its effects on parasite growth in macrophages. Parasite growth did not significantly change in the absence of LDLR or when LDL was exogenously supplemented. Lovastatin and compactin, both inhibitors of hydroxymethylglutaryl-CoA (HMG-CoA) reductase in the mevalonate pathway, significantly inhibited T. gondii growth in both wild-type and LDLR-knockout macrophages. Parasite growth was also suppressed by squalestatin, an inhibitor of squalene synthase, despite mevalonate producing isoprenoid intermediates in host cells. The present study demonstrates that lovastatin, compactin and squalestatin have anti-Toxoplasma activities and that the host cholesterol synthesis may contribute to parasite growth in macrophages.
PMID: 21187676
Tuesday, December 28, 2010
Toxoplasma gondii: humoral and cellular immune response of BALB/c mice immunized via intranasal route with rTgROP2
Rev Bras Parasitol Vet. 2010 Oct-Dec;19(4):210-6.
Toxoplasma gondii: humoral and cellular immune response of BALB/c mice immunized via intranasal route with rTgROP2
Igarashi M, Zulpo DL, Cunha IA, Barros LD, Pereira VF, Taroda A, Navarro I, Vidotto O, Vidotto MC, Jenkins MC, Garcia JL.
Laboratório de Protozoologia, Departamento de Medicina Veterinária Preventiva, Universidade de Londrina - UEL, CP 6001, CEP 86050-970Londrina - PR, Brazil. jlgarcia@uel.br.
Abstract
TgROP2 is an intracellular protein associated with rhoptries of Toxoplama gondii and an antigen component of a candidate vaccine for toxoplasmosis. The purpose of the present study was to evaluate the efficacy of rTgROP2 to stimulate humoral and cellular immune responses in BALB/c mice via intranasal injection. TgROP2 partial coding sequence was (196-561) amplified by PCR from genomic T. gondii RH strain DNA and cloned into the pTrcHis expression vector. Escherichia coli Rosetta 2 cells transformed with pTrcHis-TgROP2 showed high levels (~1 mg.mL(-1)) of recombinant protein after 4 hours of IPTG induction. Recombinant TgROP2 exhibited an apparent Mr equal to 54 kDa. In order to test immunogenicity of the recombinant protein, 10 BALB/c mice received 10 µg of rROP2 protein + 10 µg of Quil-A via intranasal injection. Doses were administered at days 0, 21, and 42. Three animals were euthanized and used to evaluate cell-ular immune response on day 62. Five (50%) and two (20%) out of ten animals produced IgG (DO mean = 0.307; cut-off = 0.240) and IgA (DO mean = 0.133, cut-off = 0.101), respectively, by ELISA on day 62. The proliferation of splenocytes revealed high stimulation index (SI) when co-cultured with 5, 10 and 15 µg.mL(-1) of rTgROP2. These results indicate that intranasal immunization with recombinant protein ROP2 plus Quil-A can elicit both cellular and humoral immune responses in BALB/c mice.
PMID: 21184696
Toxoplasma gondii: humoral and cellular immune response of BALB/c mice immunized via intranasal route with rTgROP2
Igarashi M, Zulpo DL, Cunha IA, Barros LD, Pereira VF, Taroda A, Navarro I, Vidotto O, Vidotto MC, Jenkins MC, Garcia JL.
Laboratório de Protozoologia, Departamento de Medicina Veterinária Preventiva, Universidade de Londrina - UEL, CP 6001, CEP 86050-970Londrina - PR, Brazil. jlgarcia@uel.br.
Abstract
TgROP2 is an intracellular protein associated with rhoptries of Toxoplama gondii and an antigen component of a candidate vaccine for toxoplasmosis. The purpose of the present study was to evaluate the efficacy of rTgROP2 to stimulate humoral and cellular immune responses in BALB/c mice via intranasal injection. TgROP2 partial coding sequence was (196-561) amplified by PCR from genomic T. gondii RH strain DNA and cloned into the pTrcHis expression vector. Escherichia coli Rosetta 2 cells transformed with pTrcHis-TgROP2 showed high levels (~1 mg.mL(-1)) of recombinant protein after 4 hours of IPTG induction. Recombinant TgROP2 exhibited an apparent Mr equal to 54 kDa. In order to test immunogenicity of the recombinant protein, 10 BALB/c mice received 10 µg of rROP2 protein + 10 µg of Quil-A via intranasal injection. Doses were administered at days 0, 21, and 42. Three animals were euthanized and used to evaluate cell-ular immune response on day 62. Five (50%) and two (20%) out of ten animals produced IgG (DO mean = 0.307; cut-off = 0.240) and IgA (DO mean = 0.133, cut-off = 0.101), respectively, by ELISA on day 62. The proliferation of splenocytes revealed high stimulation index (SI) when co-cultured with 5, 10 and 15 µg.mL(-1) of rTgROP2. These results indicate that intranasal immunization with recombinant protein ROP2 plus Quil-A can elicit both cellular and humoral immune responses in BALB/c mice.
PMID: 21184696
High level of soluble HLA-G in amniotic fluid is correlated with congenital transmission of Toxoplasma gondii
Clin Immunol. 2010 Dec 23. [Epub ahead of print]
High level of soluble HLA-G in amniotic fluid is correlated with congenital transmission of Toxoplasma gondii
Robert-Gangneux F, Gangneux JP, Vu N, Jaillard S, Guiguen C, Amiot L.
Laboratoire de Parasitologie, Faculté de Médecine et Centre Hospitalier Universitaire de Rennes, Rennes, France; Unité EA4427-SERAIC (Signalisation et réponse aux agents infectieux et chimiques), IRSET (Institut de Recherche en Santé Environnement Travail), Université Rennes 1, Rennes, France.
Abstract
The expression of human leukocyte antigen (HLA)-G on cytotrophoblast cells contributes to maternal-fetal tolerance. Soluble forms of HLA-G (sHLA-G) can be detected in amniotic fluid (AF) and a decrease of sHLA-G is known to be correlated to fetal loss. In this work we investigated the role of sHLA-G in the transplacental passage of the protozoan parasite Toxoplasma gondii, responsible for congenital toxoplasmosis in about 30% of fetuses when primary infection (PI) occurs during pregnancy. We determined the sHLA-G concentration in 61 AF from women with PI and 24 controls. Our results showed higher sHLA-G levels in AF from PI than in controls (p<0.001). Moreover sHLA-G level from congenitally infected fetuses (n=12) was higher than in fetus in whom congenital infection was ruled out (n=49, p<0.05). These data suggest that sHLA-G could participate in immunomodulation necessary to avoid fetal loss due to Toxoplasma infection, but that over-expression could favor congenital transmission.
Copyright © 2010. Published by Elsevier Inc.
PMID: 21185786
High level of soluble HLA-G in amniotic fluid is correlated with congenital transmission of Toxoplasma gondii
Robert-Gangneux F, Gangneux JP, Vu N, Jaillard S, Guiguen C, Amiot L.
Laboratoire de Parasitologie, Faculté de Médecine et Centre Hospitalier Universitaire de Rennes, Rennes, France; Unité EA4427-SERAIC (Signalisation et réponse aux agents infectieux et chimiques), IRSET (Institut de Recherche en Santé Environnement Travail), Université Rennes 1, Rennes, France.
Abstract
The expression of human leukocyte antigen (HLA)-G on cytotrophoblast cells contributes to maternal-fetal tolerance. Soluble forms of HLA-G (sHLA-G) can be detected in amniotic fluid (AF) and a decrease of sHLA-G is known to be correlated to fetal loss. In this work we investigated the role of sHLA-G in the transplacental passage of the protozoan parasite Toxoplasma gondii, responsible for congenital toxoplasmosis in about 30% of fetuses when primary infection (PI) occurs during pregnancy. We determined the sHLA-G concentration in 61 AF from women with PI and 24 controls. Our results showed higher sHLA-G levels in AF from PI than in controls (p<0.001). Moreover sHLA-G level from congenitally infected fetuses (n=12) was higher than in fetus in whom congenital infection was ruled out (n=49, p<0.05). These data suggest that sHLA-G could participate in immunomodulation necessary to avoid fetal loss due to Toxoplasma infection, but that over-expression could favor congenital transmission.
Copyright © 2010. Published by Elsevier Inc.
PMID: 21185786
Saturday, December 25, 2010
Toxoplasma gondii Lysine Acetyltransferase GCN5-A Functions in the Cellular Response to Alkaline Stress and Expression of Cyst Genes
PLoS Pathog. 2010 Dec 16;6(12):e1001232.
Toxoplasma gondii Lysine Acetyltransferase GCN5-A Functions in the Cellular Response to Alkaline Stress and Expression of Cyst Genes
Naguleswaran A, Elias EV, McClintick J, Edenberg HJ, Sullivan WJ.
Department of Pharmacology & Toxicology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
Abstract
Parasitic protozoa such as the apicomplexan Toxoplasma gondii progress through their life cycle in response to stimuli in the environment or host organism. Very little is known about how proliferating tachyzoites reprogram their expressed genome in response to stresses that prompt development into latent bradyzoite cysts. We have previously linked histone acetylation with the expression of stage-specific genes, but the factors involved remain to be determined. We sought to determine if GCN5, which operates as a transcriptional co-activator by virtue of its histone acetyltransferase (HAT) activity, contributed to stress-induced changes in gene expression in Toxoplasma. In contrast to other lower eukaryotes, Toxoplasma has duplicated its GCN5 lysine acetyltransferase (KAT). Disruption of the gene encoding for TgGCN5-A in type I RH strain did not produce a severe phenotype under normal culture conditions, but here we show that the TgGCN5-A null mutant is deficient in responding to alkaline pH, a common stress used to induce bradyzoite differentiation in vitro. We performed a genome-wide analysis of the Toxoplasma transcriptional response to alkaline pH stress, finding that parasites deleted for TgGCN5-A fail to up-regulate 74% of the stress response genes that are induced 2-fold or more in wild-type. Using chromatin immunoprecipitation, we verify an enrichment of TgGCN5-A at the upstream regions of genes activated by alkaline pH exposure. The TgGCN5-A knockout is also incapable of up-regulating key marker genes expressed during development of the latent cyst form, and is impaired in its ability to recover from alkaline stress. Complementation of the TgGCN5-A knockout restores the expression of these stress-induced genes and reverses the stress recovery defect. These results establish TgGCN5-A as a major contributor to the alkaline stress response in RH strain Toxoplasma.
PMID: 21179246
Toxoplasma gondii Lysine Acetyltransferase GCN5-A Functions in the Cellular Response to Alkaline Stress and Expression of Cyst Genes
Naguleswaran A, Elias EV, McClintick J, Edenberg HJ, Sullivan WJ.
Department of Pharmacology & Toxicology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
Abstract
Parasitic protozoa such as the apicomplexan Toxoplasma gondii progress through their life cycle in response to stimuli in the environment or host organism. Very little is known about how proliferating tachyzoites reprogram their expressed genome in response to stresses that prompt development into latent bradyzoite cysts. We have previously linked histone acetylation with the expression of stage-specific genes, but the factors involved remain to be determined. We sought to determine if GCN5, which operates as a transcriptional co-activator by virtue of its histone acetyltransferase (HAT) activity, contributed to stress-induced changes in gene expression in Toxoplasma. In contrast to other lower eukaryotes, Toxoplasma has duplicated its GCN5 lysine acetyltransferase (KAT). Disruption of the gene encoding for TgGCN5-A in type I RH strain did not produce a severe phenotype under normal culture conditions, but here we show that the TgGCN5-A null mutant is deficient in responding to alkaline pH, a common stress used to induce bradyzoite differentiation in vitro. We performed a genome-wide analysis of the Toxoplasma transcriptional response to alkaline pH stress, finding that parasites deleted for TgGCN5-A fail to up-regulate 74% of the stress response genes that are induced 2-fold or more in wild-type. Using chromatin immunoprecipitation, we verify an enrichment of TgGCN5-A at the upstream regions of genes activated by alkaline pH exposure. The TgGCN5-A knockout is also incapable of up-regulating key marker genes expressed during development of the latent cyst form, and is impaired in its ability to recover from alkaline stress. Complementation of the TgGCN5-A knockout restores the expression of these stress-induced genes and reverses the stress recovery defect. These results establish TgGCN5-A as a major contributor to the alkaline stress response in RH strain Toxoplasma.
PMID: 21179246
Evaluation of three novel azasterols against Toxoplasma gondii
Vet Parasitol. 2010 Dec 1. [Epub ahead of print]
Evaluation of three novel azasterols against Toxoplasma gondii
Martins-Duarte ES, Lemgruber L, Lorente SO, Gros L, Magaraci F, Gilbert IH, de Souza W, Vommaro RC.
Laboratório de Ultraestrutura Celular Herth, Meyer, Instituto de Biofísica Carlos Chagas Filho, UFRJ, CCS, Bloco G, Av. Carlo, Chagas Filho, Cidade Universitária, Ilha do Fundão, 21941-902, Rio de Janeiro, RJ, Brazil; Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Rio de Janeiro, Brazil.
Abstract
Previous studies from our group have demonstrated the high susceptibility of Toxoplasma gondii tachyzoites to the sterol analogues 22,26-azasterol and 24,25-(R,S)-epiminolanosterol. In this work we present data on testing in vitro three novel azasterols as potential agents for the treatment of toxoplasmosis. The three compounds inhibited parasite growth at micromolar concentrations, in a dose-dependent manner. Electron microscopy analysis of intracellular tachyzoites after treatment with the most effective compound showed drastic mitochondrion swelling associated with the appearance of an electron-lucent matrix and disrupted cristae. Parasite lysis also took place. The appearance of electron dense cytoplasmic structures similar to amylopectin granules distributed throughout the parasite suggests that azasterols might be inducing differentiation of those tachyzoites which were not lysed to the bradyzoite stage.
Copyright © 2010 Elsevier B.V. All rights reserved.
PMID: 21176865
Evaluation of three novel azasterols against Toxoplasma gondii
Martins-Duarte ES, Lemgruber L, Lorente SO, Gros L, Magaraci F, Gilbert IH, de Souza W, Vommaro RC.
Laboratório de Ultraestrutura Celular Herth, Meyer, Instituto de Biofísica Carlos Chagas Filho, UFRJ, CCS, Bloco G, Av. Carlo, Chagas Filho, Cidade Universitária, Ilha do Fundão, 21941-902, Rio de Janeiro, RJ, Brazil; Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Rio de Janeiro, Brazil.
Abstract
Previous studies from our group have demonstrated the high susceptibility of Toxoplasma gondii tachyzoites to the sterol analogues 22,26-azasterol and 24,25-(R,S)-epiminolanosterol. In this work we present data on testing in vitro three novel azasterols as potential agents for the treatment of toxoplasmosis. The three compounds inhibited parasite growth at micromolar concentrations, in a dose-dependent manner. Electron microscopy analysis of intracellular tachyzoites after treatment with the most effective compound showed drastic mitochondrion swelling associated with the appearance of an electron-lucent matrix and disrupted cristae. Parasite lysis also took place. The appearance of electron dense cytoplasmic structures similar to amylopectin granules distributed throughout the parasite suggests that azasterols might be inducing differentiation of those tachyzoites which were not lysed to the bradyzoite stage.
Copyright © 2010 Elsevier B.V. All rights reserved.
PMID: 21176865
Tuesday, December 21, 2010
Cell cycle-dependent, intercellular transmission of Toxoplasma gondii is accompanied by marked changes in parasite gene expression
Mol Microbiol. 2011 Jan;79(1):192-204. doi: 10.1111/j.1365-2958.2010.07441.x. Epub 2010 Nov 9.
Cell cycle-dependent, intercellular transmission of Toxoplasma gondii is accompanied by marked changes in parasite gene expression
Gaji RY, Behnke MS, Lehmann MM, White MW, Carruthers VB.
Department of Microbiology and Immunology, University of Michigan Medical School, 1150 W. Medical Center Dr., Ann Arbor, MI 48109, USA Department of Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717, USA Departments of Molecular Medicine & Global Health, University of South Florida, Tampa, FL 33612, USA.
Abstract
Intracellular microbes have evolved efficient strategies for transitioning from one cell to another in a process termed intercellular transmission. Here we show that host cell transmission of the obligate intracellular parasite Toxoplasma gondii is closely tied to specific cell cycle distributions, with egress and reinvasion occurring most proficiently by parasites in the G1 phase. We also reveal that Toxoplasma undergoes marked changes in mRNA expression when transitioning from the extracellular environment to its intracellular niche. These mRNA level changes reflect a modal switch from expression of proteins involved in invasion, motility and signal transduction in extracellular parasites to expression of metabolic and DNA replication proteins in intracellular parasites. Host cell binding and signalling associated with the discharge of parasite secretory proteins was not sufficient to induce this switch in gene expression, suggesting that the regulatory mechanisms responsible are tied to the establishment of the intracellular environment. The genes whose expression increased after parasite invasion belong to a progressive cascade known to underlie the parasite division cycle indicating that the unique relationship between the G1 phase and invasion effectively synchronizes short-term population growth. This work provides new insight into how this highly successful parasite competently transits from cell to cell.
© 2010 Blackwell Publishing Ltd.
PMID: 21166903
Cell cycle-dependent, intercellular transmission of Toxoplasma gondii is accompanied by marked changes in parasite gene expression
Gaji RY, Behnke MS, Lehmann MM, White MW, Carruthers VB.
Department of Microbiology and Immunology, University of Michigan Medical School, 1150 W. Medical Center Dr., Ann Arbor, MI 48109, USA Department of Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717, USA Departments of Molecular Medicine & Global Health, University of South Florida, Tampa, FL 33612, USA.
Abstract
Intracellular microbes have evolved efficient strategies for transitioning from one cell to another in a process termed intercellular transmission. Here we show that host cell transmission of the obligate intracellular parasite Toxoplasma gondii is closely tied to specific cell cycle distributions, with egress and reinvasion occurring most proficiently by parasites in the G1 phase. We also reveal that Toxoplasma undergoes marked changes in mRNA expression when transitioning from the extracellular environment to its intracellular niche. These mRNA level changes reflect a modal switch from expression of proteins involved in invasion, motility and signal transduction in extracellular parasites to expression of metabolic and DNA replication proteins in intracellular parasites. Host cell binding and signalling associated with the discharge of parasite secretory proteins was not sufficient to induce this switch in gene expression, suggesting that the regulatory mechanisms responsible are tied to the establishment of the intracellular environment. The genes whose expression increased after parasite invasion belong to a progressive cascade known to underlie the parasite division cycle indicating that the unique relationship between the G1 phase and invasion effectively synchronizes short-term population growth. This work provides new insight into how this highly successful parasite competently transits from cell to cell.
© 2010 Blackwell Publishing Ltd.
PMID: 21166903
Saturday, December 18, 2010
The apicoplast
Protoplasma. 2010 Dec 17. [Epub ahead of print]
The apicoplast
McFadden GI.
School of Botany, University of Melbourne, Melbourne, VIC, 3010, Australia, gim@unimelb.edu.au.
Abstract
Parasites like malaria and Toxoplasma possess a vestigial plastid homologous to the chloroplasts of plants. The plastid (known as the apicoplast) is non-photosynthetic but retains many hallmarks of its ancestry including a circular genome that it synthesises proteins from and a suite of biosynthetic pathways of cyanobacterial origin. In this review, the discovery of the apicoplast and its integration, function and purpose are explored. New insights into the apicoplast fatty acid biosynthesis pathway and some novel roles of the apicoplast in vaccine development are reviewed.
PMID: 21165662
The apicoplast
McFadden GI.
School of Botany, University of Melbourne, Melbourne, VIC, 3010, Australia, gim@unimelb.edu.au.
