Postdoc position available
Pasteur Institute in Lille (France)
A Postdoctoral position is opening at the Pasteur Institute in Lille (France) working with Dr. Gissot on the characterization of transcription factors of the apicomplexan parasite Toxoplasma gondii. The laboratory is interested in understanding the molecular mechanisms controlling differentiation and stage conversion in T. gondii. The project received funding from the French National Research Agency (ANR) for 3 years. Applicants must have a Ph.D. in biochemistry, molecular biology or related discipline, be highly motivated, and be able to critically evaluate and interpret data. Previous experience in regulation of gene expression and the wide array of techniques to examine transcription factors function will be appreciated. Experience with apicomplexan parasites is not mandatory. Ability to interact with a diverse working environment composed of parasitologists, biochemists and bioinformatic staff is required. The Pasteur Institute is located in Lille, France (1 hour from Paris, 1h30 from London and 30 mins from Brussels) and has cutting edge transcriptomics, proteomics and microscopy platforms.
Candidates should send their Curriculum Vitae and three references to:
Mathieu Gissot, PhD.
Institut Pasteur de Lille
Institut de Biologie de Lille
CNRS UMR8161
Molecular and Cellular Biology of Toxoplasma gondii
1, rue du Pr. Calmette
BP 245
59019 Lille Cedex
FRANCE
mathieu.gissot@pasteur-lille.fr
Thursday, July 30, 2009
Evolution of the Apicomplexa: where are we now?
Trends Parasitol. 2009 Jul 25. [Epub ahead of print]
Evolution of the Apicomplexa: where are we now?
Morrison DA.
Section for Parasitology, Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, 751 89 Uppsala, Sweden.
The Apicomplexa is the only large taxonomic group whose members are entirely parasitic and is, therefore, presumably of major interest to parasitologists. We might, for example, expect that we know a great deal about the biology of the group by now and that we have a clear phylogenetic framework within which to organize that knowledge. It might thus come as a surprise to learn that in terms of biodiversity, the Apicomplexa is actually the least-known group of all. Furthermore, the taxonomic framework for the Apicomplexa is rather tenuous in many respects. This situation is unlikely to change in the short term.
PMID: 19635681 [PubMed - as supplied by publisher]
Evolution of the Apicomplexa: where are we now?
Morrison DA.
Section for Parasitology, Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, 751 89 Uppsala, Sweden.
The Apicomplexa is the only large taxonomic group whose members are entirely parasitic and is, therefore, presumably of major interest to parasitologists. We might, for example, expect that we know a great deal about the biology of the group by now and that we have a clear phylogenetic framework within which to organize that knowledge. It might thus come as a surprise to learn that in terms of biodiversity, the Apicomplexa is actually the least-known group of all. Furthermore, the taxonomic framework for the Apicomplexa is rather tenuous in many respects. This situation is unlikely to change in the short term.
PMID: 19635681 [PubMed - as supplied by publisher]
Simultaneous detection of viruses and Toxoplasma gondii in cerebrospinal fluid specimens by multiplex PCR-based reverse hybridization
New Microbiol. 2009 Apr;32(2):143-6
Simultaneous detection of viruses and Toxoplasma gondii in cerebrospinal fluid specimens by multiplex polymerase chain reaction-based reverse hybridization assay
Del Prete R, Di Taranto AM, Lipsi MR, Natalicchio MI, Antonetti R, Miragliotta G.
Section of Microbiology, Department of Clinical Medicine, Immunology, Infectious Diseases, University of Bari, Italy.