Abstract
Parasites like malaria and Toxoplasma possess a vestigial plastid homologous to the chloroplasts of plants. The plastid (known as the apicoplast) is non-photosynthetic but retains many hallmarks of its ancestry including a circular genome that it synthesises proteins from and a suite of biosynthetic pathways of cyanobacterial origin. In this review, the discovery of the apicoplast and its integration, function and purpose are explored. New insights into the apicoplast fatty acid biosynthesis pathway and some novel roles of the apicoplast in vaccine development are reviewed.
PMID: 21165662
Thursday, December 16, 2010
Post-genomics resources and tools for studying apicomplexan metabolism
Trends Parasitol. 2010 Dec 8. [Epub ahead of print]
Post-genomics resources and tools for studying apicomplexan metabolism
Hung SS, Parkinson J.
Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, Ontario, Canada and Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
Abstract
The phylum Apicomplexa comprises over 5000 species of obligate intracellular parasites, many responsible for diseases that significantly impact human health and economics. To aid drug development programs, global sequencing initiatives are generating increasing numbers of apicomplexan genomes. The challenge is how best to exploit these resources to identify effective therapeutic targets. Because of its important role in growth and maintenance, much interest has centred on metabolism. However, in the absence of detailed biochemical data, reconstructing the metabolic potential from a fully sequenced genome remains problematic. In this review current resources and tools facilitating the metabolic reconstruction for apicomplexans are examined. Furthermore, how these datasets can be utilized to explore the metabolic capabilities of apicomplexans are discussed and targets for therapeutic intervention are prioritized.
Copyright © 2010 Elsevier Ltd. All rights reserved.
PMID: 21145790 [PubMed - as supplied by publisher]
Post-genomics resources and tools for studying apicomplexan metabolism
Hung SS, Parkinson J.
Program in Molecular Structure and Function, Hospital for Sick Children, Toronto, Ontario, Canada and Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
Abstract
The phylum Apicomplexa comprises over 5000 species of obligate intracellular parasites, many responsible for diseases that significantly impact human health and economics. To aid drug development programs, global sequencing initiatives are generating increasing numbers of apicomplexan genomes. The challenge is how best to exploit these resources to identify effective therapeutic targets. Because of its important role in growth and maintenance, much interest has centred on metabolism. However, in the absence of detailed biochemical data, reconstructing the metabolic potential from a fully sequenced genome remains problematic. In this review current resources and tools facilitating the metabolic reconstruction for apicomplexans are examined. Furthermore, how these datasets can be utilized to explore the metabolic capabilities of apicomplexans are discussed and targets for therapeutic intervention are prioritized.
Copyright © 2010 Elsevier Ltd. All rights reserved.
PMID: 21145790 [PubMed - as supplied by publisher]
Unusual anchor of a motor complex (MyoD-MLC2) to the plasma membrane of Toxoplasma gondii
Traffic. 2010 Dec 9. doi: 10.1111/j.1600-0854.2010.01148.x. [Epub ahead of print]
Unusual anchor of a motor complex (MyoD-MLC2) to the plasma membrane of Toxoplasma gondii
Polonais V, Foth BJ, Chinthalapudi K, Marq JB, Manstein DJ, Soldati-Favre D, Frénal K.
Department of Microbiology and Molecular Medicine, CMU, University of Geneva, 1 Rue Michel-Servet, CH-1211 Geneva 4, Switzerland Research Division for Structural Analysis, Hannover Medical School, OE8830, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany Institute für Biophysical Chemistry, Hannover Medical School, OE4350, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany.
Abstract
Toxoplasma gondii possesses 11 rather atypical myosin heavy chains. The only myosin light chain described to date is MLC1, associated to myosin A, and contributing to gliding motility. In this study, we examined the repertoire of calmodulin-like proteins in Apicomplexans, identified six putative myosin light chains and determined their subcellular localization in T. gondii and Plasmodium falciparum. MLC2, only found in coccidians, is associated to myosin D via its CaM-like domain and anchored to the plasma membrane of T. gondii via its N-terminal extension. Molecular modeling suggests that MyoD-MLC2 complex is more compact than the reported structure of Plasmodium MyoA-MTIP complex. Anchorage of this MLC2 to the plasma membrane is likely governed by palmitoylation.
© 2010 John Wiley & Sons A/S.
PMID: 21143563 [PubMed - as supplied by publisher]
Unusual anchor of a motor complex (MyoD-MLC2) to the plasma membrane of Toxoplasma gondii
Polonais V, Foth BJ, Chinthalapudi K, Marq JB, Manstein DJ, Soldati-Favre D, Frénal K.
Department of Microbiology and Molecular Medicine, CMU, University of Geneva, 1 Rue Michel-Servet, CH-1211 Geneva 4, Switzerland Research Division for Structural Analysis, Hannover Medical School, OE8830, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany Institute für Biophysical Chemistry, Hannover Medical School, OE4350, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany.
Abstract
Toxoplasma gondii possesses 11 rather atypical myosin heavy chains. The only myosin light chain described to date is MLC1, associated to myosin A, and contributing to gliding motility. In this study, we examined the repertoire of calmodulin-like proteins in Apicomplexans, identified six putative myosin light chains and determined their subcellular localization in T. gondii and Plasmodium falciparum. MLC2, only found in coccidians, is associated to myosin D via its CaM-like domain and anchored to the plasma membrane of T. gondii via its N-terminal extension. Molecular modeling suggests that MyoD-MLC2 complex is more compact than the reported structure of Plasmodium MyoA-MTIP complex. Anchorage of this MLC2 to the plasma membrane is likely governed by palmitoylation.
© 2010 John Wiley & Sons A/S.
PMID: 21143563 [PubMed - as supplied by publisher]
Phosphorylation of Immunity-Related GTPases by a Toxoplasma gondii-Secreted Kinase Promotes Macrophage Survival and Virulence
Cell Host Microbe. 2010 Dec 16;8(6):484-95.
Phosphorylation of Immunity-Related GTPases by a Toxoplasma gondii-Secreted Kinase Promotes Macrophage Survival and Virulence
Fentress SJ, Behnke MS, Dunay IR, Mashayekhi M, Rommereim LM, Fox BA, Bzik DJ, Taylor GA, Turk BE, Lichti CF, Townsend RR, Qiu W, Hui R, Beatty WL, Sibley LD.
Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Ave, St. Louis, MO 63130, USA.
Abstract
Macrophages are specialized to detect and destroy intracellular microbes and yet a number of pathogens have evolved to exploit this hostile niche. Here we demonstrate that the obligate intracellular parasite Toxoplasma gondii disarms macrophage innate clearance mechanisms by secreting a serine threonine kinase called ROP18, which binds to and phosphorylates immunity-related GTPases (IRGs). Substrate profiling of ROP18 revealed a preference for a conserved motif within switch region I of the GTPase domain, a modification predicted to disrupt IRG function. Consistent with this, expression of ROP18 was both necessary and sufficient to block recruitment of Irgb6, which was in turn required for parasite destruction. ROP18 phosphorylation of IRGs prevented clearance within inflammatory monocytes and IFN-γ-activated macrophages, conferring parasite survival in vivo and promoting virulence. IRGs are implicated in clearance of a variety of intracellular pathogens, suggesting that other virulence factors may similarly thwart this innate cellular defense mechanism.
Copyright © 2010 Elsevier Inc. All rights reserved.
PMID: 21147463 [PubMed - in process]
Phosphorylation of Immunity-Related GTPases by a Toxoplasma gondii-Secreted Kinase Promotes Macrophage Survival and Virulence
Fentress SJ, Behnke MS, Dunay IR, Mashayekhi M, Rommereim LM, Fox BA, Bzik DJ, Taylor GA, Turk BE, Lichti CF, Townsend RR, Qiu W, Hui R, Beatty WL, Sibley LD.
Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Ave, St. Louis, MO 63130, USA.
Abstract
Macrophages are specialized to detect and destroy intracellular microbes and yet a number of pathogens have evolved to exploit this hostile niche. Here we demonstrate that the obligate intracellular parasite Toxoplasma gondii disarms macrophage innate clearance mechanisms by secreting a serine threonine kinase called ROP18, which binds to and phosphorylates immunity-related GTPases (IRGs). Substrate profiling of ROP18 revealed a preference for a conserved motif within switch region I of the GTPase domain, a modification predicted to disrupt IRG function. Consistent with this, expression of ROP18 was both necessary and sufficient to block recruitment of Irgb6, which was in turn required for parasite destruction. ROP18 phosphorylation of IRGs prevented clearance within inflammatory monocytes and IFN-γ-activated macrophages, conferring parasite survival in vivo and promoting virulence. IRGs are implicated in clearance of a variety of intracellular pathogens, suggesting that other virulence factors may similarly thwart this innate cellular defense mechanism.
Copyright © 2010 Elsevier Inc. All rights reserved.
PMID: 21147463 [PubMed - in process]
Parasites paralyze cellular host defense system to promote virulence
Cell Host Microbe. 2010 Dec 16;8(6):463-4.
Parasites paralyze cellular host defense system to promote virulence
Feng CG, Sher A.
Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Abstract
To promote their survival, intracellular pathogens must confront microbicidal activities induced by interferons. In this issue of Cell Host & Microbe, Fentress et al. show that Toxoplasma gondii evades intracellular killing by deploying a virulence determinant, ROP18, which acts by directly phosphorylating and disabling an IFN-γ-inducible immunity-related GTPase involved in pathogen clearance.
Copyright © 2010 Elsevier Inc. All rights reserved.
PMID: 21147459 [PubMed - in process]
Parasites paralyze cellular host defense system to promote virulence
Feng CG, Sher A.
Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Abstract
To promote their survival, intracellular pathogens must confront microbicidal activities induced by interferons. In this issue of Cell Host & Microbe, Fentress et al. show that Toxoplasma gondii evades intracellular killing by deploying a virulence determinant, ROP18, which acts by directly phosphorylating and disabling an IFN-γ-inducible immunity-related GTPase involved in pathogen clearance.
Copyright © 2010 Elsevier Inc. All rights reserved.
PMID: 21147459 [PubMed - in process]
Acquired toxoplasmosis accompanied by facial nerve palsy in an immunocompetent 5-year-old child
J Child Neurol. 2010 Dec;25(12):1525-8.
Acquired toxoplasmosis accompanied by facial nerve palsy in an immunocompetent 5-year-old child
Galli-Tsinopoulou A, Kyrgios I, Giannopoulou EZ, Gourgoulia S, Maggana I, Katechaki E, Chatzidimitriou D, Evangeliou AE.
Department of Pediatrics, Medical School, Aristotle University, Papageorgiou General Hospital, Thessaloniki, Greece. galtsin@otenet.gr, assimina@med.auth.gr.
Abstract
Acquired toxoplasmosis, although relatively common in children, is usually asymptomatic but can also be clinically manifested by a benign and self-limited infectious mononucleosis-like syndrome. Neurological complications are very rare in immunocompetent children. The authors report a 5-year-old boy who presented with cervical lymphadenopathy because of acquired toxoplasmosis accompanied with unilateral facial nerve paralysis. Toxoplasma gondii DNA detection in blood by polymerase chain reaction, as well as elevated specific immunoglobulin M antibodies against it, established the diagnosis. Characteristic brain lesions on magnetic resonance imaging were absent and ophthalmologic examination revealed no inflammatory lesions in the retina and choroid. Treatment with pyrimethamine, sulfadiazine, and folic acid resulted in a complete recovery after 2 months of therapy. Although rare, acute facial nerve paralysis of unknown origin can be caused by acquired toxoplasmosis even in the immunocompetent pediatric population. Elevated titers of specific antibodies and the presence of parasite's DNA are key findings for the correct diagnosis.
PMID: 21148450 [PubMed - in process]
Acquired toxoplasmosis accompanied by facial nerve palsy in an immunocompetent 5-year-old child
Galli-Tsinopoulou A, Kyrgios I, Giannopoulou EZ, Gourgoulia S, Maggana I, Katechaki E, Chatzidimitriou D, Evangeliou AE.
Department of Pediatrics, Medical School, Aristotle University, Papageorgiou General Hospital, Thessaloniki, Greece. galtsin@otenet.gr, assimina@med.auth.gr.
Abstract
Acquired toxoplasmosis, although relatively common in children, is usually asymptomatic but can also be clinically manifested by a benign and self-limited infectious mononucleosis-like syndrome. Neurological complications are very rare in immunocompetent children. The authors report a 5-year-old boy who presented with cervical lymphadenopathy because of acquired toxoplasmosis accompanied with unilateral facial nerve paralysis. Toxoplasma gondii DNA detection in blood by polymerase chain reaction, as well as elevated specific immunoglobulin M antibodies against it, established the diagnosis. Characteristic brain lesions on magnetic resonance imaging were absent and ophthalmologic examination revealed no inflammatory lesions in the retina and choroid. Treatment with pyrimethamine, sulfadiazine, and folic acid resulted in a complete recovery after 2 months of therapy. Although rare, acute facial nerve paralysis of unknown origin can be caused by acquired toxoplasmosis even in the immunocompetent pediatric population. Elevated titers of specific antibodies and the presence of parasite's DNA are key findings for the correct diagnosis.
PMID: 21148450 [PubMed - in process]
Differential Effects of Three Canonical Toxoplasma Strains on Gene Expression in Human Neuroepithelial Cells
Infect Immun. 2010 Dec 13. [Epub ahead of print]
Differential Effects of Three Canonical Toxoplasma Strains on Gene Expression in Human Neuroepithelial Cells
Xiao J, Jones-Brando L, Talbot CC Jr, Yolken RH.
The Stanley Division of Developmental Neurovirology, Institute for Basic Biomedical Sciences, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Abstract
Strain type is one of the key factors suspected to play a role in determing the outcome of Toxoplasma infection. In this study, we examined the transcriptional profile of human neuroepithelioma cells in response to representative strains of Toxoplasma using microarray analysis to characterize the strain-specific host cell response. The study of neural cells is of interest in light of the ability of Toxoplasma to infect the brain and to establish persistent infection within the central nervous system. We found that the extent of the expression changes varied considerably among the three strains. Neuroepithelial cells infected with Toxoplasma type I exhibited the highest level of differential gene expression, whereas type II infected cells had a substantially smaller number of genes which were differentially expressed. Cells infected with type III exhibited intermediate effects on gene expression. The three strains also differed in the individual genes and gene pathways which were altered following cellular infection. For example, gene ontology (GO) analysis indicated that type I infection largely affects genes related to central nervous system while type III infection largely alters genes which affect nucleotide metabolism; type II infection does not alter expression of a clearly defined set of genes. Moreover, Ingenuity pathway analysis (IPA) suggested the three lineages differ in the ability to manipulate their host, e.g. they employ different strategies to avoid, deflect, or subvert host defense mechanisms. These observed differences may explain some of the variation in the neurobiological effects of different strains of Toxoplasma on infected individuals.
PMID: 21149591 [PubMed - as supplied by publisher]
Differential Effects of Three Canonical Toxoplasma Strains on Gene Expression in Human Neuroepithelial Cells
Xiao J, Jones-Brando L, Talbot CC Jr, Yolken RH.
The Stanley Division of Developmental Neurovirology, Institute for Basic Biomedical Sciences, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Abstract
Strain type is one of the key factors suspected to play a role in determing the outcome of Toxoplasma infection. In this study, we examined the transcriptional profile of human neuroepithelioma cells in response to representative strains of Toxoplasma using microarray analysis to characterize the strain-specific host cell response. The study of neural cells is of interest in light of the ability of Toxoplasma to infect the brain and to establish persistent infection within the central nervous system. We found that the extent of the expression changes varied considerably among the three strains. Neuroepithelial cells infected with Toxoplasma type I exhibited the highest level of differential gene expression, whereas type II infected cells had a substantially smaller number of genes which were differentially expressed. Cells infected with type III exhibited intermediate effects on gene expression. The three strains also differed in the individual genes and gene pathways which were altered following cellular infection. For example, gene ontology (GO) analysis indicated that type I infection largely affects genes related to central nervous system while type III infection largely alters genes which affect nucleotide metabolism; type II infection does not alter expression of a clearly defined set of genes. Moreover, Ingenuity pathway analysis (IPA) suggested the three lineages differ in the ability to manipulate their host, e.g. they employ different strategies to avoid, deflect, or subvert host defense mechanisms. These observed differences may explain some of the variation in the neurobiological effects of different strains of Toxoplasma on infected individuals.
PMID: 21149591 [PubMed - as supplied by publisher]
Anti-inflammatory effects of resveratrol, curcumin and simvastatin in acute small intestinal inflammation
PLoS One. 2010 Dec 3;5(12):e15099.
Anti-inflammatory effects of resveratrol, curcumin and simvastatin in acute small intestinal inflammation
Bereswill S, Muñoz M, Fischer A, Plickert R, Haag LM, Otto B, Kühl AA, Loddenkemper C, Göbel UB, Heimesaat MM.
Institut für Mikrobiologie und Hygiene, Charité - Universitätsmedizin Berlin, Berlin, Germany.
Abstract
BACKGROUND: The health beneficial effects of Resveratrol, Curcumin and Simvastatin have been demonstrated in various experimental models of inflammation. We investigated the potential anti-inflammatory and immunomodulatory mechanisms of the above mentioned compounds in a murine model of hyper-acute Th1-type ileitis following peroral infection with Toxoplasma gondii.
METHODOLOGY/PRINCIPAL FINDINGS: Here we show that after peroral administration of Resveratrol, Curcumin or Simvastatin, mice were protected from ileitis development and survived the acute phase of inflammation whereas all Placebo treated controls died. In particular, Resveratrol treatment resulted in longer-term survival. Resveratrol, Curcumin or Simvastatin treated animals displayed significantly increased numbers of regulatory T cells and augmented intestinal epithelial cell proliferation/regeneration in the ileum mucosa compared to placebo control animals. In contrast, mucosal T lymphocyte and neutrophilic granulocyte numbers in treated mice were reduced. In addition, levels of the anti-inflammatory cytokine IL-10 in ileum, mesenteric lymph nodes and spleen were increased whereas pro-inflammatory cytokine expression (IL-23p19, IFN-γ, TNF-α, IL-6, MCP-1) was found to be significantly lower in the ileum of treated animals as compared to Placebo controls. Furthermore, treated animals displayed not only fewer pro-inflammatory enterobacteria and enterococci but also higher anti-inflammatory lactobacilli and bifidobacteria loads. Most importantly, treatment with all three compounds preserved intestinal barrier functions as indicated by reduced bacterial translocation rates into spleen, liver, kidney and blood.
CONCLUSION/SIGNIFICANCE: Oral treatment with Resveratrol, Curcumin or Simvastatin ameliorates acute small intestinal inflammation by down-regulating Th1-type immune responses and prevents bacterial translocation by maintaining gut barrier function. These findings provide novel and potential prophylaxis and treatment options of patients with inflammatory bowel diseases.
PMID: 21151942 [PubMed - in process]
Anti-inflammatory effects of resveratrol, curcumin and simvastatin in acute small intestinal inflammation
Bereswill S, Muñoz M, Fischer A, Plickert R, Haag LM, Otto B, Kühl AA, Loddenkemper C, Göbel UB, Heimesaat MM.
Institut für Mikrobiologie und Hygiene, Charité - Universitätsmedizin Berlin, Berlin, Germany.
Abstract
BACKGROUND: The health beneficial effects of Resveratrol, Curcumin and Simvastatin have been demonstrated in various experimental models of inflammation. We investigated the potential anti-inflammatory and immunomodulatory mechanisms of the above mentioned compounds in a murine model of hyper-acute Th1-type ileitis following peroral infection with Toxoplasma gondii.
METHODOLOGY/PRINCIPAL FINDINGS: Here we show that after peroral administration of Resveratrol, Curcumin or Simvastatin, mice were protected from ileitis development and survived the acute phase of inflammation whereas all Placebo treated controls died. In particular, Resveratrol treatment resulted in longer-term survival. Resveratrol, Curcumin or Simvastatin treated animals displayed significantly increased numbers of regulatory T cells and augmented intestinal epithelial cell proliferation/regeneration in the ileum mucosa compared to placebo control animals. In contrast, mucosal T lymphocyte and neutrophilic granulocyte numbers in treated mice were reduced. In addition, levels of the anti-inflammatory cytokine IL-10 in ileum, mesenteric lymph nodes and spleen were increased whereas pro-inflammatory cytokine expression (IL-23p19, IFN-γ, TNF-α, IL-6, MCP-1) was found to be significantly lower in the ileum of treated animals as compared to Placebo controls. Furthermore, treated animals displayed not only fewer pro-inflammatory enterobacteria and enterococci but also higher anti-inflammatory lactobacilli and bifidobacteria loads. Most importantly, treatment with all three compounds preserved intestinal barrier functions as indicated by reduced bacterial translocation rates into spleen, liver, kidney and blood.
CONCLUSION/SIGNIFICANCE: Oral treatment with Resveratrol, Curcumin or Simvastatin ameliorates acute small intestinal inflammation by down-regulating Th1-type immune responses and prevents bacterial translocation by maintaining gut barrier function. These findings provide novel and potential prophylaxis and treatment options of patients with inflammatory bowel diseases.
PMID: 21151942 [PubMed - in process]
Regulation of chemokine responses in intestinal epithelial cells by stress and Toxoplasma gondii infection
Parasite Immunol. 2011 Jan;33(1):12-24. doi: 10.1111/j.1365-3024.2010.01248.x.
Regulation of chemokine responses in intestinal epithelial cells by stress and Toxoplasma gondii infection
Gopal R, Birdsell D, Monroy FP.
Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ, USA.
Abstract
Infection with Toxoplasma gondii induces chemokine up-regulation in several cell types. Here, we investigated the role of stress products (norepinephrine, NE) on chemokine production in mouse intestinal epithelial cells (IECs). Purified IECs were used to determine the expression levels of chemokines by real-time PCR. There was significantly increased expression in CCL2, CCL3, CCL5, CXCL2, CXCL9 and CXCL10 in IECs following peroral infection with T. gondii (INF) on day eight post-infection (PI) compared to infected mice subjected to cold-water stress (INF+CWS). In vitro studies using the MODE-K cell line showed increased chemokine mRNA and protein expression in infected but not in cells exposed to parasite antigen. Down-regulation of chemokine expression was more pronounced when active infection was used in combination with NE. Chemokine receptor expression was increased in IECs isolated from INF and decreased in the INF+CWS group. In MODE-K cells, there was decreased mRNA expression of chemokine receptors when incubated with β-adrenergic antagonists. Neither, adrenergic antagonists blocked the effect of infection on chemokine receptor expression. Cold-water stress was able to decrease expression of chemokines and their receptors in IECs in vivo and in vitro. Cold-water stress-mediated modulation of innate intestinal responses are beneficial in C57BL/6 mice during T. gondii infection.
© 2010 Blackwell Publishing Ltd.
PMID: 21155839 [PubMed - in process]
Regulation of chemokine responses in intestinal epithelial cells by stress and Toxoplasma gondii infection
Gopal R, Birdsell D, Monroy FP.
Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ, USA.
Abstract
Infection with Toxoplasma gondii induces chemokine up-regulation in several cell types. Here, we investigated the role of stress products (norepinephrine, NE) on chemokine production in mouse intestinal epithelial cells (IECs). Purified IECs were used to determine the expression levels of chemokines by real-time PCR. There was significantly increased expression in CCL2, CCL3, CCL5, CXCL2, CXCL9 and CXCL10 in IECs following peroral infection with T. gondii (INF) on day eight post-infection (PI) compared to infected mice subjected to cold-water stress (INF+CWS). In vitro studies using the MODE-K cell line showed increased chemokine mRNA and protein expression in infected but not in cells exposed to parasite antigen. Down-regulation of chemokine expression was more pronounced when active infection was used in combination with NE. Chemokine receptor expression was increased in IECs isolated from INF and decreased in the INF+CWS group. In MODE-K cells, there was decreased mRNA expression of chemokine receptors when incubated with β-adrenergic antagonists. Neither, adrenergic antagonists blocked the effect of infection on chemokine receptor expression. Cold-water stress was able to decrease expression of chemokines and their receptors in IECs in vivo and in vitro. Cold-water stress-mediated modulation of innate intestinal responses are beneficial in C57BL/6 mice during T. gondii infection.
© 2010 Blackwell Publishing Ltd.
PMID: 21155839 [PubMed - in process]
Thursday, December 09, 2010
Immunome Res. 2010 Dec 3;6(1):12. [Epub ahead of print]
Human immunome, bioinformatic analyses using HLA supermotifs and the parasite genome, binding assays, studies of human T cell responses, and immunization of HLA-A*1101 transgenic mice including novel adjuvants provide a foundation for HLA-A03 restricted CD8+T cell epitope based, adjuvanted vaccine protective against Toxoplasma gondii
Cong H, Mui EJ, Witola WH, Sidney J, Alexander J, Sette A, Maewal A, McLeod R.
Abstract
ABSTRACT:
BACKGROUND: Toxoplasmosis causes loss of life, cognitive and motor function, and sight. A vaccine is greatly needed to prevent this disease. The purpose of this study was to use an immmunosense approach to develop a foundation for development of vaccines to protect humans with the HLA-A03 supertype. Three peptides had been identified with high binding scores for HLA-A03 supertypes using bioinformatic algorhythms, high measured binding affinity for HLA-A03 supertype molecules, and ability to elicit IFN-gamma production by human HLA-A03 supertype peripheral blood CD8+ T cells from seropositive but not seronegative persons.
RESULTS: Herein, when these peptides were administered with the universal CD4+T cell epitope PADRE (AKFVAAWTLKAAA) and formulated as lipopeptides, or administered with GLA-SE either alone, or with PAM2Cys added, we found we successfully created preparations that induced IFN-gamma and reduced parasite burden in HLA-A*1101(an HLA-A03 supertype allele) transgenic mice. GLA-SE is a novel emulsified synthetic TLR4 ligand that is known to facilitate development of T Helper 1 cell (TH1) responses. Then, so our peptides would include those expressed in tachyzoites, bradyzoites and sporozoites from both Type I and II parasites, we used our approaches which had identified the initial peptides. We identified additional peptides using bioinformatics, binding affinity assays, and study of responses of HLA-A03 human cells. Lastly, we found that immunization of HLA-A*1101 transgenic mice with all the pooled peptides administered with PADRE, GLA-SE, and Pam2Cys is an effective way to elicit IFN-gamma producing CD8+ splenic T cells and protection. Immunizations included the following peptides together: KSFKDILPK (SAG1224-232); AMLTAFFLR (GRA6164-172); RSFKDLLKK (GRA7134-142); STFWPCLLR (SAG2C13-21); SSAYVFSVK(SPA250-258); and AVVSLLRLLK(SPA89-98). This immunization elicited robust protection, measured as reduced parasite burden using a luciferase transfected parasite, luciferin, this novel, HLA transgenic mouse model, and imaging with a Xenogen camera.
CONCLUSIONS: Toxoplasma gondii peptides elicit HLA-A03 restricted, IFN-gamma producing, CD8+ T cells in humans and mice. These peptides administered with adjuvants reduce parasite burden in HLA-A*1101 transgenic mice. This work provides a foundation for immunosense based vaccines. It also defines novel adjuvants for newly identified peptides for vaccines to prevent toxoplasmosis in those with HLA-A03 supertype alleles.
PMID: 21129215 [PubMed - as supplied by publisher]
Human immunome, bioinformatic analyses using HLA supermotifs and the parasite genome, binding assays, studies of human T cell responses, and immunization of HLA-A*1101 transgenic mice including novel adjuvants provide a foundation for HLA-A03 restricted CD8+T cell epitope based, adjuvanted vaccine protective against Toxoplasma gondii
Cong H, Mui EJ, Witola WH, Sidney J, Alexander J, Sette A, Maewal A, McLeod R.
Abstract
ABSTRACT:
BACKGROUND: Toxoplasmosis causes loss of life, cognitive and motor function, and sight. A vaccine is greatly needed to prevent this disease. The purpose of this study was to use an immmunosense approach to develop a foundation for development of vaccines to protect humans with the HLA-A03 supertype. Three peptides had been identified with high binding scores for HLA-A03 supertypes using bioinformatic algorhythms, high measured binding affinity for HLA-A03 supertype molecules, and ability to elicit IFN-gamma production by human HLA-A03 supertype peripheral blood CD8+ T cells from seropositive but not seronegative persons.
RESULTS: Herein, when these peptides were administered with the universal CD4+T cell epitope PADRE (AKFVAAWTLKAAA) and formulated as lipopeptides, or administered with GLA-SE either alone, or with PAM2Cys added, we found we successfully created preparations that induced IFN-gamma and reduced parasite burden in HLA-A*1101(an HLA-A03 supertype allele) transgenic mice. GLA-SE is a novel emulsified synthetic TLR4 ligand that is known to facilitate development of T Helper 1 cell (TH1) responses. Then, so our peptides would include those expressed in tachyzoites, bradyzoites and sporozoites from both Type I and II parasites, we used our approaches which had identified the initial peptides. We identified additional peptides using bioinformatics, binding affinity assays, and study of responses of HLA-A03 human cells. Lastly, we found that immunization of HLA-A*1101 transgenic mice with all the pooled peptides administered with PADRE, GLA-SE, and Pam2Cys is an effective way to elicit IFN-gamma producing CD8+ splenic T cells and protection. Immunizations included the following peptides together: KSFKDILPK (SAG1224-232); AMLTAFFLR (GRA6164-172); RSFKDLLKK (GRA7134-142); STFWPCLLR (SAG2C13-21); SSAYVFSVK(SPA250-258); and AVVSLLRLLK(SPA89-98). This immunization elicited robust protection, measured as reduced parasite burden using a luciferase transfected parasite, luciferin, this novel, HLA transgenic mouse model, and imaging with a Xenogen camera.
CONCLUSIONS: Toxoplasma gondii peptides elicit HLA-A03 restricted, IFN-gamma producing, CD8+ T cells in humans and mice. These peptides administered with adjuvants reduce parasite burden in HLA-A*1101 transgenic mice. This work provides a foundation for immunosense based vaccines. It also defines novel adjuvants for newly identified peptides for vaccines to prevent toxoplasmosis in those with HLA-A03 supertype alleles.
PMID: 21129215 [PubMed - as supplied by publisher]
Parasitic, fungal and prion zoonoses: An expanding universe of candidates for human disease
Clin Microbiol Infect. 2010 Dec 4. doi: 10.1111/j.1469-0691.2010.03442.x. [Epub ahead of print]
Parasitic, fungal and prion zoonoses: An expanding universe of candidates for human disease
Akritidis N.
Head, Internal Medicine Department, General Hospital "G. Hatzikosta" of Ioannina, Greece.
Abstract
Zoonotic infections have emerged as a burden for millions of people in recent years, due to re-emerging or novel pathogens often causing outbreaks in the developing world in the presence of inadequate public health infrastructure. Among zoonotic infections, parasitic pathogens are the ones that affect millions of humans worldwide, while furthermore are at risk of developing disease which is often chronic. The present review discusses the global effect of protozoan pathogens as Leishmania sp., Trypanosoma sp., and Toxoplasma sp., as well as helminthic pathogens as Echinococcus sp., Fasciola sp., and Trichinella sp. The zoonotic aspects of agents that are not essentially zoonotic are also discussed. The review further focuses on the zoonotic dynamics of fungal pathogens and prion diseases as observed in recent years, in an evolving environment that has developed novel patient target groups for agents that were previously considered obscure or of minimal significance.
Copyright © 2010 European Society of Clinical Microbiology and Infectious Diseases.
PMID: 21129103 [PubMed - as supplied by publisher]
Parasitic, fungal and prion zoonoses: An expanding universe of candidates for human disease
Akritidis N.
Head, Internal Medicine Department, General Hospital "G. Hatzikosta" of Ioannina, Greece.
Abstract
Zoonotic infections have emerged as a burden for millions of people in recent years, due to re-emerging or novel pathogens often causing outbreaks in the developing world in the presence of inadequate public health infrastructure. Among zoonotic infections, parasitic pathogens are the ones that affect millions of humans worldwide, while furthermore are at risk of developing disease which is often chronic. The present review discusses the global effect of protozoan pathogens as Leishmania sp., Trypanosoma sp., and Toxoplasma sp., as well as helminthic pathogens as Echinococcus sp., Fasciola sp., and Trichinella sp. The zoonotic aspects of agents that are not essentially zoonotic are also discussed. The review further focuses on the zoonotic dynamics of fungal pathogens and prion diseases as observed in recent years, in an evolving environment that has developed novel patient target groups for agents that were previously considered obscure or of minimal significance.
Copyright © 2010 European Society of Clinical Microbiology and Infectious Diseases.
PMID: 21129103 [PubMed - as supplied by publisher]
T cell production of matrix metalloproteases and inhibition of parasite clearance by TIMP-1 during chronic Toxoplasma infection in the brain
ASN Neuro. 2010 Dec 6. [Epub ahead of print]
T cell production of matrix metalloproteases and inhibition of parasite clearance by TIMP-1 during chronic Toxoplasma infection in the brain
Clark RT, Nance JP, Noor S, Wilson EH.
Abstract
Chronic infection with the intracellular protozoan parasite Toxoplasma gondii leads to tissue remodeling in the brain and a continuous requirement for peripheral leukocyte migration within the CNS. In this study we investigated the role of matrix metalloproteases (MMPs) and their inhibitors for T cell migration into the infected brain. Increased expression of two key molecules, MMP-8 and MMP-10, along with their inhibitor, Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) was observed in the CNS following infection. Analysis of infiltrating lymphocytes demonstrated MMP-8 and -10 production by CD4+ and CD8+ T cells. In addition, infiltrating T cells and CNS resident astrocytes increased their expression of TIMP-1 following infection. TIMP-1 deficient mice had a decrease in perivascular accumulation of lymphocyte populations yet an increase in the proportion of CD4+ T cells that had trafficked into the CNS. This was accompanied by a reduction in parasite burden in the brain. Together, these findings demonstrate a role for MMPs and TIMP-1 in the trafficking of lymphocytes into the CNS during chronic infection in the brain.
PMID: 21133852 [PubMed - as supplied by publisher]Free Article
T cell production of matrix metalloproteases and inhibition of parasite clearance by TIMP-1 during chronic Toxoplasma infection in the brain
Clark RT, Nance JP, Noor S, Wilson EH.
Abstract
Chronic infection with the intracellular protozoan parasite Toxoplasma gondii leads to tissue remodeling in the brain and a continuous requirement for peripheral leukocyte migration within the CNS. In this study we investigated the role of matrix metalloproteases (MMPs) and their inhibitors for T cell migration into the infected brain. Increased expression of two key molecules, MMP-8 and MMP-10, along with their inhibitor, Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) was observed in the CNS following infection. Analysis of infiltrating lymphocytes demonstrated MMP-8 and -10 production by CD4+ and CD8+ T cells. In addition, infiltrating T cells and CNS resident astrocytes increased their expression of TIMP-1 following infection. TIMP-1 deficient mice had a decrease in perivascular accumulation of lymphocyte populations yet an increase in the proportion of CD4+ T cells that had trafficked into the CNS. This was accompanied by a reduction in parasite burden in the brain. Together, these findings demonstrate a role for MMPs and TIMP-1 in the trafficking of lymphocytes into the CNS during chronic infection in the brain.
PMID: 21133852 [PubMed - as supplied by publisher]Free Article
Saturday, December 04, 2010
Impact of protozoan cell death on parasite-host interactions and pathogenesis
Parasit Vectors. 2010 Dec 2;3(1):116. [Epub ahead of print]
Impact of protozoan cell death on parasite-host interactions and pathogenesis
Luder CG, Campos-Salinas J, Gonzalez-Rey E, van Zandbergen G.
Abstract
ABSTRACT: PCD in protozoan parasites has emerged as a fascinating field of parasite biology. This not only relates to the underlying mechanisms and their evolutionary implications but also to the impact on the parasite-host interactions within mammalian hosts and arthropod vectors. During recent years, common functions of apoptosis and autophagy in protozoa and during parasitic infections have emerged. Here, we review how distinct cell death pathways in Trypanosoma, Leishmania, Plasmodium or Toxoplasma may contribute to regulation of parasite cell densities in vectors and mammalian hosts, to differentiation of parasites, to stress responses, and to modulation of the host immunity. The examples provided indicate crucial roles of PCD in parasite biology. The existence of PCD pathways in these organisms and the identification as being critical for parasite biology and parasite-host interactions could serve as a basis for developing new anti-parasitic drugs that take advantage of these pathways.
PMID: 21126352 [PubMed - as supplied by publisher]
Impact of protozoan cell death on parasite-host interactions and pathogenesis
Luder CG, Campos-Salinas J, Gonzalez-Rey E, van Zandbergen G.
Abstract
ABSTRACT: PCD in protozoan parasites has emerged as a fascinating field of parasite biology. This not only relates to the underlying mechanisms and their evolutionary implications but also to the impact on the parasite-host interactions within mammalian hosts and arthropod vectors. During recent years, common functions of apoptosis and autophagy in protozoa and during parasitic infections have emerged. Here, we review how distinct cell death pathways in Trypanosoma, Leishmania, Plasmodium or Toxoplasma may contribute to regulation of parasite cell densities in vectors and mammalian hosts, to differentiation of parasites, to stress responses, and to modulation of the host immunity. The examples provided indicate crucial roles of PCD in parasite biology. The existence of PCD pathways in these organisms and the identification as being critical for parasite biology and parasite-host interactions could serve as a basis for developing new anti-parasitic drugs that take advantage of these pathways.
PMID: 21126352 [PubMed - as supplied by publisher]
Ciliate pellicular proteome identifies novel protein families with characteristic repeat motifs that are common to Alveolates
Mol Biol Evol. 2010 Dec 2. [Epub ahead of print]
Ciliate pellicular proteome identifies novel protein families with characteristic repeat motifs that are common to Alveolates
Gould SB, Kraft LG, van Dooren GG, Goodman CD, Ford KL, Cassin AM, Bacic A, McFadden GI, Waller RF.
School of Botany, University of Melbourne, 3010 Victoria, Australia.
Abstract
The pellicles of alveolates (ciliates, apicomplexans and dinoflagellates) share a common organization, yet perform very divergent functions including motility, host cell invasion and armor. The alveolate pellicle consists of a system of flattened membrane sacs (alveoli, which are the defining feature of the group) below the plasma-membrane that is supported by a membrane skeleton as well as a network of microtubules and other filamentous elements. We recently showed that a family of proteins, alveolins, are common and unique to this pellicular structure in alveolates. To identify additional proteins that contribute to this structure, a pellicle proteome study was conducted for the ciliate Tetrahymena thermophila. We found 1173 proteins associated with this structure, 45% (529 proteins) of which represented novel proteins without matches to other functionally characterized proteins. Expression of four newly identified T. thermophila pellicular proteins as GFP-fusion proteins confirmed pellicular location, and one new protein located in the oral apparatus. Bioinformatic analysis revealed that 21% of the putative pellicular proteins, predominantly the novel proteins, contained highly repetitive regions with strong amino acid biases for particular residues (K, E, Q, L, I & V). When the T. thermophila novel proteins were compared to apicomplexan genomic data, 278 proteins with high sequence similarity were identified, suggesting that many of these putative pellicular components are shared between the alveolates. Of these shared proteins, 126 contained the distinctive repeat regions. Localization of two such proteins in Toxoplasma gondii confirmed their role in the pellicle, and in doing so identified two new proteins of the apicomplexan invasive structure - the apical complex. Screening broadly for these repetitive domains in genomic data revealed large and actively evolving families of such proteins in alveolates, suggesting that these proteins might underpin the diversity and utility of their unique pellicular structure.