The lack of rapidity and the low sensitivity and specificity of traditional laboratory methods limits their usefulness in the laboratory diagnosis of viral central nervous system (CNS) infections. This study describes the use of a commercially available multiplex polymerase chain reaction (mPCR)-based reverse hybridization assay (RHA) for the simultaneous detection of the genomes of 8 viruses and Toxoplasma gondii in cerebrospinal fluids (CSF) from 181 patients suspected of having viral meningitis. Twenty-two/181 (12.15%) CSF samples resulted positive by mPCR. Eighteen/22 were positive for 1 viral pathogen, whereas a dual infection was detected in 4/22 samples. Epstein-Barr virus (EBV) was the most commonly detected virus (6/22), followed by herpes simplex virus type-1 (HSV-1) (5/22) and -2 (HSV-2) (4/22). Cytomegalovirus (CMV), human herpesvirus-6 (HHV-6), and Epstein-Barr virus (EBV) were detected in 1 specimen each. Two CSF samples were co-infected by HSV-1/HSV-2, 1 sample by HHV-6/T. gondii, and 1 sample by EBV/EV, respectively. Our data support the usefulness of mPCR as a rapid molecular method for the simultaneous detection of major viral pathogens and T. gondii in aseptic meningitis also to allow the earlier application of specific antiviral therapy.
PMID: 19579690 [PubMed - indexed for MEDLINE]
Simultaneous detection of viruses and Toxoplasma gondii in cerebrospinal fluid specimens by multiplex polymerase chain reaction-based reverse hybridization assay
Del Prete R, Di Taranto AM, Lipsi MR, Natalicchio MI, Antonetti R, Miragliotta G.
Section of Microbiology, Department of Clinical Medicine, Immunology, Infectious Diseases, University of Bari, Italy.
The lack of rapidity and the low sensitivity and specificity of traditional laboratory methods limits their usefulness in the laboratory diagnosis of viral central nervous system (CNS) infections. This study describes the use of a commercially available multiplex polymerase chain reaction (mPCR)-based reverse hybridization assay (RHA) for the simultaneous detection of the genomes of 8 viruses and Toxoplasma gondii in cerebrospinal fluids (CSF) from 181 patients suspected of having viral meningitis. Twenty-two/181 (12.15%) CSF samples resulted positive by mPCR. Eighteen/22 were positive for 1 viral pathogen, whereas a dual infection was detected in 4/22 samples. Epstein-Barr virus (EBV) was the most commonly detected virus (6/22), followed by herpes simplex virus type-1 (HSV-1) (5/22) and -2 (HSV-2) (4/22). Cytomegalovirus (CMV), human herpesvirus-6 (HHV-6), and Epstein-Barr virus (EBV) were detected in 1 specimen each. Two CSF samples were co-infected by HSV-1/HSV-2, 1 sample by HHV-6/T. gondii, and 1 sample by EBV/EV, respectively. Our data support the usefulness of mPCR as a rapid molecular method for the simultaneous detection of major viral pathogens and T. gondii in aseptic meningitis also to allow the earlier application of specific antiviral therapy.
PMID: 19579690 [PubMed - indexed for MEDLINE]
Resonance assignments for Toxo proteins
Biomol NMR Assign. 2009 Jun;3(1). Epub 2009 Feb 27.
Complete resonance assignments for the MIC2 associated protein from Toxoplasma gondii
Liu B, Sawmynaden K, Marchant J, Simpson P, Matthews S.
Division of Molecular Biosciences, Department of Life Sciences, Imperial College London, South Kensington, London, SW7 2AZ, UK.
Toxoplasma gondii is an obligate parasite that infects most warm blood animals. Micronemal proteins actively involves in the invasion process, where TgMIC2 and TgM2AP complex plays vital roles. Complete NMR assignments for major fragment of TgM2AP were successfully obtained.
Publication Types:
Research Support, Non-U.S. Gov't
PMID: 19636952 [PubMed - in process]
Complete NMR assignments for the second EGF domain of MIC6 from Toxoplasma gondii and re-assignment in complex with the galectin-like domain of MIC1
Sawmynaden K, Saouros S, Marchant J, Simpson P, Matthews S.
Division of Molecular Biosciences, Department of Life Sciences, Imperial College London, South Kensington, London, SW7 2AZ, UK.