PMID: 21127172 [PubMed - as supplied by publisher]
Ciliate pellicular proteome identifies novel protein families with characteristic repeat motifs that are common to Alveolates
Gould SB, Kraft LG, van Dooren GG, Goodman CD, Ford KL, Cassin AM, Bacic A, McFadden GI, Waller RF.
School of Botany, University of Melbourne, 3010 Victoria, Australia.
Abstract
The pellicles of alveolates (ciliates, apicomplexans and dinoflagellates) share a common organization, yet perform very divergent functions including motility, host cell invasion and armor. The alveolate pellicle consists of a system of flattened membrane sacs (alveoli, which are the defining feature of the group) below the plasma-membrane that is supported by a membrane skeleton as well as a network of microtubules and other filamentous elements. We recently showed that a family of proteins, alveolins, are common and unique to this pellicular structure in alveolates. To identify additional proteins that contribute to this structure, a pellicle proteome study was conducted for the ciliate Tetrahymena thermophila. We found 1173 proteins associated with this structure, 45% (529 proteins) of which represented novel proteins without matches to other functionally characterized proteins. Expression of four newly identified T. thermophila pellicular proteins as GFP-fusion proteins confirmed pellicular location, and one new protein located in the oral apparatus. Bioinformatic analysis revealed that 21% of the putative pellicular proteins, predominantly the novel proteins, contained highly repetitive regions with strong amino acid biases for particular residues (K, E, Q, L, I & V). When the T. thermophila novel proteins were compared to apicomplexan genomic data, 278 proteins with high sequence similarity were identified, suggesting that many of these putative pellicular components are shared between the alveolates. Of these shared proteins, 126 contained the distinctive repeat regions. Localization of two such proteins in Toxoplasma gondii confirmed their role in the pellicle, and in doing so identified two new proteins of the apicomplexan invasive structure - the apical complex. Screening broadly for these repetitive domains in genomic data revealed large and actively evolving families of such proteins in alveolates, suggesting that these proteins might underpin the diversity and utility of their unique pellicular structure.
PMID: 21127172 [PubMed - as supplied by publisher]
Friday, December 03, 2010
T. gondii RP Promoters & Knockdown Reveal Molecular Pathways Associated with Proliferation and Cell-Cycle Arrest
PLoS One. 2010 Nov 22;5(11):e14057.
T. gondii RP Promoters & Knockdown Reveal Molecular Pathways Associated with Proliferation and Cell-Cycle Arrest
Hutson SL, Mui E, Kinsley K, Witola WH, Behnke MS, El Bissati K, Muench SP, Rohrman B, Liu SR, Wollmann R, Ogata Y, Sarkeshik A, Yates JR, McLeod R.
Department of Surgery (Ophthalmology), The University of Chicago, Chicago, Illinois, United States of America.
Abstract
Molecular pathways regulating rapid proliferation and persistence are fundamental for pathogens but are not elucidated fully in Toxoplasma gondii. Promoters of T. gondii ribosomal proteins (RPs) were analyzed by EMSAs and ChIP. One RP promoter domain, known to bind an Apetela 2, bound to nuclear extract proteins. Promoter domains appeared to associate with histone acetyl transferases. To study effects of a RP gene's regulation in T. gondii, mutant parasites (Δrps13) were engineered with integration of tetracycline repressor (TetR) response elements in a critical location in the rps13 promoter and transfection of a yellow fluorescent-tetracycline repressor (YFP-TetR). This permitted conditional knockdown of rps13 expression in a tightly regulated manner. Δrps13 parasites were studied in the presence (+ATc) or absence of anhydrotetracycline (-ATc) in culture. -ATc, transcription of the rps13 gene and expression of RPS13 protein were markedly diminished, with concomitant cessation of parasite replication. Study of Δrps13 expressing Myc-tagged RPL22, -ATc, showed RPL22 diminished but at a slower rate. Quantitation of RNA showed diminution of 18S RNA. Depletion of RPS13 caused arrest of parasites in the G1 cell cycle phase, thereby stopping parasite proliferation. Transcriptional differences ±ATc implicate molecules likely to function in regulation of these processes. In vitro, -ATc, Δrps13 persists for months and the proliferation phenotype can be rescued with ATc. In vivo, however, Δrps13 could only be rescued when ATc was given simultaneously and not at any time after 1 week, even when L-NAME and ATc were administered. Immunization with Δrps13 parasites protects mice completely against subsequent challenge with wildtype clonal Type 1 parasites, and robustly protects mice against wildtype clonal Type 2 parasites. Our results demonstrate that G1 arrest by ribosomal protein depletion is associated with persistence of T. gondii in a model system in vitro and immunization with Δrps13 protects mice against subsequent challenge with wildtype parasites.
PMID: 21124925 [PubMed - as supplied by publisher]
T. gondii RP Promoters & Knockdown Reveal Molecular Pathways Associated with Proliferation and Cell-Cycle Arrest
Hutson SL, Mui E, Kinsley K, Witola WH, Behnke MS, El Bissati K, Muench SP, Rohrman B, Liu SR, Wollmann R, Ogata Y, Sarkeshik A, Yates JR, McLeod R.
Department of Surgery (Ophthalmology), The University of Chicago, Chicago, Illinois, United States of America.
Abstract
Molecular pathways regulating rapid proliferation and persistence are fundamental for pathogens but are not elucidated fully in Toxoplasma gondii. Promoters of T. gondii ribosomal proteins (RPs) were analyzed by EMSAs and ChIP. One RP promoter domain, known to bind an Apetela 2, bound to nuclear extract proteins. Promoter domains appeared to associate with histone acetyl transferases. To study effects of a RP gene's regulation in T. gondii, mutant parasites (Δrps13) were engineered with integration of tetracycline repressor (TetR) response elements in a critical location in the rps13 promoter and transfection of a yellow fluorescent-tetracycline repressor (YFP-TetR). This permitted conditional knockdown of rps13 expression in a tightly regulated manner. Δrps13 parasites were studied in the presence (+ATc) or absence of anhydrotetracycline (-ATc) in culture. -ATc, transcription of the rps13 gene and expression of RPS13 protein were markedly diminished, with concomitant cessation of parasite replication. Study of Δrps13 expressing Myc-tagged RPL22, -ATc, showed RPL22 diminished but at a slower rate. Quantitation of RNA showed diminution of 18S RNA. Depletion of RPS13 caused arrest of parasites in the G1 cell cycle phase, thereby stopping parasite proliferation. Transcriptional differences ±ATc implicate molecules likely to function in regulation of these processes. In vitro, -ATc, Δrps13 persists for months and the proliferation phenotype can be rescued with ATc. In vivo, however, Δrps13 could only be rescued when ATc was given simultaneously and not at any time after 1 week, even when L-NAME and ATc were administered. Immunization with Δrps13 parasites protects mice completely against subsequent challenge with wildtype clonal Type 1 parasites, and robustly protects mice against wildtype clonal Type 2 parasites. Our results demonstrate that G1 arrest by ribosomal protein depletion is associated with persistence of T. gondii in a model system in vitro and immunization with Δrps13 protects mice against subsequent challenge with wildtype parasites.
PMID: 21124925 [PubMed - as supplied by publisher]
Thursday, December 02, 2010
Brain parasites and suicide
Psychol Rep. 2010 Oct;107(2):424.
Brain parasites and suicide
Lester D.
Psychology Program, The Richard Stockton College of New Jersey, Jimmie Leeds Road, Pomona, NJ 08240-0195, USA. lesterd@stockton.edu
Abstract
In a sample of 20 European nations, the prevalence of the brain parasite Toxoplasma gondii was positively associated with national suicide rates for men and women.
PMID: 21117467 [PubMed - in process]
Brain parasites and suicide
Lester D.
Psychology Program, The Richard Stockton College of New Jersey, Jimmie Leeds Road, Pomona, NJ 08240-0195, USA. lesterd@stockton.edu
Abstract
In a sample of 20 European nations, the prevalence of the brain parasite Toxoplasma gondii was positively associated with national suicide rates for men and women.
PMID: 21117467 [PubMed - in process]
Role of the NF-{kappa}B transcription factor c-Rel in the generation of CD8+ T-cell responses to Toxoplasma gondii
Int Immunol. 2010 Nov;22(11):851-61.
Role of the NF-{kappa}B transcription factor c-Rel in the generation of CD8+ T-cell responses to Toxoplasma gondii
Jordan KA, Dupont CD, Tait ED, Liou HC, Hunter CA.
Department of Pathobiology, University of Pennsylvania, 380 South University Avenue, Philadelphia, PA 19104, USA.
Abstract
The nuclear factor κB transcription factor c-Rel is exclusively expressed in immune cells and plays a role in numerous cellular functions including proliferation, survival and production of chemokines and cytokines. c-Rel has also been implicated in the regulation of multiple genes involved in innate and adaptive immune responses to the intracellular protozoan parasite Toxoplasma gondii, in particular IL-12. To better understand how this transcription factor controls the CD8(+) T-cell response to this organism, wild-type (WT) and c-Rel(-/-) mice were challenged with a replication-deficient strain of T. gondii that expresses the model antigen ovalbumin (OVA). These studies revealed that c-Rel was required for optimal primary expansion of OVA-specific CD8(+) T cells and that immunized c-Rel-deficient mice were susceptible to challenge with a virulent strain of T. gondii. However, when c-Rel(-/-) cells specific for OVA were adoptively transferred into a WT recipient, or c-Rel(-/-) mice were treated with IL-12 at the time of immunization, there was no apparent proliferative defect. Surprisingly, upon secondary challenge, antigen-specific CD8(+) T cells in c-Rel(-/-) mice expanded to a much greater degree in terms of frequency as well as numbers when compared with WT mice. Despite this, the cytokine responses of c-Rel(-/-) mice remained defective, consistent with their susceptibility to secondary challenge. Together, these results indicate that in this infection model, the major influence of c-Rel in generation of CD8(+) T-cell responses is through its regulation of the inflammatory environment, rather than playing a substantial T-cell-intrinsic role.
PMID: 21118906 [PubMed - in process]
Role of the NF-{kappa}B transcription factor c-Rel in the generation of CD8+ T-cell responses to Toxoplasma gondii
Jordan KA, Dupont CD, Tait ED, Liou HC, Hunter CA.
Department of Pathobiology, University of Pennsylvania, 380 South University Avenue, Philadelphia, PA 19104, USA.
Abstract
The nuclear factor κB transcription factor c-Rel is exclusively expressed in immune cells and plays a role in numerous cellular functions including proliferation, survival and production of chemokines and cytokines. c-Rel has also been implicated in the regulation of multiple genes involved in innate and adaptive immune responses to the intracellular protozoan parasite Toxoplasma gondii, in particular IL-12. To better understand how this transcription factor controls the CD8(+) T-cell response to this organism, wild-type (WT) and c-Rel(-/-) mice were challenged with a replication-deficient strain of T. gondii that expresses the model antigen ovalbumin (OVA). These studies revealed that c-Rel was required for optimal primary expansion of OVA-specific CD8(+) T cells and that immunized c-Rel-deficient mice were susceptible to challenge with a virulent strain of T. gondii. However, when c-Rel(-/-) cells specific for OVA were adoptively transferred into a WT recipient, or c-Rel(-/-) mice were treated with IL-12 at the time of immunization, there was no apparent proliferative defect. Surprisingly, upon secondary challenge, antigen-specific CD8(+) T cells in c-Rel(-/-) mice expanded to a much greater degree in terms of frequency as well as numbers when compared with WT mice. Despite this, the cytokine responses of c-Rel(-/-) mice remained defective, consistent with their susceptibility to secondary challenge. Together, these results indicate that in this infection model, the major influence of c-Rel in generation of CD8(+) T-cell responses is through its regulation of the inflammatory environment, rather than playing a substantial T-cell-intrinsic role.
PMID: 21118906 [PubMed - in process]
Discovery of Potent and Selective Inhibitors of Calcium-Dependent Protein Kinase 1 (CDPK1) from C. parvum and T. gondii
ACS Med Chem Lett. 2010 Oct 14;1(7):331-335.
Discovery of Potent and Selective Inhibitors of Calcium-Dependent Protein Kinase 1 (CDPK1) from C. parvum and T. gondii
Murphy RC, Ojo KK, Larson ET, Castellanos-Gonzalez A, Perera BG, Keyloun KR, Kim JE, Bhandari JG, Muller NR, Verlinde CL, White AC, Merritt EA, Van Voorhis WC, Maly DJ.
Department of Chemistry, University of Washington.
Abstract
The protozoans Cryptosporidium parvum and Toxoplasma gondii are parasites of major health concern to humans. Both parasites contain a group of calcium-dependent protein kinases (CDPKs), which are found in plants and ciliates but not in humans or fungi. Here we describe a series of potent inhibitors that target CDPK1 in C. parvum (CpCDPK1) and T. gondii (TgCDPK1). These inhibitors are highly selective for CpCDPK1 and TgCDPK1 over the mammalian kinases SRC and ABL. Furthermore, they are able to block an early stage of C. parvum invasion of HCT-8 host cells, which is similar to their effects on T. gondii invasion of human fibroblasts.
PMID: 21116453 [PubMed]
Discovery of Potent and Selective Inhibitors of Calcium-Dependent Protein Kinase 1 (CDPK1) from C. parvum and T. gondii
Murphy RC, Ojo KK, Larson ET, Castellanos-Gonzalez A, Perera BG, Keyloun KR, Kim JE, Bhandari JG, Muller NR, Verlinde CL, White AC, Merritt EA, Van Voorhis WC, Maly DJ.
Department of Chemistry, University of Washington.
Abstract
The protozoans Cryptosporidium parvum and Toxoplasma gondii are parasites of major health concern to humans. Both parasites contain a group of calcium-dependent protein kinases (CDPKs), which are found in plants and ciliates but not in humans or fungi. Here we describe a series of potent inhibitors that target CDPK1 in C. parvum (CpCDPK1) and T. gondii (TgCDPK1). These inhibitors are highly selective for CpCDPK1 and TgCDPK1 over the mammalian kinases SRC and ABL. Furthermore, they are able to block an early stage of C. parvum invasion of HCT-8 host cells, which is similar to their effects on T. gondii invasion of human fibroblasts.
PMID: 21116453 [PubMed]
Saturday, November 27, 2010
The antibiotic monensin causes cell cycle disruption of Toxoplasma gondii, mediated through the DNA repair enzyme TgMSH-1
Antimicrob Agents Chemother. 2010 Nov 22. [Epub ahead of print]
The antibiotic monensin causes cell cycle disruption of Toxoplasma gondii, mediated through the DNA repair enzyme TgMSH-1
Lavine MD, Arrizabalaga G.
Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Life Sciences South Room 142, Moscow, ID 83843, USA.
Abstract
Monensin is a polyether ionophore antibiotic that is widely used in the control of coccidia in animals. Despite its significance in veterinary medicine, little is known about its mode of action and potential mechanisms of resistance in coccidian parasites. Here we show that monensin causes accumulation of the coccidian Toxoplasma gondii at an apparent late S-phase cell cycle checkpoint. In addition, experiments utilizing a monensin-resistant T. gondii mutant show that this effect of monensin is dependent on the function of a mitochondrial homologue of the Mut-S DNA damage repair enzyme (TgMSH-1). Furthermore, the same TgMSH-1 dependent cell cycle disruption is observed with the anti-parasitic ionophore salinomycin and the DNA alkylating agent methyl nitrosourea. Our results suggest a novel mechanism for the mode of action of monensin and salynomicin on coccidial parasites, in which the drug activates a MSH-1 dependent cell cycle checkpoint by an unknown mechanism, ultimately leading to the death of the parasite. This model would indicate that cell cycle disruption is an important mediator of drug susceptibility and resistance to ionophoric antibiotics in coccidian parasites.
PMID: 21098240 [PubMed - as supplied by publisher]
The antibiotic monensin causes cell cycle disruption of Toxoplasma gondii, mediated through the DNA repair enzyme TgMSH-1
Lavine MD, Arrizabalaga G.
Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Life Sciences South Room 142, Moscow, ID 83843, USA.
Abstract
Monensin is a polyether ionophore antibiotic that is widely used in the control of coccidia in animals. Despite its significance in veterinary medicine, little is known about its mode of action and potential mechanisms of resistance in coccidian parasites. Here we show that monensin causes accumulation of the coccidian Toxoplasma gondii at an apparent late S-phase cell cycle checkpoint. In addition, experiments utilizing a monensin-resistant T. gondii mutant show that this effect of monensin is dependent on the function of a mitochondrial homologue of the Mut-S DNA damage repair enzyme (TgMSH-1). Furthermore, the same TgMSH-1 dependent cell cycle disruption is observed with the anti-parasitic ionophore salinomycin and the DNA alkylating agent methyl nitrosourea. Our results suggest a novel mechanism for the mode of action of monensin and salynomicin on coccidial parasites, in which the drug activates a MSH-1 dependent cell cycle checkpoint by an unknown mechanism, ultimately leading to the death of the parasite. This model would indicate that cell cycle disruption is an important mediator of drug susceptibility and resistance to ionophoric antibiotics in coccidian parasites.
PMID: 21098240 [PubMed - as supplied by publisher]
Intracellular transport of Toxoplasma gondii through the blood-brain barrier
J Neuroimmunol. 2010 Nov 22. [Epub ahead of print]
Intracellular transport of Toxoplasma gondii through the blood-brain barrier
Lachenmaier SM, Deli MA, Meissner M, Liesenfeld O.
Institute of Microbiology and Hygiene, Charité-Medical School Berlin, Campus Benjamin Franklin, Berlin, Germany.
Abstract
Toxoplasma gondii establishes latent infection in the central nervous system of immunocompentent hosts. Toxoplasmic encephalitis is a life threatening reactivation of latent infection in the brain of immunocompromised patients. To further understand the mechanisms of entry into the brain of T. gondii we investigated host molecules and cells involved in the passage of the parasite through the blood-brain barrier. First, using microarrays brain endothelial cells were found to upregulate, among others, chemokines and adhesion molecules following infection with tachyzoites. Using flow cytometry we observed upregulated ICAM-1 expression on the surface of brain endothelial cells following infection; ICAM-1 expression was further increased after pre-incubation with IFN-γ. Compared to RH tachyzoites, ME49 tachyzoites induced a stronger upregulation of ICAM-1 and an earlier and stronger IL-6 and MCP-1 secretion by brain endothelial cells. Using an in vitro coculture model of the BBB (primary glia cells and brain endothelial cells) we found a stronger migration of infected antigen-presenting cells compared to lymphocytes (4.63% vs. 0.6% of all cells) across the BBB. Among all antigen-presenting cells CD11b(+)/CD11c(+) cells showed the highest infection rate, whereas the majority of infected cells that migrated through the blood-brain barrier were CD11b(+)/CD11c(-) cells. Infection of PBMCs with type I or type II Toxoplasma strains resulted in similar patterns of cell migration across the in vitro BBB model. In conclusion, these results suggest that T. gondii modulates gene expression of brain endothelial cells to promote its own migration through the blood-brain barrier in a 'Trojan horse' manner. Cells expressing CD11b either with or without CD11c are likely candidate cells for the intracellular transport of T. gondii across the BBB. T. gondii type I and type II strains induced similar migration patterns of antigen-presenting cells across the in vitro BBB.