Toxoplasma gondii is the causative agent of toxoplasmosis. Here we present a complete set of NMR assignments for the second EGF domain from microneme protein 6 and its re-assignment in complex with the galectin-like domain from microneme protein 1.
Publication Types:
Research Support, Non-U.S. Gov't
PMID: 19636901 [PubMed - in process]
Complete resonance assignment of the galectin-like domain of MIC1 from Toxoplasma gondii in complex with the second EGF domain from MIC6 and the backbone assignment in complex with the third EGF domain
Saouros S, Sawmynaden K, Marchant J, Simpson P, Matthews S.
Division of Molecular Biosciences, Imperial College London, South Kensington Campus, London, SW7 2AZ, UK.
Microneme protein complexes are important for invasion of host cells by Toxoplasma gondii. We report the resonance assignment of the galectin-like domain of microneme protein 1 in complexes with the second and third EGF domains from microneme protein 6.
Publication Types:
Research Support, Non-U.S. Gov't
PMID: 19636898 [PubMed - in process]
Complete resonance assignment of the first and second apple domains of MIC4 from Toxoplasma gondii, using a new NMRView-based assignment aid
Marchant J, Sawmynaden K, Saouros S, Simpson P, Matthews S.
Division of Molecular Biosciences, Department of Life Sciences, Imperial College London, South Kensington, London, SW7 2AZ, UK.
Microneme protein 4 is involved in cell binding by the important parasite Toxoplasma gondii. We present here the backbone and side-chain assignments of the first two apple domains together with a new graphical aid for their assignment using NMRView.
Publication Types:
Research Support, Non-U.S. Gov't
PMID: 19636884 [PubMed - in process]
Complete resonance assignments for the MIC2 associated protein from Toxoplasma gondii
Liu B, Sawmynaden K, Marchant J, Simpson P, Matthews S.
Division of Molecular Biosciences, Department of Life Sciences, Imperial College London, South Kensington, London, SW7 2AZ, UK.
Toxoplasma gondii is an obligate parasite that infects most warm blood animals. Micronemal proteins actively involves in the invasion process, where TgMIC2 and TgM2AP complex plays vital roles. Complete NMR assignments for major fragment of TgM2AP were successfully obtained.
Publication Types:
Research Support, Non-U.S. Gov't
PMID: 19636952 [PubMed - in process]
Complete NMR assignments for the second EGF domain of MIC6 from Toxoplasma gondii and re-assignment in complex with the galectin-like domain of MIC1
Sawmynaden K, Saouros S, Marchant J, Simpson P, Matthews S.
Division of Molecular Biosciences, Department of Life Sciences, Imperial College London, South Kensington, London, SW7 2AZ, UK.
Toxoplasma gondii is the causative agent of toxoplasmosis. Here we present a complete set of NMR assignments for the second EGF domain from microneme protein 6 and its re-assignment in complex with the galectin-like domain from microneme protein 1.
Publication Types:
Research Support, Non-U.S. Gov't
PMID: 19636901 [PubMed - in process]
Complete resonance assignment of the galectin-like domain of MIC1 from Toxoplasma gondii in complex with the second EGF domain from MIC6 and the backbone assignment in complex with the third EGF domain
Saouros S, Sawmynaden K, Marchant J, Simpson P, Matthews S.
Division of Molecular Biosciences, Imperial College London, South Kensington Campus, London, SW7 2AZ, UK.
Microneme protein complexes are important for invasion of host cells by Toxoplasma gondii. We report the resonance assignment of the galectin-like domain of microneme protein 1 in complexes with the second and third EGF domains from microneme protein 6.
Publication Types:
Research Support, Non-U.S. Gov't
PMID: 19636898 [PubMed - in process]
Complete resonance assignment of the first and second apple domains of MIC4 from Toxoplasma gondii, using a new NMRView-based assignment aid
Marchant J, Sawmynaden K, Saouros S, Simpson P, Matthews S.