Copyright © 2010 Elsevier B.V. All rights reserved.
PMID: 21106256 [PubMed - as supplied by publisher]
Intracellular transport of Toxoplasma gondii through the blood-brain barrier
Lachenmaier SM, Deli MA, Meissner M, Liesenfeld O.
Institute of Microbiology and Hygiene, Charité-Medical School Berlin, Campus Benjamin Franklin, Berlin, Germany.
Abstract
Toxoplasma gondii establishes latent infection in the central nervous system of immunocompentent hosts. Toxoplasmic encephalitis is a life threatening reactivation of latent infection in the brain of immunocompromised patients. To further understand the mechanisms of entry into the brain of T. gondii we investigated host molecules and cells involved in the passage of the parasite through the blood-brain barrier. First, using microarrays brain endothelial cells were found to upregulate, among others, chemokines and adhesion molecules following infection with tachyzoites. Using flow cytometry we observed upregulated ICAM-1 expression on the surface of brain endothelial cells following infection; ICAM-1 expression was further increased after pre-incubation with IFN-γ. Compared to RH tachyzoites, ME49 tachyzoites induced a stronger upregulation of ICAM-1 and an earlier and stronger IL-6 and MCP-1 secretion by brain endothelial cells. Using an in vitro coculture model of the BBB (primary glia cells and brain endothelial cells) we found a stronger migration of infected antigen-presenting cells compared to lymphocytes (4.63% vs. 0.6% of all cells) across the BBB. Among all antigen-presenting cells CD11b(+)/CD11c(+) cells showed the highest infection rate, whereas the majority of infected cells that migrated through the blood-brain barrier were CD11b(+)/CD11c(-) cells. Infection of PBMCs with type I or type II Toxoplasma strains resulted in similar patterns of cell migration across the in vitro BBB model. In conclusion, these results suggest that T. gondii modulates gene expression of brain endothelial cells to promote its own migration through the blood-brain barrier in a 'Trojan horse' manner. Cells expressing CD11b either with or without CD11c are likely candidate cells for the intracellular transport of T. gondii across the BBB. T. gondii type I and type II strains induced similar migration patterns of antigen-presenting cells across the in vitro BBB.
Copyright © 2010 Elsevier B.V. All rights reserved.
PMID: 21106256 [PubMed - as supplied by publisher]
Protective immunity induced by Toxoplasma gondii rhoptry protein 16 (ROP16) against toxoplasmosis in mice
Clin Vaccine Immunol. 2010 Nov 24. [Epub ahead of print]
Protective immunity induced by Toxoplasma gondii rhoptry protein 16 (ROP16) against toxoplasmosis in mice
Yuan ZG, Zhang XX, He XH, Petersen E, Zhou DH, He Y, Lin RQ, Li XZ, Chen XL, Shi XR, Zhong XL, Zhang B, Zhu XQ.
State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, CAAS, Lanzhou, Gansu Province 730046, PR China; Department of Parasitology, College of Veterinary Medicine, South China Agricultural University, Guangzhou, Guangdong Province 510642, PR China; College of Agriculture, South China Agricultural University, 483 Wushan Street, Tianhe District, Guangzhou, Guangdong Province 510642, PR China; Department of Infectious Diseases, Clinical Institute, and Institute of Medical Microbiology and Immunology, Faculty of Health Sciences, Aarhus University, Aarhus, Denmark; College of Animal Science and Technology, Yunnan Agricultural University, Kunming, Yunnan Province 650201, PR China.
Abstract
Toxoplasma gondii can infect a large variety of domestic and wild animals and human beings, sometimes causing severe pathology. Rhoptries are involved in T. gondii invasion and host cell interaction, and have been implicated as important virulence factors. In this study, we constructed a DNA vaccine expressing rhoptry protein 16 (ROP16) of T. gondii, and evaluated the immune responses it induced in Kunming mice. The gene sequence encoding ROP16 was inserted into the eukaryotic expression vector pVAX I. We immunized Kunming mice intramuscularly. After immunization, we evaluated the immune response using lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged lethally. The results showed that mice immunized with pVAX-ROP16 developed a high level of specific antibody responses against T. gondii ROP16 expressed in Escherichia coli, a strong lymphoproliferative response, and significant levels of IFN-γ, IL-2, IL-4 and IL-10 production, compared with other mice immunized with either empty plasmid or phosphate-buffered saline, respectively. The results showed that pVAX-ROP16 induces significant humoral and cellular Th1 immune responses. After lethal challenge, the mice immunized with the pVAX-ROP16 showed a significantly (P values < 0.05) prolonged survival time (21.6 ± 9.9 days) compared with control mice which died within 7 days of challenge. Our data demonstrate, for the first time, that ROP16 triggered a strong humoral and cellular response against T. gondii, and that ROP16 is a promising vaccine candidate against toxoplasmosis, worth further development.
PMID: 21106780 [PubMed - as supplied by publisher]
Protective immunity induced by Toxoplasma gondii rhoptry protein 16 (ROP16) against toxoplasmosis in mice
Yuan ZG, Zhang XX, He XH, Petersen E, Zhou DH, He Y, Lin RQ, Li XZ, Chen XL, Shi XR, Zhong XL, Zhang B, Zhu XQ.
State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, CAAS, Lanzhou, Gansu Province 730046, PR China; Department of Parasitology, College of Veterinary Medicine, South China Agricultural University, Guangzhou, Guangdong Province 510642, PR China; College of Agriculture, South China Agricultural University, 483 Wushan Street, Tianhe District, Guangzhou, Guangdong Province 510642, PR China; Department of Infectious Diseases, Clinical Institute, and Institute of Medical Microbiology and Immunology, Faculty of Health Sciences, Aarhus University, Aarhus, Denmark; College of Animal Science and Technology, Yunnan Agricultural University, Kunming, Yunnan Province 650201, PR China.
Abstract
Toxoplasma gondii can infect a large variety of domestic and wild animals and human beings, sometimes causing severe pathology. Rhoptries are involved in T. gondii invasion and host cell interaction, and have been implicated as important virulence factors. In this study, we constructed a DNA vaccine expressing rhoptry protein 16 (ROP16) of T. gondii, and evaluated the immune responses it induced in Kunming mice. The gene sequence encoding ROP16 was inserted into the eukaryotic expression vector pVAX I. We immunized Kunming mice intramuscularly. After immunization, we evaluated the immune response using lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged lethally. The results showed that mice immunized with pVAX-ROP16 developed a high level of specific antibody responses against T. gondii ROP16 expressed in Escherichia coli, a strong lymphoproliferative response, and significant levels of IFN-γ, IL-2, IL-4 and IL-10 production, compared with other mice immunized with either empty plasmid or phosphate-buffered saline, respectively. The results showed that pVAX-ROP16 induces significant humoral and cellular Th1 immune responses. After lethal challenge, the mice immunized with the pVAX-ROP16 showed a significantly (P values < 0.05) prolonged survival time (21.6 ± 9.9 days) compared with control mice which died within 7 days of challenge. Our data demonstrate, for the first time, that ROP16 triggered a strong humoral and cellular response against T. gondii, and that ROP16 is a promising vaccine candidate against toxoplasmosis, worth further development.
PMID: 21106780 [PubMed - as supplied by publisher]
NALP1 Influences Susceptibility to Human Congenital Toxoplasmosis, Pro-Inflammatory Cytokine Response and Fate of T. gondii-Infected Monocytic Cells
Infect Immun. 2010 Nov 22. [Epub ahead of print]
NALP1 Influences Susceptibility to Human Congenital Toxoplasmosis, Pro-Inflammatory Cytokine Response and Fate of T. gondii-Infected Monocytic Cells
Witola WH, Mui E, Hargrave A, Liu S, Hypolite M, Montpetit A, Cavailles P, Bisanz C, Cesbron-Delauw MF, Fournié GJ, McLeod R.
Departments of Surgery (Ophthalmology) and Pediatrics (Infectious Disease), The University of Chicago, Chicago, Illinois 60637, USA; Laboratoire Adaptation et Pathogénie des Micro-organismes, CNRS UMR 5163, Université Joseph Fourier GRENOBLE 1, Institut Jean Roget, BP 170, 38042 Grenoble cedex 9, France; Centre d'Innovation, Génome Québec, Montréal, Québec H3A 1A4, Canada; INSERM, U563, F-31000 Toulouse, France and University Toulouse III Paul Sabatier, F-31000 Toulouse, France.
Abstract
NALP1 is a member of the NOD-like receptor (NLR) family proteins that form inflammasomes. Upon cellular infection or stress, inflammasomes are activated, triggering maturation of pro-inflammatory cytokines and downstream cellular signaling mediated through the MyD88 adaptor. Toxoplasma gondii is an obligate intracellular parasite that stimulates production of high levels of pro-inflammatory cytokines important in innate immunity. Herein, susceptibility alleles for human congenital toxoplasmosis in NALP1 gene were identified. To investigate the role of the NALP1 inflammasome during infection with T. gondii, we genetically engineered a human monocytic cell line for NALP1 gene knockdown by RNA interference. NALP1 silencing attenuated progression of T. gondii infection, with accelerated host cell death and eventual cell disintegration. In line with this observation, up-regulation of pro-inflammatory cytokines IL-1β, IL-18 and IL-12 with T. gondii infection was not observed in monocytic cells with NALP1 knockdown. These findings suggest that the NALP1 inflammasome is critical for mediating innate immune responses to T. gondii infection and pathogenesis. Although there have been recent advances in understanding the potent activity of inflammasomes in directing innate immune responses to disease, to our knowledge, this is the first report on the crucial role of NALP1 inflammasome in the pathogenesis of T. gondii infections in humans.
PMID: 21098108 [PubMed - as supplied by publisher]
NALP1 Influences Susceptibility to Human Congenital Toxoplasmosis, Pro-Inflammatory Cytokine Response and Fate of T. gondii-Infected Monocytic Cells
Witola WH, Mui E, Hargrave A, Liu S, Hypolite M, Montpetit A, Cavailles P, Bisanz C, Cesbron-Delauw MF, Fournié GJ, McLeod R.
Departments of Surgery (Ophthalmology) and Pediatrics (Infectious Disease), The University of Chicago, Chicago, Illinois 60637, USA; Laboratoire Adaptation et Pathogénie des Micro-organismes, CNRS UMR 5163, Université Joseph Fourier GRENOBLE 1, Institut Jean Roget, BP 170, 38042 Grenoble cedex 9, France; Centre d'Innovation, Génome Québec, Montréal, Québec H3A 1A4, Canada; INSERM, U563, F-31000 Toulouse, France and University Toulouse III Paul Sabatier, F-31000 Toulouse, France.
Abstract
NALP1 is a member of the NOD-like receptor (NLR) family proteins that form inflammasomes. Upon cellular infection or stress, inflammasomes are activated, triggering maturation of pro-inflammatory cytokines and downstream cellular signaling mediated through the MyD88 adaptor. Toxoplasma gondii is an obligate intracellular parasite that stimulates production of high levels of pro-inflammatory cytokines important in innate immunity. Herein, susceptibility alleles for human congenital toxoplasmosis in NALP1 gene were identified. To investigate the role of the NALP1 inflammasome during infection with T. gondii, we genetically engineered a human monocytic cell line for NALP1 gene knockdown by RNA interference. NALP1 silencing attenuated progression of T. gondii infection, with accelerated host cell death and eventual cell disintegration. In line with this observation, up-regulation of pro-inflammatory cytokines IL-1β, IL-18 and IL-12 with T. gondii infection was not observed in monocytic cells with NALP1 knockdown. These findings suggest that the NALP1 inflammasome is critical for mediating innate immune responses to T. gondii infection and pathogenesis. Although there have been recent advances in understanding the potent activity of inflammasomes in directing innate immune responses to disease, to our knowledge, this is the first report on the crucial role of NALP1 inflammasome in the pathogenesis of T. gondii infections in humans.
PMID: 21098108 [PubMed - as supplied by publisher]
UNC93B1 is essential for TLR11 activation and IL-12 dependent host resistance to Toxoplasma gondii
J Biol Chem. 2010 Nov 19. [Epub ahead of print]
UNC93B1 is essential for TLR11 activation and IL-12 dependent host resistance to Toxoplasma gondii
Pifer R, Benson A, Sturge CR, Yarovinsky F.
University of Texas Southwestern Medical Center at Dallas, United States.
Abstract
Toll-like receptor (TLR) activation relies on biochemical recognition of microbial molecules and localization of the TLR within specific cellular compartments. Cell surface TLRs largely recognize bacterial membrane components, and intracellular TLRs are exclusively involved in sensing nucleic acids. Here we show that TLR11, an innate sensor for the Toxoplasma protein profilin, is an intracellular receptor that resides in the endoplasmic reticulum. The twelve membrane-spanning endoplasmic reticulum-resident protein UNC93B1 interacts directly with TLR11 and regulates the activation of dendritic cells (DC) in response to T. gondii profilin and parasitic infection in vivo. A deficiency in functional UNC93B1 protein abolished TLR11-dependent IL-12 secretion by DC, attenuated Th1 responses against T. gondii, and dramatically enhanced susceptibility to the parasite. Our results reveal that the association with UNC93B1 and the intracellular localization of TLRs is not a unique feature of nucleic acid-sensing TLRs but is also essential for TLR11-dependent recognition of T. gondii profilin and for host protection against this parasite.
PMID: 21097503 [PubMed - as supplied by publisher]
UNC93B1 is essential for TLR11 activation and IL-12 dependent host resistance to Toxoplasma gondii
Pifer R, Benson A, Sturge CR, Yarovinsky F.
University of Texas Southwestern Medical Center at Dallas, United States.
Abstract
Toll-like receptor (TLR) activation relies on biochemical recognition of microbial molecules and localization of the TLR within specific cellular compartments. Cell surface TLRs largely recognize bacterial membrane components, and intracellular TLRs are exclusively involved in sensing nucleic acids. Here we show that TLR11, an innate sensor for the Toxoplasma protein profilin, is an intracellular receptor that resides in the endoplasmic reticulum. The twelve membrane-spanning endoplasmic reticulum-resident protein UNC93B1 interacts directly with TLR11 and regulates the activation of dendritic cells (DC) in response to T. gondii profilin and parasitic infection in vivo. A deficiency in functional UNC93B1 protein abolished TLR11-dependent IL-12 secretion by DC, attenuated Th1 responses against T. gondii, and dramatically enhanced susceptibility to the parasite. Our results reveal that the association with UNC93B1 and the intracellular localization of TLRs is not a unique feature of nucleic acid-sensing TLRs but is also essential for TLR11-dependent recognition of T. gondii profilin and for host protection against this parasite.
PMID: 21097503 [PubMed - as supplied by publisher]
Towards an immunosense vaccine to prevent toxoplasmosis: Protective Toxoplasma gondii epitopes restricted by HLA-A*0201
Vaccine. 2010 Nov 20. [Epub ahead of print]
Towards an immunosense vaccine to prevent toxoplasmosis: Protective Toxoplasma gondii epitopes restricted by HLA-A*0201
Cong H, Mui EJ, Witola WH, Sidney J, Alexander J, Sette A, Maewal A, McLeod R.
Department of Surgery, The University of Chicago, 5841 S. Maryland Ave., MC 2114, Chicago, IL 60637, USA.
Abstract
The ideal vaccine to protect against toxoplasmosis in humans would include antigens that elicit a protective T helper cell type 1 immune response, and generate long-lived IFN-γ-producing CD8(+) T cells. Herein, we utilized a predictive algorithm to identify candidate HLA-A02 supertype epitopes from Toxoplasma gondii proteins. Thirteen peptides elicited production of IFN-γ from PBMC of HLA-A02 supertype persons seropositive for T. gondii infection but not from seronegative controls. These peptides displayed high-affinity binding to HLA-A02 proteins. Immunization of HLA-A*0201 transgenic mice with these pooled peptides, with a universal CD4(+) epitope peptide called PADRE, formulated with adjuvant GLA-SE, induced CD8(+) T cell IFN-γ production and protected against parasite challenge. Peptides identified in this study provide candidates for inclusion in immunosense epitope-based vaccines.
Copyright © 2010. Published by Elsevier Ltd.
PMID: 21095258 [PubMed - as supplied by publisher]
Towards an immunosense vaccine to prevent toxoplasmosis: Protective Toxoplasma gondii epitopes restricted by HLA-A*0201
Cong H, Mui EJ, Witola WH, Sidney J, Alexander J, Sette A, Maewal A, McLeod R.
Department of Surgery, The University of Chicago, 5841 S. Maryland Ave., MC 2114, Chicago, IL 60637, USA.
Abstract
The ideal vaccine to protect against toxoplasmosis in humans would include antigens that elicit a protective T helper cell type 1 immune response, and generate long-lived IFN-γ-producing CD8(+) T cells. Herein, we utilized a predictive algorithm to identify candidate HLA-A02 supertype epitopes from Toxoplasma gondii proteins. Thirteen peptides elicited production of IFN-γ from PBMC of HLA-A02 supertype persons seropositive for T. gondii infection but not from seronegative controls. These peptides displayed high-affinity binding to HLA-A02 proteins. Immunization of HLA-A*0201 transgenic mice with these pooled peptides, with a universal CD4(+) epitope peptide called PADRE, formulated with adjuvant GLA-SE, induced CD8(+) T cell IFN-γ production and protected against parasite challenge. Peptides identified in this study provide candidates for inclusion in immunosense epitope-based vaccines.
Copyright © 2010. Published by Elsevier Ltd.
PMID: 21095258 [PubMed - as supplied by publisher]
Evaluation of the immune response induced by DNA vaccine cocktail expressing complete SAG1 and ROP2 genes against toxoplasmosis
Vaccine. 2010 Nov 20. [Epub ahead of print]
Evaluation of the immune response induced by DNA vaccine cocktail expressing complete SAG1 and ROP2 genes against toxoplasmosis
Hoseinian Khosroshahi K, Ghaffarifar F, D'Souza S, Sharifi Z, Dalimi A.
Dept. Parasitology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Abstract
Toxoplasma gondii, the pathogen of toxoplasmosis, can infect most mammals and birds. The high incidence and severe or lethal damages of toxoplasmosis clearly indicate the need for the development of a more effective vaccine. We constructed a DNA cocktail, containing plasmids encoding the full-length SAG1 and ROP2 genes of T. gondii and evaluated its immune response and protective efficacy in comparison with single-gene vaccines and control groups. We immunized BALB/c mice intramuscularly three times. DNA cocktail elicited IgG and IFN-γ, TNF-α and IL-2 greater than single-gene plasmids and increased survival time against a lethal challenge with the highly virulent T. gondii RH strain. The current study shows that pc-SAG1+ pc-ROP2 as a cocktail DNA vaccine produces higher Th1 immune response than single-gene plasmids and cocktail DNA is effective to prime an enhanced and balanced specific immunity.
Copyright © 2010. Published by Elsevier Ltd.
PMID: 21095254 [PubMed - as supplied by publisher]
Evaluation of the immune response induced by DNA vaccine cocktail expressing complete SAG1 and ROP2 genes against toxoplasmosis
Hoseinian Khosroshahi K, Ghaffarifar F, D'Souza S, Sharifi Z, Dalimi A.