Division of Molecular Biosciences, Department of Life Sciences, Imperial College London, South Kensington, London, SW7 2AZ, UK.
Microneme protein 4 is involved in cell binding by the important parasite Toxoplasma gondii. We present here the backbone and side-chain assignments of the first two apple domains together with a new graphical aid for their assignment using NMRView.
Publication Types:
Research Support, Non-U.S. Gov't
PMID: 19636884 [PubMed - in process]
Artemisone and Artemiside Control Acute and Reactivated Toxoplasmosis in the Murine Model
Antimicrob Agents Chemother. 2009 Jul 27. [Epub ahead of print]
Artemisone and Artemiside Control Acute and Reactivated Toxoplasmosis in the Murine Model
Dunay IR, Chan WC, Haynes RK, Sibley LD.
Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, Missouri 63110, USA; Department of Chemistry, Open Laboratory of Chemical Biology, Institute of Molecular Technology for Drug Discovery and Synthesis, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, PRC.
Immunocompromised patients are at risk to develop toxoplasmosis and although chemotherapy is available, standard treatments are often complicated by severe side effects. Artemisinin is a new highly potent antimalarial drug that has activity against Toxoplasma gondii in vitro. However, artemisinin derivatives have previously been ineffective in vivo using a rat model of toxoplasmosis. In the present study, the efficacy of several new artemisinin derivates was investigated for treatment of mice infected with the parasite Toxoplasma gondii. Artemiside and artemisone displayed better inhibition than either artemisinin or artesunate against the parasite in vitro. Artemiside and artemisone treatment controlled parasite replication in vivo and mice survived the acute infection. In a murine model of reactivated toxoplasmosis, both drugs increased survival, although artemiside was more effective. These results indicate that these newer derivatives of artemisinin may have potential for treatment of toxoplasmosis.
PMID: 19635951 [PubMed - as supplied by publisher]
Artemisone and Artemiside Control Acute and Reactivated Toxoplasmosis in the Murine Model
Dunay IR, Chan WC, Haynes RK, Sibley LD.
Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, Missouri 63110, USA; Department of Chemistry, Open Laboratory of Chemical Biology, Institute of Molecular Technology for Drug Discovery and Synthesis, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, PRC.
Immunocompromised patients are at risk to develop toxoplasmosis and although chemotherapy is available, standard treatments are often complicated by severe side effects. Artemisinin is a new highly potent antimalarial drug that has activity against Toxoplasma gondii in vitro. However, artemisinin derivatives have previously been ineffective in vivo using a rat model of toxoplasmosis. In the present study, the efficacy of several new artemisinin derivates was investigated for treatment of mice infected with the parasite Toxoplasma gondii. Artemiside and artemisone displayed better inhibition than either artemisinin or artesunate against the parasite in vitro. Artemiside and artemisone treatment controlled parasite replication in vivo and mice survived the acute infection. In a murine model of reactivated toxoplasmosis, both drugs increased survival, although artemiside was more effective. These results indicate that these newer derivatives of artemisinin may have potential for treatment of toxoplasmosis.
PMID: 19635951 [PubMed - as supplied by publisher]
Antimicrobial and immunoregulatory properties of human tryptophan 2,3-dioxygenase
Eur J Immunol. 2009 Jul 27. [Epub ahead of print]
Antimicrobial and immunoregulatory properties of human tryptophan 2,3-dioxygenase
Schmidt SK, Müller A, Heseler K, Woite C, Spekker K, Mackenzie CR, Däubener W.
Institute of Medical Microbiology and Hospital Hygiene, Heinrich-Heine-University Düsseldorf, Germany.