Dept. Parasitology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Abstract
Toxoplasma gondii, the pathogen of toxoplasmosis, can infect most mammals and birds. The high incidence and severe or lethal damages of toxoplasmosis clearly indicate the need for the development of a more effective vaccine. We constructed a DNA cocktail, containing plasmids encoding the full-length SAG1 and ROP2 genes of T. gondii and evaluated its immune response and protective efficacy in comparison with single-gene vaccines and control groups. We immunized BALB/c mice intramuscularly three times. DNA cocktail elicited IgG and IFN-γ, TNF-α and IL-2 greater than single-gene plasmids and increased survival time against a lethal challenge with the highly virulent T. gondii RH strain. The current study shows that pc-SAG1+ pc-ROP2 as a cocktail DNA vaccine produces higher Th1 immune response than single-gene plasmids and cocktail DNA is effective to prime an enhanced and balanced specific immunity.
Copyright © 2010. Published by Elsevier Ltd.
PMID: 21095254 [PubMed - as supplied by publisher]
Segmental Meningomyelitis in 2 Cats Caused by Toxoplasma gondii
J Vet Intern Med. 2010 Nov 23. doi: 10.1111/j.1939-1676.2010.0635.x. [Epub ahead of print]
Segmental Meningomyelitis in 2 Cats Caused by Toxoplasma gondii
Alves L, Gorgas D, Vandevelde M, Gandini G, Henke D.
Department of Clinical Veterinary Medicine, Division of Clinical Neurology, Vetsuisse Faculty, University of Berne, Berne, Switzerland Division of Clinical Radiology, Vetsuisse Faculty, University of Berne, Berne, Switzerland Department of Veterinary Clinics, Faculty of Veterinary Medicine, University of Bologna, Bologna, Italy.
PMID: 21092003 [PubMed - as supplied by publisher]
Segmental Meningomyelitis in 2 Cats Caused by Toxoplasma gondii
Alves L, Gorgas D, Vandevelde M, Gandini G, Henke D.
Department of Clinical Veterinary Medicine, Division of Clinical Neurology, Vetsuisse Faculty, University of Berne, Berne, Switzerland Division of Clinical Radiology, Vetsuisse Faculty, University of Berne, Berne, Switzerland Department of Veterinary Clinics, Faculty of Veterinary Medicine, University of Bologna, Bologna, Italy.
PMID: 21092003 [PubMed - as supplied by publisher]
Monday, November 22, 2010
Evaluation of three recombinant multi-antigenic vaccines composed of surface and secretory antigens of Toxoplasma gondii
Vaccine. 2010 Nov 15. [Epub ahead of print]
Evaluation of three recombinant multi-antigenic vaccines composed of surface and secretory antigens of Toxoplasma gondii in murine models of experimental toxoplasmosis
Dziadek B, Gatkowska J, Brzostek A, Dziadek J, Dzitko K, Grzybowski M, Dlugonska H.
Department of Immunoparasitology, University of Lodz, Lodz, Poland.
Abstract
The great clinical and economical impact of Toxoplasma gondii infections makes the development of an effective vaccine for controlling toxoplasmosis an extremely important aim. In the present study, we evaluate the protective and immunogenic properties of three recombinant subunit vaccines composed of rROP2+rGRA4+rSAG1, rROP2+rROP4+rGRA4 and rROP2+rROP4+rSAG1 proteins of T. gondii in an experimental toxoplasmosis model in the C3H/HeJ and C57BL/6 mouse strains. All three recombinant vaccines induced partial protection as measured by the reduction of brain cyst burden following challenge with five tissue cysts of the low virulence DX T. gondii strain. The level of protection was dependent on the antigen composition of the vaccine and the genetic background of the laboratory animals. The strongest protection against chronic toxoplasmosis was induced in both C3H/HeJ and C57BL/6 mice by the mixture of rhoptry proteins rROP2 and rROP4 combined with tachyzoite major protein SAG1. The average parasite burden in these groups of mice was reduced by 71% and 90%, respectively, compared to non-vaccinated mice. The observed protective effect was related to the vaccine-induced cellular and humoral immune responses, as measured by the antigen-induced release of the Th1 cytokines IFN-γ and IL-2, the antigen-stimulated proliferation of spleen cells of vaccinated animals in comparison to control animals and the development of systemic antigen-specific IgG1 and IgG2a (C3H/HeJ) or IgG2c (C57BL/6) antibodies. Our studies show that recombinant rROP2, rROP4, rGRA4 and rSAG1 antigens may be promising candidates for a subunit vaccine against toxoplasmosis. Additionally, we demonstrate that the ideal composition of vaccine antigens can be equally effective in mice with different genetic backgrounds and variable levels of innate resistance to toxoplasmosis, resulting in strong protection against T. gondii invasion.
Copyright © 2010. Published by Elsevier Ltd.
PMID: 21087690 [PubMed - as supplied by publisher]
Evaluation of three recombinant multi-antigenic vaccines composed of surface and secretory antigens of Toxoplasma gondii in murine models of experimental toxoplasmosis
Dziadek B, Gatkowska J, Brzostek A, Dziadek J, Dzitko K, Grzybowski M, Dlugonska H.
Department of Immunoparasitology, University of Lodz, Lodz, Poland.
Abstract
The great clinical and economical impact of Toxoplasma gondii infections makes the development of an effective vaccine for controlling toxoplasmosis an extremely important aim. In the present study, we evaluate the protective and immunogenic properties of three recombinant subunit vaccines composed of rROP2+rGRA4+rSAG1, rROP2+rROP4+rGRA4 and rROP2+rROP4+rSAG1 proteins of T. gondii in an experimental toxoplasmosis model in the C3H/HeJ and C57BL/6 mouse strains. All three recombinant vaccines induced partial protection as measured by the reduction of brain cyst burden following challenge with five tissue cysts of the low virulence DX T. gondii strain. The level of protection was dependent on the antigen composition of the vaccine and the genetic background of the laboratory animals. The strongest protection against chronic toxoplasmosis was induced in both C3H/HeJ and C57BL/6 mice by the mixture of rhoptry proteins rROP2 and rROP4 combined with tachyzoite major protein SAG1. The average parasite burden in these groups of mice was reduced by 71% and 90%, respectively, compared to non-vaccinated mice. The observed protective effect was related to the vaccine-induced cellular and humoral immune responses, as measured by the antigen-induced release of the Th1 cytokines IFN-γ and IL-2, the antigen-stimulated proliferation of spleen cells of vaccinated animals in comparison to control animals and the development of systemic antigen-specific IgG1 and IgG2a (C3H/HeJ) or IgG2c (C57BL/6) antibodies. Our studies show that recombinant rROP2, rROP4, rGRA4 and rSAG1 antigens may be promising candidates for a subunit vaccine against toxoplasmosis. Additionally, we demonstrate that the ideal composition of vaccine antigens can be equally effective in mice with different genetic backgrounds and variable levels of innate resistance to toxoplasmosis, resulting in strong protection against T. gondii invasion.
Copyright © 2010. Published by Elsevier Ltd.
PMID: 21087690 [PubMed - as supplied by publisher]
Friday, November 19, 2010
New details on the fine structure of the rhoptry of Toxoplasma gondii
Microsc Res Tech. 2010 Nov 17. [Epub ahead of print]
New details on the fine structure of the rhoptry of Toxoplasma gondii
Lemgruber L, Lupetti P, De Souza W, Vommaro RC.
Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil.
Abstract
Rhoptries are organelles that have important, complex roles in Apicomplexa biology. During Toxoplasma gondii infection, these organelles take part in several essential and complex processes that include host cell entry and parasite development. Using different electron microscopy techniques, we characterized the fine morphology of the rhoptries of two of the most important life stages of T. gondii: the tachyzoite and the bradyzoite forms. The observed tachyzoite and bradyzoite rhoptries had delimited regions characterized by a dark and electron-dense neck, an amorphous and less electron-dense bulb, and a region of intermediate electron density, which connects the bulb to the neck. Metal replicas of frozen-fractured tachyzoites showed intramembranous particles of different densities and sizes on the fractured faces of rhoptry membranes. Both in tachyzoites and bradyzoites, the intramembranous particles were arranged in distinctive parallel arrays that decorated most part of these organelles. Tubulo-vesicular subcompartments and free particles within the rhoptry lumen were observed on freeze-fractured replicas. Cryo-fixed, deep-etched samples showed several pore-like structures localized in the bulb portion. No obvious evidence was found of a possible connection between rhoptries and micronemes. Microsc. Res. Tech., 2010. © 2010 Wiley-Liss, Inc.
PMID: 21086448 [PubMed - as supplied by publisher]
New details on the fine structure of the rhoptry of Toxoplasma gondii
Lemgruber L, Lupetti P, De Souza W, Vommaro RC.
Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil.
Abstract
Rhoptries are organelles that have important, complex roles in Apicomplexa biology. During Toxoplasma gondii infection, these organelles take part in several essential and complex processes that include host cell entry and parasite development. Using different electron microscopy techniques, we characterized the fine morphology of the rhoptries of two of the most important life stages of T. gondii: the tachyzoite and the bradyzoite forms. The observed tachyzoite and bradyzoite rhoptries had delimited regions characterized by a dark and electron-dense neck, an amorphous and less electron-dense bulb, and a region of intermediate electron density, which connects the bulb to the neck. Metal replicas of frozen-fractured tachyzoites showed intramembranous particles of different densities and sizes on the fractured faces of rhoptry membranes. Both in tachyzoites and bradyzoites, the intramembranous particles were arranged in distinctive parallel arrays that decorated most part of these organelles. Tubulo-vesicular subcompartments and free particles within the rhoptry lumen were observed on freeze-fractured replicas. Cryo-fixed, deep-etched samples showed several pore-like structures localized in the bulb portion. No obvious evidence was found of a possible connection between rhoptries and micronemes. Microsc. Res. Tech., 2010. © 2010 Wiley-Liss, Inc.
PMID: 21086448 [PubMed - as supplied by publisher]
Tuesday, November 09, 2010
Regions of intrinsic disorder help identify a novel nuclear localization signal in Toxoplasma gondii histone acetyltransferase TgGCN5-B
Mol Biochem Parasitol. 2010 Nov 2. [Epub ahead of print]
Regions of intrinsic disorder help identify a novel nuclear localization signal in Toxoplasma gondii histone acetyltransferase TgGCN5-B
Dixon SE, Bhatti MM, Uversky VN, Dunker AK, Sullivan WJ Jr.
Department of Pharmacology and Toxicology.
Abstract
We have previously shown that protozoan parasites, such as Toxoplasma gondii, contain a high prevalence of intrinsically disordered regions in their predicted proteins. Here, we determine that both TgGCN5-family histone acetyltransferases (HATs) contain unusually high levels of intrinsic disorder. A previously identified basic-rich nuclear localization signal (NLS) in the N-terminus of TgGCN5-A is located within such a region of predicted disorder, but this NLS is not conserved in TgGCN5-B. We therefore analyzed the intrinsically disordered regions of TgGCN5-B for basic-rich sequences that could be indicative of a functional NLS, and this led to the identification of a novel NLS for TgGCN5-B, RPAENKKRGR. The functionality of the GCN5-B NLS was validated experimentally and has predictive value. These studies demonstrate that basic-rich sequences within regions predicted to be intrinsically disordered constitute criteria for a candidate NLS.
Copyright © 2010. Published by Elsevier B.V.
PMID: 21055425 [PubMed - as supplied by publisher]
Regions of intrinsic disorder help identify a novel nuclear localization signal in Toxoplasma gondii histone acetyltransferase TgGCN5-B
Dixon SE, Bhatti MM, Uversky VN, Dunker AK, Sullivan WJ Jr.
Department of Pharmacology and Toxicology.
Abstract
We have previously shown that protozoan parasites, such as Toxoplasma gondii, contain a high prevalence of intrinsically disordered regions in their predicted proteins. Here, we determine that both TgGCN5-family histone acetyltransferases (HATs) contain unusually high levels of intrinsic disorder. A previously identified basic-rich nuclear localization signal (NLS) in the N-terminus of TgGCN5-A is located within such a region of predicted disorder, but this NLS is not conserved in TgGCN5-B. We therefore analyzed the intrinsically disordered regions of TgGCN5-B for basic-rich sequences that could be indicative of a functional NLS, and this led to the identification of a novel NLS for TgGCN5-B, RPAENKKRGR. The functionality of the GCN5-B NLS was validated experimentally and has predictive value. These studies demonstrate that basic-rich sequences within regions predicted to be intrinsically disordered constitute criteria for a candidate NLS.
Copyright © 2010. Published by Elsevier B.V.
PMID: 21055425 [PubMed - as supplied by publisher]
Sunday, November 07, 2010
Full-parasites: database of full-length cDNAs of apicomplexa parasites, 2010 update
Nucleic Acids Res. 2010 Nov 4. [Epub ahead of print]
Full-parasites: database of full-length cDNAs of apicomplexa parasites, 2010 update
Tuda J, Mongan AE, Tolba ME, Imada M, Yamagishi J, Xuan X, Wakaguri H, Sugano S, Sugimoto C, Suzuki Y.
Faculty of Medicine, Sam Ratulangi University, Kampus Unsrat, Bahu Manado, 95115, Indonesia, Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan, Department of Parasitology, Assiut University, Assiut, 71515, Egypt, Department of Tropical Medicine and Parasitology School of Medicine, Keio University, 35 Shinanomachi Sinjuku, Tokyo 160-8582, National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho west 2-13, Obihiro, Hokkaido 080-8555 and Research Center for Zoonosis Control, Hokkaido University, North 20, West 10 Kita-ku, Sapporo 001-0020, Japan.
Abstract
Full-Parasites (http://fullmal.hgc.jp/) is a transcriptome database of apicomplexa parasites, which include Plasmodium and Toxoplasma species. The latest version of Full-Parasites contains a total of 105 786 EST sequences from 12 parasites, of which 5925 full-length cDNAs have been completely sequenced. Full-Parasites also contain more than 30 million transcription start sites (TSS) for Plasmodium falciparum (Pf) and Toxoplasma gondii (Tg), which were identified using our novel oligo-capping-based protocol. Various types of cDNA data resources were interconnected with our original database functionalities. Specifically, in this update, we have included two unique RNA-Seq data sets consisting of 730 million mapped RNA-Seq tags. One is a dataset of 16 time-lapse experiments of cultured bradyzoite differentiation for Tg. The other dataset includes 31 clinical samples of Pf. Parasite RNA was extracted together with host human RNA, and the extracted mixed RNA was used for RNA sequencing, with the expectation that gene expression information from the host and parasite would be simultaneously represented. By providing the largest unique full-length cDNA and dynamic transcriptome data, Full-Parasites is useful for understanding host-parasite interactions and will help to eventually elucidate how monophyletic organisms have evolved to become parasites by adopting complex life cycles.
PMID: 21051343 [PubMed - as supplied by publisher]
Full-parasites: database of full-length cDNAs of apicomplexa parasites, 2010 update
Tuda J, Mongan AE, Tolba ME, Imada M, Yamagishi J, Xuan X, Wakaguri H, Sugano S, Sugimoto C, Suzuki Y.
Faculty of Medicine, Sam Ratulangi University, Kampus Unsrat, Bahu Manado, 95115, Indonesia, Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan, Department of Parasitology, Assiut University, Assiut, 71515, Egypt, Department of Tropical Medicine and Parasitology School of Medicine, Keio University, 35 Shinanomachi Sinjuku, Tokyo 160-8582, National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho west 2-13, Obihiro, Hokkaido 080-8555 and Research Center for Zoonosis Control, Hokkaido University, North 20, West 10 Kita-ku, Sapporo 001-0020, Japan.
Abstract
Full-Parasites (http://fullmal.hgc.jp/) is a transcriptome database of apicomplexa parasites, which include Plasmodium and Toxoplasma species. The latest version of Full-Parasites contains a total of 105 786 EST sequences from 12 parasites, of which 5925 full-length cDNAs have been completely sequenced. Full-Parasites also contain more than 30 million transcription start sites (TSS) for Plasmodium falciparum (Pf) and Toxoplasma gondii (Tg), which were identified using our novel oligo-capping-based protocol. Various types of cDNA data resources were interconnected with our original database functionalities. Specifically, in this update, we have included two unique RNA-Seq data sets consisting of 730 million mapped RNA-Seq tags. One is a dataset of 16 time-lapse experiments of cultured bradyzoite differentiation for Tg. The other dataset includes 31 clinical samples of Pf. Parasite RNA was extracted together with host human RNA, and the extracted mixed RNA was used for RNA sequencing, with the expectation that gene expression information from the host and parasite would be simultaneously represented. By providing the largest unique full-length cDNA and dynamic transcriptome data, Full-Parasites is useful for understanding host-parasite interactions and will help to eventually elucidate how monophyletic organisms have evolved to become parasites by adopting complex life cycles.
PMID: 21051343 [PubMed - as supplied by publisher]
The immunity-related GTPases in mammals: a fast-evolving cell-autonomous resistance system against intracellular pathogens
Mamm Genome. 2010 Oct 30. [Epub ahead of print]
The immunity-related GTPases in mammals: a fast-evolving cell-autonomous resistance system against intracellular pathogens
Hunn JP, Feng CG, Sher A, Howard JC.
Institute for Genetics, University of Cologne, Zuelpicher Str. 47a, 50674, Cologne, Germany.
Abstract
The immunity-related GTPases (IRGs) belong to the family of large, interferon-inducible GTPases and constitute a cell-autonomous resistance system essential for the control of vacuolar pathogens like Toxoplasma gondii in mice. Recent results demonstrated that numerous IRG members accumulate collaboratively at the parasitophorous vacuole of invading T. gondii leading to the destruction of the vacuole and the parasite and subsequent necrotic host cell death. Complex regulatory interactions between different IRG proteins are necessary for these processes. Disturbance of this finely balanced system, e.g., by single genetic deficiency for the important negative regulator Irgm1 or the autophagic regulator Atg5, leads to spontaneous activation of the effector IRG proteins when induced by IFNγ. This activation has cytotoxic consequences resulting in a severe lymphopenia, macrophage defects, and failure of the adaptive immune system in Irgm1-deficient mice. However, alternative functions in phagosome maturation and induction of autophagy have been proposed for Irgm1. The IRG system has been studied primarily in mice, but IRG genes are present throughout the mammalian lineage. Interestingly, the number, type, and diversity of genes present differ greatly even between closely related species, probably reflecting intimate host-pathogen coevolution driven by an armed race between the IRG resistance proteins and pathogen virulence factors. IRG proteins are targets for polymorphic T. gondii virulence factors, and genetic variation in the IRG system between different mouse strains correlates with resistance and susceptibility to virulent T. gondii strains.
PMID: 21052678 [PubMed - as supplied by publisher]
The immunity-related GTPases in mammals: a fast-evolving cell-autonomous resistance system against intracellular pathogens
Hunn JP, Feng CG, Sher A, Howard JC.
Institute for Genetics, University of Cologne, Zuelpicher Str. 47a, 50674, Cologne, Germany.