In mammals the regulation of local tryptophan concentrations by the interferon-gamma inducible enzyme indoleamine 2,3-dioxygenase (IDO) is a prominent antimicrobial and immunoregulatory effector mechanism. Here, we show for the first time that another tryptophan degrading enzyme, the liver specific tryptophan 2,3-dioxygenase (TDO), is also capable of mediating antimicrobial and immunoregulatory effects.Using a tetracycline inducible eukaryotic system, we were able to express recombinant TDO protein, which exhibits functional properties of native TDO. We found that HeLa cells expressing recombinant TDO were capable of inhibiting the growth of bacteria (Staphylococcus aureus), parasites (Toxoplasma gondii) and viruses (herpes simplex virus). These TDO-mediated antimicrobial effects could be blocked by the addition of tryptophan.In addition we observed that, similar to IDO-positive cells, TDO-positive cells, were capable of inhibiting anti CD3-driven T cell proliferation and IFN- production. Furthermore, TDO-positive cells also restricted alloantigen induced T cell activation.Here, we describe for the first time that TDO mediates antimicrobial and immunoregulatory effects and suggest that TDO-dependent inhibition of T cell growth might be involved in the immunotolerance observed in vivo during allogeneic liver transplantation.
PMID: 19637229 [PubMed - as supplied by publisher]
Antimicrobial and immunoregulatory properties of human tryptophan 2,3-dioxygenase
Schmidt SK, Müller A, Heseler K, Woite C, Spekker K, Mackenzie CR, Däubener W.
Institute of Medical Microbiology and Hospital Hygiene, Heinrich-Heine-University Düsseldorf, Germany.
In mammals the regulation of local tryptophan concentrations by the interferon-gamma inducible enzyme indoleamine 2,3-dioxygenase (IDO) is a prominent antimicrobial and immunoregulatory effector mechanism. Here, we show for the first time that another tryptophan degrading enzyme, the liver specific tryptophan 2,3-dioxygenase (TDO), is also capable of mediating antimicrobial and immunoregulatory effects.Using a tetracycline inducible eukaryotic system, we were able to express recombinant TDO protein, which exhibits functional properties of native TDO. We found that HeLa cells expressing recombinant TDO were capable of inhibiting the growth of bacteria (Staphylococcus aureus), parasites (Toxoplasma gondii) and viruses (herpes simplex virus). These TDO-mediated antimicrobial effects could be blocked by the addition of tryptophan.In addition we observed that, similar to IDO-positive cells, TDO-positive cells, were capable of inhibiting anti CD3-driven T cell proliferation and IFN- production. Furthermore, TDO-positive cells also restricted alloantigen induced T cell activation.Here, we describe for the first time that TDO mediates antimicrobial and immunoregulatory effects and suggest that TDO-dependent inhibition of T cell growth might be involved in the immunotolerance observed in vivo during allogeneic liver transplantation.
PMID: 19637229 [PubMed - as supplied by publisher]
Wednesday, July 29, 2009
Postdoctoral Position Available - Parsons Lab
Postdoctoral Position Available
Seattle Biomedical Research Institute Toxoplasma Cell Biology
The Parsons lab at Seattle Biomedical Research Institute has an opening for a postdoctoral scientist to join the team studying protein trafficking to the Toxoplasma apicoplast. The apicoplast is a relict chloroplast found in most apicomplexan parasites, and the enzymes it contains are of high interest as drugs targets for several diseases caused by these pathogens. We are studying the sequences and machinery required to route nuclearly encoded proteins to the apicoplast, using a combinations of fluorescence microscopy, molecular biology, cell culture, and protein analysis. Candidates who have recently received their Ph.D. and have a strong background in molecular or cell biology are encouraged to apply. An interest in infectious diseases is desirable.
SBRI is a non-profit research institute that is solely focused on infectious diseases of global importance, and provides an excellent environment in which to learn more about pathogens and disease. The Parsons lab also conducts research on African trypanosomes, and other labs work on agents such as Plasmodium, Leishmania, HIV, pathogenic fungi, and mycobacteria. We provide competitive benefits for postdoctoral scientists. SBRI is affiliated with the University of Washington and is located in a modern facility just a few miles away in downtown Seattle. It has grown to more than 250 staff members, and partners with key collaborators around the globe. To find out more, visit our website at www.sbri.org. To apply visit the jobs page (drop down menu at left).