Abstract
The immunity-related GTPases (IRGs) belong to the family of large, interferon-inducible GTPases and constitute a cell-autonomous resistance system essential for the control of vacuolar pathogens like Toxoplasma gondii in mice. Recent results demonstrated that numerous IRG members accumulate collaboratively at the parasitophorous vacuole of invading T. gondii leading to the destruction of the vacuole and the parasite and subsequent necrotic host cell death. Complex regulatory interactions between different IRG proteins are necessary for these processes. Disturbance of this finely balanced system, e.g., by single genetic deficiency for the important negative regulator Irgm1 or the autophagic regulator Atg5, leads to spontaneous activation of the effector IRG proteins when induced by IFNγ. This activation has cytotoxic consequences resulting in a severe lymphopenia, macrophage defects, and failure of the adaptive immune system in Irgm1-deficient mice. However, alternative functions in phagosome maturation and induction of autophagy have been proposed for Irgm1. The IRG system has been studied primarily in mice, but IRG genes are present throughout the mammalian lineage. Interestingly, the number, type, and diversity of genes present differ greatly even between closely related species, probably reflecting intimate host-pathogen coevolution driven by an armed race between the IRG resistance proteins and pathogen virulence factors. IRG proteins are targets for polymorphic T. gondii virulence factors, and genetic variation in the IRG system between different mouse strains correlates with resistance and susceptibility to virulent T. gondii strains.
PMID: 21052678 [PubMed - as supplied by publisher]
Thursday, November 04, 2010
Dysregulation of the inflammatory response to the parasite, Toxoplasma gondii, in P2X(7) receptor-deficient mice
Int J Parasitol. 2010 Oct 30. [Epub ahead of print]
Dysregulation of the inflammatory response to the parasite, Toxoplasma gondii, in P2X(7) receptor-deficient mice
Miller CM, Zakrzewski AM, Ikin RJ, Boulter NR, Katrib M, Lees MP, Fuller SJ, Wiley JS, Smith NC.
Institute for the Biotechnology of Infectious Diseases, University of Technology, Sydney, Broadway, NSW, 2007, Australia.
Abstract
The P2X(7) receptor (P2X(7)R) is a two transmembrane receptor that is highly expressed on the surface of immune cells. Loss of function polymorphisms in this receptor have been linked to increased susceptibility to intracellular pathogens. P2X(7)R gene knockout (P2X(7)R(-/-); on a C57Bl/6J background), C57Bl/6J and BALB/c mice were infected with the avirulent ME49 strain of the intracellular parasite, Toxoplasma gondii, and susceptibility determined by monitoring weight loss. P2X(7)R(-/-) mice lost significantly more weight than C57Bl/6J mice from day 8 p.i.. C57Bl/6J, in turn, lost significantly more weight than BALB/c mice. Thus, by day 10 p.i., P2X(7)R(-/-) mice had lost 5.7 ± 0.7% of their weight versus 2.4 ± 0.6% for C57Bl/6J mice, whereas BALB/c mice had gained 1.9 ± 0.5%; by day 12 p.i., P2X(7)R(-/-) mice had lost 15.1 ± 0.6%, C57Bl/6J had lost 10.1 ± 0.8% and BALB/c had lost 4.8±0.8% of their weight. Neither parasite burden nor liver pathology was greater in the P2X(7)R(-/-) mice than in C57Bl/6J mice but BALB/c mice had significantly smaller numbers of parasites and less pathology in their livers than these strains. Absence of the P2X(7) receptor did not affect IFN-γ, IL-12, IL-1β, monocyte chemoattractant protein-1 (MCP-1) or TNF production. However, both P2X(7)R(-/-) and C57Bl/6J mice produced more IL-1 β and TNF than BALB/c mice. There was one important point of differentiation between the P2X(7)R(-/-) and C57Bl/6J mice, namely the significantly enhanced and prolonged production of nitric oxide, accompanied by delayed production of IL-10 in the P2X(7)R-deficient mice.
Copyright © 2010. Published by Elsevier Ltd.
PMID: 21044631 [PubMed - as supplied by publisher]
Dysregulation of the inflammatory response to the parasite, Toxoplasma gondii, in P2X(7) receptor-deficient mice
Miller CM, Zakrzewski AM, Ikin RJ, Boulter NR, Katrib M, Lees MP, Fuller SJ, Wiley JS, Smith NC.
Institute for the Biotechnology of Infectious Diseases, University of Technology, Sydney, Broadway, NSW, 2007, Australia.
Abstract
The P2X(7) receptor (P2X(7)R) is a two transmembrane receptor that is highly expressed on the surface of immune cells. Loss of function polymorphisms in this receptor have been linked to increased susceptibility to intracellular pathogens. P2X(7)R gene knockout (P2X(7)R(-/-); on a C57Bl/6J background), C57Bl/6J and BALB/c mice were infected with the avirulent ME49 strain of the intracellular parasite, Toxoplasma gondii, and susceptibility determined by monitoring weight loss. P2X(7)R(-/-) mice lost significantly more weight than C57Bl/6J mice from day 8 p.i.. C57Bl/6J, in turn, lost significantly more weight than BALB/c mice. Thus, by day 10 p.i., P2X(7)R(-/-) mice had lost 5.7 ± 0.7% of their weight versus 2.4 ± 0.6% for C57Bl/6J mice, whereas BALB/c mice had gained 1.9 ± 0.5%; by day 12 p.i., P2X(7)R(-/-) mice had lost 15.1 ± 0.6%, C57Bl/6J had lost 10.1 ± 0.8% and BALB/c had lost 4.8±0.8% of their weight. Neither parasite burden nor liver pathology was greater in the P2X(7)R(-/-) mice than in C57Bl/6J mice but BALB/c mice had significantly smaller numbers of parasites and less pathology in their livers than these strains. Absence of the P2X(7) receptor did not affect IFN-γ, IL-12, IL-1β, monocyte chemoattractant protein-1 (MCP-1) or TNF production. However, both P2X(7)R(-/-) and C57Bl/6J mice produced more IL-1 β and TNF than BALB/c mice. There was one important point of differentiation between the P2X(7)R(-/-) and C57Bl/6J mice, namely the significantly enhanced and prolonged production of nitric oxide, accompanied by delayed production of IL-10 in the P2X(7)R-deficient mice.
Copyright © 2010. Published by Elsevier Ltd.
PMID: 21044631 [PubMed - as supplied by publisher]
Crystallization and preliminary X-ray structural analysis of nucleoside triphosphate hydrolases from Neospora caninum and Toxoplasma gondii
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2010 Nov 1;66(Pt 11):1445-8. Epub 2010 Oct 28.
Crystallization and preliminary X-ray structural analysis of nucleoside triphosphate hydrolases from Neospora caninum and Toxoplasma gondii
Matoba K, Shiba T, Takeuchi T, Sibley LD, Seiki M, Kikyo F, Horiuchi T, Asai T, Harada S.
Department of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto 606-8585, Japan.
Abstract
The nucleoside triphosphate hydrolases that are produced by Neospora caninum (NcNTPase) and Toxoplasma gondii (TgNTPase-I) have a different physiological function from the ubiquitous ecto-ATPases. The recombinant enzymes were crystallized at 293 K using polyethylene glycol 3350 as a precipitant and X-ray diffraction data sets were collected for NcNTPase (to 2.8 Å resolution) and TgNTPase-I (to 3.1 Å resolution) at 100 K using synchrotron radiation. The crystals of NcNTPase and TgNTPase-I belonged to the orthorhombic space group I222 (unit-cell parameters a = 93.6, b = 140.8, c = 301.1 Å) and the monoclinic space group P2(1) (unit-cell parameters a = 87.1, b = 123.5, c = 120.2 Å, β = 96.6°), respectively, with two NcNTPase (V(M) = 3.7 Å(3) Da(-1)) and four TgNTPase-I (V(M) = 2.7 Å(3) Da(-1)) molecules per asymmetric unit. SAD phasing trials using a data set (λ = 0.97904 Å) collected from a crystal of selenomethionylated NcNTPase gave an initial electron-density map of sufficient quality to build a molecular model of NcNTPase.
PMID: 21045291 [PubMed - in process]
Crystallization and preliminary X-ray structural analysis of nucleoside triphosphate hydrolases from Neospora caninum and Toxoplasma gondii
Matoba K, Shiba T, Takeuchi T, Sibley LD, Seiki M, Kikyo F, Horiuchi T, Asai T, Harada S.
Department of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto 606-8585, Japan.
Abstract
The nucleoside triphosphate hydrolases that are produced by Neospora caninum (NcNTPase) and Toxoplasma gondii (TgNTPase-I) have a different physiological function from the ubiquitous ecto-ATPases. The recombinant enzymes were crystallized at 293 K using polyethylene glycol 3350 as a precipitant and X-ray diffraction data sets were collected for NcNTPase (to 2.8 Å resolution) and TgNTPase-I (to 3.1 Å resolution) at 100 K using synchrotron radiation. The crystals of NcNTPase and TgNTPase-I belonged to the orthorhombic space group I222 (unit-cell parameters a = 93.6, b = 140.8, c = 301.1 Å) and the monoclinic space group P2(1) (unit-cell parameters a = 87.1, b = 123.5, c = 120.2 Å, β = 96.6°), respectively, with two NcNTPase (V(M) = 3.7 Å(3) Da(-1)) and four TgNTPase-I (V(M) = 2.7 Å(3) Da(-1)) molecules per asymmetric unit. SAD phasing trials using a data set (λ = 0.97904 Å) collected from a crystal of selenomethionylated NcNTPase gave an initial electron-density map of sufficient quality to build a molecular model of NcNTPase.
PMID: 21045291 [PubMed - in process]
An insertional trap for coditional gene expression in Toxoplasma gondii: identification of TAF250 as an essential gene
Mol Biochem Parasitol. 2010 Oct 27. [Epub ahead of print]
An insertional trap for coditional gene expression in Toxoplasma gondii: identification of TAF250 as an essential gene
Jammallo L, Eidell K, Davis PH, Dufort FJ, Cronin C, Thirugnanam S, Chiles TC, Roos DS, Gubbels MJ.
Department of Biology, Boston College, Chestnut Hill, MA.
Abstract
Toxoplasmosis is characterized by fast lytic replication cycles leading to severe tissue lesions. Successful host cell invasion is essential for pathogenesis. The division cycle of Toxoplasma gondii is characterized by an unusual cell cycle progression and a distinct internal budding mechanism. To identify essential genes involved in the lytic cycle we devised an insertional gene trapping strategy using the Tet-transactivator system. In essence, a random, active promoter is displaced with a tetracycline regulatable promoter, which if in an essential gene, will result in a conditionally lethal phenotype upon tetracycline addition. We isolated eight mutants with growth defects, two of which displayed modest invasion defects, one of which had an additional cell cycle defect. The trapped loci were identified using expression microarrays, exploiting the tetracycline dependent expression of the trapped genes. In mutant 3.3H6 we identified TCP-1, a component of the chaperonin protein folding machinery under the control of the Tet promoter. However, this gene was not critical for growth of mutant 3.3H6. Subsequently, we identified a suppressor gene encoding a protein with a hypothetical function by guided cosmid complementation. In mutant 4.3B13, we identified TAF250, an RNA polymerase II complex component, as the trapped, essential gene. Furthermore, by mapping the plasmid insertion boundaries we identified multiple genomic rearrangements, which hint at a potential replication dependent DNA repair mechanism. Furthermore, these rearrangements provide an explanation for inconsistent locus rescue results observed by molecular biological approaches. Taken together, we have added an approach to identify and study essential genes in Toxoplasma.
An insertional trap for coditional gene expression in Toxoplasma gondii: identification of TAF250 as an essential gene
Jammallo L, Eidell K, Davis PH, Dufort FJ, Cronin C, Thirugnanam S, Chiles TC, Roos DS, Gubbels MJ.
Department of Biology, Boston College, Chestnut Hill, MA.
Abstract
Toxoplasmosis is characterized by fast lytic replication cycles leading to severe tissue lesions. Successful host cell invasion is essential for pathogenesis. The division cycle of Toxoplasma gondii is characterized by an unusual cell cycle progression and a distinct internal budding mechanism. To identify essential genes involved in the lytic cycle we devised an insertional gene trapping strategy using the Tet-transactivator system. In essence, a random, active promoter is displaced with a tetracycline regulatable promoter, which if in an essential gene, will result in a conditionally lethal phenotype upon tetracycline addition. We isolated eight mutants with growth defects, two of which displayed modest invasion defects, one of which had an additional cell cycle defect. The trapped loci were identified using expression microarrays, exploiting the tetracycline dependent expression of the trapped genes. In mutant 3.3H6 we identified TCP-1, a component of the chaperonin protein folding machinery under the control of the Tet promoter. However, this gene was not critical for growth of mutant 3.3H6. Subsequently, we identified a suppressor gene encoding a protein with a hypothetical function by guided cosmid complementation. In mutant 4.3B13, we identified TAF250, an RNA polymerase II complex component, as the trapped, essential gene. Furthermore, by mapping the plasmid insertion boundaries we identified multiple genomic rearrangements, which hint at a potential replication dependent DNA repair mechanism. Furthermore, these rearrangements provide an explanation for inconsistent locus rescue results observed by molecular biological approaches. Taken together, we have added an approach to identify and study essential genes in Toxoplasma.
Thursday, October 28, 2010
A novel multifunctional oligonucleotide microarray for Toxoplasma gondii
BMC Genomics. 2010 Oct 25;11(1):603. [Epub ahead of print]
A novel multifunctional oligonucleotide microarray for Toxoplasma gondii
Bahl A, Davis PH, Behnke M, Dzierszinski F, Jagalur M, Chen F, Shanmugam D, White M, Kulp D, Roos DS.
Abstract
ABSTRACT:
BACKGROUND: Microarrays are invaluable tools for genome interrogation, SNP detection, and expression analysis, among other applications. Such broad capabilities would be of value to many pathogen research communities, although the development and use of genome-scale microarrays is often a prohibitively expensive undertaking.
RESULTS: Taking advantage of available genome sequences and annotation for Toxoplasma gondii (the causative agent of opportunistic illness in immunocompromised individuals) and Plasmodium falciparum (a related parasite responsible for severe human malaria), we designed a single oligonucleotide microarray capable of supporting a wide range of applications at relatively low cost, including genome-wide expression profiling for Toxoplasma, and single-nucleotide polymorphism (SNP)-based genotyping of both T. gondii and P. falciparum. Expression profiling of the three clonotypic lineages dominating T. gondii populations in North America and Europe provides a first comprehensive view of the parasite transcriptome, revealing that ~49% of all annotated genes are expressed in parasite tachyzoites (the acutely lytic stage responsible for pathogenesis). The relatively small genomes of P. falciparum (23 Mb) and T. gondii (65 Mb), and the ability to isolate DNA from haploid stages, coupled with a careful analysis of signals from overlapping probes, permits a novel design enabling high confidence genotyping using only four probes per SNP. This strategy permits resolution of recombination points in the clonal progeny of sexual crosses, allowing detailed mapping of phenotypic traits. Recent sequencing of additional T. gondii isolates identifies >620K new SNPs, including >11K that intersect with expression profiling probes, yielding additional markers for genotyping studies, and further validating the utility of a combined expression profiling/genotyping array design. Additional applications facilitating gene and transcript discovery, statistical analysis of expression profiles, etc. are also discussed.
CONCLUSIONS: In addition to providing an initial global view of the T. gondii transcriptome across major lineages and permitting detailed resolution of recombination points in a historical sexual cross, the multifunctional nature of this array also allowed opportunities to exploit probes for purposes beyond their intended use, enhancing analyses. This array is in widespread use by the T. gondii research community, and several aspects of the design strategy are likely to be useful for other pathogens.
PMID: 20974003 [PubMed - as supplied by publisher]
A novel multifunctional oligonucleotide microarray for Toxoplasma gondii
Bahl A, Davis PH, Behnke M, Dzierszinski F, Jagalur M, Chen F, Shanmugam D, White M, Kulp D, Roos DS.
Abstract
ABSTRACT:
BACKGROUND: Microarrays are invaluable tools for genome interrogation, SNP detection, and expression analysis, among other applications. Such broad capabilities would be of value to many pathogen research communities, although the development and use of genome-scale microarrays is often a prohibitively expensive undertaking.
RESULTS: Taking advantage of available genome sequences and annotation for Toxoplasma gondii (the causative agent of opportunistic illness in immunocompromised individuals) and Plasmodium falciparum (a related parasite responsible for severe human malaria), we designed a single oligonucleotide microarray capable of supporting a wide range of applications at relatively low cost, including genome-wide expression profiling for Toxoplasma, and single-nucleotide polymorphism (SNP)-based genotyping of both T. gondii and P. falciparum. Expression profiling of the three clonotypic lineages dominating T. gondii populations in North America and Europe provides a first comprehensive view of the parasite transcriptome, revealing that ~49% of all annotated genes are expressed in parasite tachyzoites (the acutely lytic stage responsible for pathogenesis). The relatively small genomes of P. falciparum (23 Mb) and T. gondii (65 Mb), and the ability to isolate DNA from haploid stages, coupled with a careful analysis of signals from overlapping probes, permits a novel design enabling high confidence genotyping using only four probes per SNP. This strategy permits resolution of recombination points in the clonal progeny of sexual crosses, allowing detailed mapping of phenotypic traits. Recent sequencing of additional T. gondii isolates identifies >620K new SNPs, including >11K that intersect with expression profiling probes, yielding additional markers for genotyping studies, and further validating the utility of a combined expression profiling/genotyping array design. Additional applications facilitating gene and transcript discovery, statistical analysis of expression profiles, etc. are also discussed.
CONCLUSIONS: In addition to providing an initial global view of the T. gondii transcriptome across major lineages and permitting detailed resolution of recombination points in a historical sexual cross, the multifunctional nature of this array also allowed opportunities to exploit probes for purposes beyond their intended use, enhancing analyses. This array is in widespread use by the T. gondii research community, and several aspects of the design strategy are likely to be useful for other pathogens.
PMID: 20974003 [PubMed - as supplied by publisher]
Thursday, October 21, 2010
Generation of a Neutralizing Human Monoclonal Antibody Fab Fragment to SAG1 of Toxoplasma gondii Tachyzoites
Infect Immun. 2010 Oct 18. [Epub ahead of print]
Generation of a Neutralizing Human Monoclonal Antibody Fab Fragment to SAG1 of Toxoplasma gondii Tachyzoites
Fu YF, Feng M, Ohnishi K, Kimura T, Itoh J, Cheng XJ, Tachibana H.
Department of Medical Microbiology and Parasitology, Fudan University School of Medicine, Shanghai 200032, China; Department of Infectious Diseases, Tokyo Metropolitan Bokutoh General Hospital, Sumida-Ku, Tokyo 130-8575, Japan; Department of Infectious Diseases and Teaching and Research Support Center, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan.
Abstract
A combinatorial immunoglobulin gene library was constructed from lymphocytes in peripheral blood of a patient with toxoplasmosis and screened for production of human monoclonal antibody Fab fragments to recombinant SAG1 of Toxoplasma gondii. Two Fab clones, Tox203 and Tox1403, which consisted of a common heavy chain and different light chains, showed positive staining on the entire surface of tachyzoites in confocal microscopy. Sequence analysis of the heavy chain gene revealed that the closest germline V segments were VH3-23. The germline D segment was D1-7, and the closest germline J segment was JH4. In the light chain genes, the closest germline V segment was Vκ1-17 with the Jκ1 or Jκ4 segments. The dissociation constants of these Fab fragments with recombinant SAG1 were 3.09x10(-9) M for Tox203 and 2.01x10(-8) M for Tox1403, indicating that the affinity of Tox203 was 7 times higher than that of Tox1403. Preincubation of T. gondii tachyzoites with Tox203 significantly inhibited their attachment to cultured MDBK cells. Passive immunization of mice with Tox203 also significantly reduced mortality after challenge with T. gondii tachyzoites. This is the first report of bacterial expression of human monoclonal antibody Fab fragments to SAG1 of T. gondii. These results also demonstrate that human Fab fragments to SAG1 might be applicable for immunoprophylaxis of toxoplasmosis.