SBRI is an Equal Opportunity Employer.
Sunday, July 26, 2009
History of the discovery of the life cycle of Toxoplasma gondii
Int J Parasitol. 2009 Jul 1;39(8):877-82
Comment in:
Int J Parasitol. 2009 Jul 1;39(8):859-60.
History of the discovery of the life cycle of Toxoplasma gondii
Dubey JP.
United States Department of Agriculture, Animal and Natural Resources Institute, Beltsville, MD 20705-2350, USA. jitender.dubey@ars.usda.gov
It has been 100 years since the discovery of Toxoplasma gondii in 1908. Its full life cycle was not discovered until 1970 when it was found that it is a coccidian parasite of cats with all non-feline warm blooded animals (including humans) as intermediate hosts. The discovery of the environmentally resistant stage of the parasite, the oocyst, made it possible to explain its worldwide prevalence. In the present paper, events associated with the discovery of its life cycle are recalled.
PMID: 19630138 [PubMed - in process]
Comment in:
Int J Parasitol. 2009 Jul 1;39(8):859-60.
History of the discovery of the life cycle of Toxoplasma gondii
Dubey JP.
United States Department of Agriculture, Animal and Natural Resources Institute, Beltsville, MD 20705-2350, USA. jitender.dubey@ars.usda.gov
It has been 100 years since the discovery of Toxoplasma gondii in 1908. Its full life cycle was not discovered until 1970 when it was found that it is a coccidian parasite of cats with all non-feline warm blooded animals (including humans) as intermediate hosts. The discovery of the environmentally resistant stage of the parasite, the oocyst, made it possible to explain its worldwide prevalence. In the present paper, events associated with the discovery of its life cycle are recalled.
PMID: 19630138 [PubMed - in process]
A history of studies that examine the interactions of Toxoplasma with its host cell: Emphasis on in vitro models
Int J Parasitol. 2009 Jul 1;39(8):903-14
A history of studies that examine the interactions of Toxoplasma with its host cell: Emphasis on in vitro models
Boyle JP, Radke JR.
Department of Biological Sciences, University of Pittsburgh, PA 15260, USA. boylej@pitt.edu
This review is a historical look at work carried out over the past 50 years examining interactions of Toxoplasma with the host cell and attempts to focus on some of the seminal experiments in the field. This early work formed the foundation for more recent studies aimed at identifying the host and parasite factors mediating key Toxoplasma-host cell interactions. We focus especially on those studies that were performed in vitro and provide discussions of the following general areas: (i) establishment of the parasitophorous vacuole, (ii) the requirement of specific host cell molecules for parasite replication, (iii) the scenarios under which the host cell can resist parasite replication and/or persistence, (iv) host species-specific and host strain-specific responses to Toxoplasma infection, and (v) Toxoplasma-induced immune modulation.
PMID: 19630139 [PubMed - in process]
A history of studies that examine the interactions of Toxoplasma with its host cell: Emphasis on in vitro models
Boyle JP, Radke JR.
Department of Biological Sciences, University of Pittsburgh, PA 15260, USA. boylej@pitt.edu
This review is a historical look at work carried out over the past 50 years examining interactions of Toxoplasma with the host cell and attempts to focus on some of the seminal experiments in the field. This early work formed the foundation for more recent studies aimed at identifying the host and parasite factors mediating key Toxoplasma-host cell interactions. We focus especially on those studies that were performed in vitro and provide discussions of the following general areas: (i) establishment of the parasitophorous vacuole, (ii) the requirement of specific host cell molecules for parasite replication, (iii) the scenarios under which the host cell can resist parasite replication and/or persistence, (iv) host species-specific and host strain-specific responses to Toxoplasma infection, and (v) Toxoplasma-induced immune modulation.
PMID: 19630139 [PubMed - in process]
Toxoplasma gondii: 25 years and 25 major advances for the field
Int J Parasitol. 2009 Jul 1;39(8):935-46
Toxoplasma gondii: 25 years and 25 major advances for the field
Boothroyd JC.
Department of Microbiology and Immunology, Stanford University School of Medicine, CA 94305-5124, USA. john.boothroyd@stanford.edu
This article is an attempt to identify the most significant highlights of Toxoplasma research over the last 25 years. It has been a period of enormous progress and the top 25 most significant advances, in the view of this author, are described. These range from the bench to the bedside and represent a tremendous body of work from countless investigators. And, having laid out so much that has been discovered, it is impossible not to also reflect on the challenges that lie ahead. These, too, are briefly discussed. Finally, while every effort has been made to view the field as a whole, the molecular biology background of the author almost certainly will have skewed the relative importance attached to past and future advances. Despite this, it is hoped that the reader will agree with, or at least not disagree too strongly with, most of the choices presented here.
Publication Types:
Research Support, N.I.H., Extramural
PMID: 19630140 [PubMed - in process]
Toxoplasma gondii: 25 years and 25 major advances for the field
Boothroyd JC.
Department of Microbiology and Immunology, Stanford University School of Medicine, CA 94305-5124, USA. john.boothroyd@stanford.edu
This article is an attempt to identify the most significant highlights of Toxoplasma research over the last 25 years. It has been a period of enormous progress and the top 25 most significant advances, in the view of this author, are described. These range from the bench to the bedside and represent a tremendous body of work from countless investigators. And, having laid out so much that has been discovered, it is impossible not to also reflect on the challenges that lie ahead. These, too, are briefly discussed. Finally, while every effort has been made to view the field as a whole, the molecular biology background of the author almost certainly will have skewed the relative importance attached to past and future advances. Despite this, it is hoped that the reader will agree with, or at least not disagree too strongly with, most of the choices presented here.
Publication Types:
Research Support, N.I.H., Extramural
PMID: 19630140 [PubMed - in process]
Thursday, July 23, 2009
Synthesis and Evaluation of 4-Acyl-2-thiazolylhydrazone Derivatives for Anti-Toxoplasma Efficacy in Vitro
J Med Chem. 2009 Jul 20. [Epub ahead of print]
Synthesis and Evaluation of 4-Acyl-2-thiazolylhydrazone Derivatives for Anti-Toxoplasma Efficacy in Vitro
Chimenti F, Bizzarri B, Bolasco A, Secci D, Chimenti P, Carradori S, Granese A, Rivanera D, Frishberg N, Bordón C, Jones-Brando L.
Dipartimento di Chimica e Tecnologie del Farmaco.
A new series of 4-acyl-2-thiazolylhydrazone derivatives was synthesized and screened for its in vitro activity against Toxoplasma gondii. We evaluated parasite growth inhibition and cytotoxicity, inhibition of replication, and inhibition of parasite invasion of host cells. The biological results indicated that some substances had an antiproliferative effect against intracellular T. gondii tachyzoites cultivated in vitro.
PMID: 19618935 [PubMed - as supplied by publisher]
Synthesis and Evaluation of 4-Acyl-2-thiazolylhydrazone Derivatives for Anti-Toxoplasma Efficacy in Vitro
Chimenti F, Bizzarri B, Bolasco A, Secci D, Chimenti P, Carradori S, Granese A, Rivanera D, Frishberg N, Bordón C, Jones-Brando L.
Dipartimento di Chimica e Tecnologie del Farmaco.
A new series of 4-acyl-2-thiazolylhydrazone derivatives was synthesized and screened for its in vitro activity against Toxoplasma gondii. We evaluated parasite growth inhibition and cytotoxicity, inhibition of replication, and inhibition of parasite invasion of host cells. The biological results indicated that some substances had an antiproliferative effect against intracellular T. gondii tachyzoites cultivated in vitro.