PMID: 20956568 [PubMed - as supplied by publisher]
Generation of a Neutralizing Human Monoclonal Antibody Fab Fragment to SAG1 of Toxoplasma gondii Tachyzoites
Fu YF, Feng M, Ohnishi K, Kimura T, Itoh J, Cheng XJ, Tachibana H.
Department of Medical Microbiology and Parasitology, Fudan University School of Medicine, Shanghai 200032, China; Department of Infectious Diseases, Tokyo Metropolitan Bokutoh General Hospital, Sumida-Ku, Tokyo 130-8575, Japan; Department of Infectious Diseases and Teaching and Research Support Center, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan.
Abstract
A combinatorial immunoglobulin gene library was constructed from lymphocytes in peripheral blood of a patient with toxoplasmosis and screened for production of human monoclonal antibody Fab fragments to recombinant SAG1 of Toxoplasma gondii. Two Fab clones, Tox203 and Tox1403, which consisted of a common heavy chain and different light chains, showed positive staining on the entire surface of tachyzoites in confocal microscopy. Sequence analysis of the heavy chain gene revealed that the closest germline V segments were VH3-23. The germline D segment was D1-7, and the closest germline J segment was JH4. In the light chain genes, the closest germline V segment was Vκ1-17 with the Jκ1 or Jκ4 segments. The dissociation constants of these Fab fragments with recombinant SAG1 were 3.09x10(-9) M for Tox203 and 2.01x10(-8) M for Tox1403, indicating that the affinity of Tox203 was 7 times higher than that of Tox1403. Preincubation of T. gondii tachyzoites with Tox203 significantly inhibited their attachment to cultured MDBK cells. Passive immunization of mice with Tox203 also significantly reduced mortality after challenge with T. gondii tachyzoites. This is the first report of bacterial expression of human monoclonal antibody Fab fragments to SAG1 of T. gondii. These results also demonstrate that human Fab fragments to SAG1 might be applicable for immunoprophylaxis of toxoplasmosis.
PMID: 20956568 [PubMed - as supplied by publisher]
Toxoplasma gondii primary infection in renal transplant recipients. Two case reports and literature review
Transpl Int. 2010 Oct 19. doi: 10.1111/j.1432-2277.2010.01173.x. [Epub ahead of print]
Toxoplasma gondii primary infection in renal transplant recipients. Two case reports and literature review
Martina MN, Cervera C, Esforzado N, Linares L, Torregrosa V, Sanclemente G, Hoyo I, Cofan F, Oppenheimer F, Miro JM, Campistol JM, Moreno A.
Renal Transplant Unit, Hospital Clinic of Barcelona, IDIBAPS, University of Barcelona, Barcelona, Spain Infectious Diseases Service, Hospital Clinic of Barcelona, IDIBAPS, University of Barcelona, Barcelona, Spain.
Abstract
Toxoplasmosis after solid organ transplantation is a complication associated with high morbidity and mortality. Universal prophylaxis with trimethoprim-sulfamethoxazole (TMP-SMX) is effective to prevent post-transplant toxoplasmosis. We report two cases of renal transplant recipients with negative antibodies against Toxoplasma gondii pretransplant who developed toxoplasmosis after TMP-SMX discontinuation. We have also performed a review of published cases of primary toxoplasmosis after renal transplantation. Of 20 cases reviewed, 11 were male and the mean age was 37.8 years (SD = 13.8). Donor serology for T. gondii was determined in 15 donors, two of them (13%) with negative immunoglobulin (Ig)G and four (27%) with positive IgG and IgM antibodies. Fever was present in 85% of primary toxoplasmosis and hematologic abnormalities were observed in 69% of the cases. Ten patients died (50%). All patients with fatal outcomes had clinical evidence of toxoplasmosis during the early post-transplant period (first 90 days), while no patient with late toxoplasmosis died (P = 0.003). Primary toxoplasmosis is associated with high mortality rates and TMP-SMX prophylaxis can delay the onset of symptoms resulting in an improvement of prognosis.
© 2010 The Authors. Transplant International © 2010 European Society for Organ Transplantation.
PMID: 20955469 [PubMed - as supplied by publisher]
Toxoplasma gondii primary infection in renal transplant recipients. Two case reports and literature review
Martina MN, Cervera C, Esforzado N, Linares L, Torregrosa V, Sanclemente G, Hoyo I, Cofan F, Oppenheimer F, Miro JM, Campistol JM, Moreno A.
Renal Transplant Unit, Hospital Clinic of Barcelona, IDIBAPS, University of Barcelona, Barcelona, Spain Infectious Diseases Service, Hospital Clinic of Barcelona, IDIBAPS, University of Barcelona, Barcelona, Spain.
Abstract
Toxoplasmosis after solid organ transplantation is a complication associated with high morbidity and mortality. Universal prophylaxis with trimethoprim-sulfamethoxazole (TMP-SMX) is effective to prevent post-transplant toxoplasmosis. We report two cases of renal transplant recipients with negative antibodies against Toxoplasma gondii pretransplant who developed toxoplasmosis after TMP-SMX discontinuation. We have also performed a review of published cases of primary toxoplasmosis after renal transplantation. Of 20 cases reviewed, 11 were male and the mean age was 37.8 years (SD = 13.8). Donor serology for T. gondii was determined in 15 donors, two of them (13%) with negative immunoglobulin (Ig)G and four (27%) with positive IgG and IgM antibodies. Fever was present in 85% of primary toxoplasmosis and hematologic abnormalities were observed in 69% of the cases. Ten patients died (50%). All patients with fatal outcomes had clinical evidence of toxoplasmosis during the early post-transplant period (first 90 days), while no patient with late toxoplasmosis died (P = 0.003). Primary toxoplasmosis is associated with high mortality rates and TMP-SMX prophylaxis can delay the onset of symptoms resulting in an improvement of prognosis.
© 2010 The Authors. Transplant International © 2010 European Society for Organ Transplantation.
PMID: 20955469 [PubMed - as supplied by publisher]
Intracellular Protozoan Parasites of Humans: The Role of Molecular Chaperones in Development and Pathogenesis
Protein Pept Lett. 2010 Oct 18. [Epub ahead of print]
Intracellular Protozoan Parasites of Humans: The Role of Molecular Chaperones in Development and Pathogenesis
Shonhai A, Maier AG, Przyborski JM, Blatch GL.
Department of Biochemistry & Microbiology, University of Zululand, Kwadlangezwa, South Africa. G.Blatch@ru.ac.za.
Abstract
Certain kinetoplastid (Leishmania spp. and Tryapnosoma cruzi) and apicomplexan parasites (Plasmodium falciparum and Toxoplasma gondii) are capable of invading human cells as part of their pathology. These parasites appear to have evolved a relatively expanded or diverse complement of genes encoding molecular chaperones. The gene families encoding heat shock protein 90 (Hsp90) and heat shock protein 70 (Hsp70) chaperones show significant expansion and diversity (especially for Leishmania spp. and T. cruzi), and in particular the Hsp40 family appears to be an extreme example of phylogenetic radiation. In general, Hsp40 proteins act as co-chaperones of Hsp70 chaperones, forming protein folding pathways that integrate with Hsp90 to ensure proteostasis in the cell. It is tempting to speculate that the diverse environmental insults that these parasites endure have resulted in the evolutionary selection of a diverse and expanded chaperone network. Hsp90 is involved in development and growth of all of these intracellular parasites, and so far represents the strongest candidate as a target for chemotherapeutic interventions. While there have been some excellent studies on the molecular and cell biology of Hsp70 proteins, relatively little is known about the biological function of Hsp70-Hsp40 interactions in these intracellular parasites. This review focuses on intracellular protozoan parasites of humans, and provides a critique of the role of heat shock proteins in development and pathogenesis, especially the molecular chaperones Hsp90, Hsp70 and Hsp40.
PMID: 20955165 [PubMed - as supplied by publisher]
Intracellular Protozoan Parasites of Humans: The Role of Molecular Chaperones in Development and Pathogenesis
Shonhai A, Maier AG, Przyborski JM, Blatch GL.
Department of Biochemistry & Microbiology, University of Zululand, Kwadlangezwa, South Africa. G.Blatch@ru.ac.za.
Abstract
Certain kinetoplastid (Leishmania spp. and Tryapnosoma cruzi) and apicomplexan parasites (Plasmodium falciparum and Toxoplasma gondii) are capable of invading human cells as part of their pathology. These parasites appear to have evolved a relatively expanded or diverse complement of genes encoding molecular chaperones. The gene families encoding heat shock protein 90 (Hsp90) and heat shock protein 70 (Hsp70) chaperones show significant expansion and diversity (especially for Leishmania spp. and T. cruzi), and in particular the Hsp40 family appears to be an extreme example of phylogenetic radiation. In general, Hsp40 proteins act as co-chaperones of Hsp70 chaperones, forming protein folding pathways that integrate with Hsp90 to ensure proteostasis in the cell. It is tempting to speculate that the diverse environmental insults that these parasites endure have resulted in the evolutionary selection of a diverse and expanded chaperone network. Hsp90 is involved in development and growth of all of these intracellular parasites, and so far represents the strongest candidate as a target for chemotherapeutic interventions. While there have been some excellent studies on the molecular and cell biology of Hsp70 proteins, relatively little is known about the biological function of Hsp70-Hsp40 interactions in these intracellular parasites. This review focuses on intracellular protozoan parasites of humans, and provides a critique of the role of heat shock proteins in development and pathogenesis, especially the molecular chaperones Hsp90, Hsp70 and Hsp40.
PMID: 20955165 [PubMed - as supplied by publisher]
Sunday, October 17, 2010
Concerted Action of Two Formins in Gliding Motility and Host Cell Invasion by Toxoplasma gondii.
PLoS Pathog. 2010 Oct 7;6(10). pii: e1001132.
Concerted Action of Two Formins in Gliding Motility and Host Cell Invasion by Toxoplasma gondii.
Daher W, Plattner F, Carlier MF, Soldati-Favre D.
Department of Microbiology and Molecular Medicine, CMU, University of Geneva, Geneva, Switzerland.
Abstract
The invasive forms of apicomplexan parasites share a conserved form of gliding motility that powers parasite migration across biological barriers, host cell invasion and egress from infected cells. Previous studies have established that the duration and direction of gliding motility are determined by actin polymerization; however, regulators of actin dynamics in apicomplexans remain poorly characterized. In the absence of a complete ARP2/3 complex, the formin homology 2 domain containing proteins and the accessory protein profilin are presumed to orchestrate actin polymerization during host cell invasion. Here, we have undertaken the biochemical and functional characterization of two Toxoplasma gondii formins and established that they act in concert as actin nucleators during invasion. The importance of TgFRM1 for parasite motility has been assessed by conditional gene disruption. The contribution of each formin individually and jointly was revealed by an approach based upon the expression of dominant mutants with modified FH2 domains impaired in actin binding but still able to dimerize with their respective endogenous formin. These mutated FH2 domains were fused to the ligand-controlled destabilization domain (DD-FKBP) to achieve conditional expression. This strategy proved unique in identifying the non-redundant and critical roles of both formins in invasion. These findings provide new insights into how controlled actin polymerization drives the directional movement required for productive penetration of parasites into host cells.
PMID: 20949068 [PubMed - in process]
Concerted Action of Two Formins in Gliding Motility and Host Cell Invasion by Toxoplasma gondii.
Daher W, Plattner F, Carlier MF, Soldati-Favre D.
Department of Microbiology and Molecular Medicine, CMU, University of Geneva, Geneva, Switzerland.
Abstract
The invasive forms of apicomplexan parasites share a conserved form of gliding motility that powers parasite migration across biological barriers, host cell invasion and egress from infected cells. Previous studies have established that the duration and direction of gliding motility are determined by actin polymerization; however, regulators of actin dynamics in apicomplexans remain poorly characterized. In the absence of a complete ARP2/3 complex, the formin homology 2 domain containing proteins and the accessory protein profilin are presumed to orchestrate actin polymerization during host cell invasion. Here, we have undertaken the biochemical and functional characterization of two Toxoplasma gondii formins and established that they act in concert as actin nucleators during invasion. The importance of TgFRM1 for parasite motility has been assessed by conditional gene disruption. The contribution of each formin individually and jointly was revealed by an approach based upon the expression of dominant mutants with modified FH2 domains impaired in actin binding but still able to dimerize with their respective endogenous formin. These mutated FH2 domains were fused to the ligand-controlled destabilization domain (DD-FKBP) to achieve conditional expression. This strategy proved unique in identifying the non-redundant and critical roles of both formins in invasion. These findings provide new insights into how controlled actin polymerization drives the directional movement required for productive penetration of parasites into host cells.
PMID: 20949068 [PubMed - in process]
Wednesday, October 13, 2010
Chronic infection with Toxoplasma gondii causes myenteric neuroplasticity of the jejunum in rats
Auton Neurosci. 2010 Oct 5. [Epub ahead of print]
Chronic infection with Toxoplasma gondii causes myenteric neuroplasticity of the jejunum in rats
Hermes-Uliana C, Pereira-Severi LS, Luerdes RB, Franco CL, da Silva AV, Araújo EJ, Sant'ana DD.
Programa de Pós-Graduação em Ciência Animal, Universidade Paranaense, (UNIPAR), Paraná, Brazil.
Abstract
Toxoplasma gondii is an aetiological agent of toxoplasmosis, which commonly causes diarrhoea in a number of species. This observation and the parasite's affinity for the nervous tissue support the theory that T. gondii infection may affect the myenteric neurons. The aim of this study was to evaluate the changes caused by T. gondii (genotype III) in the myenteric neurons of the jejunum in rats. Fifteen rats were distributed into three groups: control (CG), inoculated for 30days (G30) and inoculated for 90days (G90). Rats from the G30 and G90 groups received an oral inoculum with 500 oocysts from a genotype III (M7741) T. gondii strain. At 180days of age, all animals were anaesthetised and euthanised. Whole mounts were stained by using Giemsa (total population) and NADPH-diaphorase (nitrergic subpopulation) histochemistry. Maintenance of the width, length, area and neuronal density was observed; there was neuronal atrophy in the G30 group and a tendency to hypertrophy in the G90 group. Rats inoculated orally with sporulated oocysts did not show clinical illness or macroscopic or microscopic lesions, as do the majority of animal species. Therefore, infection was confirmed by a serum agglutination test; 30days of infection caused increased weight gain and atrophy of myenteric neurons. At 90days post-infection, weight gain became normal, and myenteric neurons hypertrophied.
PMID: 20932812 [PubMed - as supplied by publisher]
Chronic infection with Toxoplasma gondii causes myenteric neuroplasticity of the jejunum in rats
Hermes-Uliana C, Pereira-Severi LS, Luerdes RB, Franco CL, da Silva AV, Araújo EJ, Sant'ana DD.
Programa de Pós-Graduação em Ciência Animal, Universidade Paranaense, (UNIPAR), Paraná, Brazil.
Abstract
Toxoplasma gondii is an aetiological agent of toxoplasmosis, which commonly causes diarrhoea in a number of species. This observation and the parasite's affinity for the nervous tissue support the theory that T. gondii infection may affect the myenteric neurons. The aim of this study was to evaluate the changes caused by T. gondii (genotype III) in the myenteric neurons of the jejunum in rats. Fifteen rats were distributed into three groups: control (CG), inoculated for 30days (G30) and inoculated for 90days (G90). Rats from the G30 and G90 groups received an oral inoculum with 500 oocysts from a genotype III (M7741) T. gondii strain. At 180days of age, all animals were anaesthetised and euthanised. Whole mounts were stained by using Giemsa (total population) and NADPH-diaphorase (nitrergic subpopulation) histochemistry. Maintenance of the width, length, area and neuronal density was observed; there was neuronal atrophy in the G30 group and a tendency to hypertrophy in the G90 group. Rats inoculated orally with sporulated oocysts did not show clinical illness or macroscopic or microscopic lesions, as do the majority of animal species. Therefore, infection was confirmed by a serum agglutination test; 30days of infection caused increased weight gain and atrophy of myenteric neurons. At 90days post-infection, weight gain became normal, and myenteric neurons hypertrophied.
PMID: 20932812 [PubMed - as supplied by publisher]
Comprehensive proteomic analysis of membrane proteins in toxoplasma gondii
Mol Cell Proteomics. 2010 Oct 10. [Epub ahead of print]
Comprehensive proteomic analysis of membrane proteins in toxoplasma gondii
Che FY, Madrid-Aliste C, Burd B, Zhang H, Nieves E, Kim K, Fiser A, Angeletti RH, Weiss LM.
Albert Einstein College of Medicine, United States.
Abstract
Toxoplasma gondii (T.gondii) is an obligate intracellular protozoan parasite that is important human and animal pathogen. Experimental information on T.gondii membrane proteins is limited, and the majority of gen predictions with predicted transmembrane motifs are of unknown function. A systematic analysis of the membrane proteome of T.gondii is important not only for understanding this parasite's invasion mechanism(s), but also for the discovery of potentail drug targets and new preventative and therapeutic strategies. Here we report a comprehensive analysis of the membrane proteome of T.gondii, employing three proteomics strategies: 1D gel LC-MSMS annlysis (one dimensional gel electrophoresis liquid chromatography tandem mass spectrometry), biotin labeling in conjunction with 1D gel LC-MSMS analysis, and a novel strategy that combines Three Layer "Sandwich" gel Electrophoresis (TLSGE) with multidimensional protein identification technology (MudPIT). A total of 2, 241 T.gondii proteins with at least one predicted transmembrane segment were identified and grouped into 841 sequentially non-redundant protein clusters, which account for 21.8% of the predicted transmembrane protein clusters in the T.gondii genmoe. A large portion (42%) of the identified T. gondii membrane proteins are hypothetical proteins. Furthermore, many of the membrane proteins validated by mass spectrometry are unique to T.gondii or to the Apicomplexa, providing a set of gene predictions ripe for experimental investigation, and potentially siutable targets for the development of therapeutic strategies.
PMID: 20935347 [PubMed - as supplied by publisher]
Comprehensive proteomic analysis of membrane proteins in toxoplasma gondii
Che FY, Madrid-Aliste C, Burd B, Zhang H, Nieves E, Kim K, Fiser A, Angeletti RH, Weiss LM.
Albert Einstein College of Medicine, United States.
Abstract
Toxoplasma gondii (T.gondii) is an obligate intracellular protozoan parasite that is important human and animal pathogen. Experimental information on T.gondii membrane proteins is limited, and the majority of gen predictions with predicted transmembrane motifs are of unknown function. A systematic analysis of the membrane proteome of T.gondii is important not only for understanding this parasite's invasion mechanism(s), but also for the discovery of potentail drug targets and new preventative and therapeutic strategies. Here we report a comprehensive analysis of the membrane proteome of T.gondii, employing three proteomics strategies: 1D gel LC-MSMS annlysis (one dimensional gel electrophoresis liquid chromatography tandem mass spectrometry), biotin labeling in conjunction with 1D gel LC-MSMS analysis, and a novel strategy that combines Three Layer "Sandwich" gel Electrophoresis (TLSGE) with multidimensional protein identification technology (MudPIT). A total of 2, 241 T.gondii proteins with at least one predicted transmembrane segment were identified and grouped into 841 sequentially non-redundant protein clusters, which account for 21.8% of the predicted transmembrane protein clusters in the T.gondii genmoe. A large portion (42%) of the identified T. gondii membrane proteins are hypothetical proteins. Furthermore, many of the membrane proteins validated by mass spectrometry are unique to T.gondii or to the Apicomplexa, providing a set of gene predictions ripe for experimental investigation, and potentially siutable targets for the development of therapeutic strategies.
PMID: 20935347 [PubMed - as supplied by publisher]
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