PMID: 19618935 [PubMed - as supplied by publisher]
Indoleamine 2,3-dioxygenase is involved in the defense against Neospora caninum in human and bovine cells
Infect Immun. 2009 Jul 20. [Epub ahead of print]
Indoleamine 2,3-dioxygenase is involved in the defense against Neospora caninum in human and bovine cells
Spekker K, Czesla M, Ince V, Heseler K, Schmidt SK, Schares G, Däubener W.
Institute of Medical Microbiology and Hospital Hygiene, Heinrich-Heine-Universität, D-40225 Düsseldorf, Germany; Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Epidemiology, D-16868 Wusterhausen, Germany.
Neospora caninum is an apicomplexa parasite closely related to Toxoplasma gondii. In nature this parasite is found especially in dogs and cattle, but may also infect other livestock. As an obligate intracellular parasite, Neospora caninum growth is mainly controlled by the cell-mediated immune response. During infection the cytokine interferon-gamma (IFN-gamma) plays a prominent role in regulating the growth of Neospora caninum in natural and also experimental diseases. The present study indicates that the induction of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) is responsible for the inhibition of parasite growth, mediated by IFN-gamma activated bovine fibroblasts and endothelial cells. This antiparasitic effect could be abrogated by the supplementation of tryptophan as well as by the IDO-specific inhibitor 1-L-methyltryptophan. In conclusion, our data show that human and bovine cells use the identical effector mechanism to control the growth of Neospora caninum.
PMID: 19620347 [PubMed - as supplied by publisher]
Indoleamine 2,3-dioxygenase is involved in the defense against Neospora caninum in human and bovine cells
Spekker K, Czesla M, Ince V, Heseler K, Schmidt SK, Schares G, Däubener W.
Institute of Medical Microbiology and Hospital Hygiene, Heinrich-Heine-Universität, D-40225 Düsseldorf, Germany; Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Epidemiology, D-16868 Wusterhausen, Germany.
Neospora caninum is an apicomplexa parasite closely related to Toxoplasma gondii. In nature this parasite is found especially in dogs and cattle, but may also infect other livestock. As an obligate intracellular parasite, Neospora caninum growth is mainly controlled by the cell-mediated immune response. During infection the cytokine interferon-gamma (IFN-gamma) plays a prominent role in regulating the growth of Neospora caninum in natural and also experimental diseases. The present study indicates that the induction of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) is responsible for the inhibition of parasite growth, mediated by IFN-gamma activated bovine fibroblasts and endothelial cells. This antiparasitic effect could be abrogated by the supplementation of tryptophan as well as by the IDO-specific inhibitor 1-L-methyltryptophan. In conclusion, our data show that human and bovine cells use the identical effector mechanism to control the growth of Neospora caninum.
PMID: 19620347 [PubMed - as supplied by publisher]
Wednesday, July 22, 2009
New roles for perforins and proteases in apicomplexan egress
Cell Microbiol. 2009 Jul 13. [Epub ahead of print]
New roles for perforins and proteases in apicomplexan egress
Roiko MS, Carruthers VB.
Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan 48109 U.S.A.
Summary Egress is a pivotal step in the lifecycle of intracellular pathogens initiating the transition from an expiring host cell to a fresh target cell. While much attention has been focused on understanding cell invasion by intracellular pathogens, recent work is providing a new appreciation of mechanisms and therapeutic potential of microbial egress. This review highlights recent insight into cell egress by apicomplexan parasites and emerging contributions of membranolytic and proteolytic secretory products, along with host proteases. New findings suggest that Toxoplasma gondii secretes a pore-forming protein, TgPLP1, during egress that facilitates parasite escape from the cell by perforating the parasitophorous membrane. Also, in a cascade of proteolytic events, Plasmodium falciparum late stage schizonts activate and secrete a subtilisin, PfSUB1, which processes enigmatic putative proteases called SERAs that contribute to merozoite egress. A new report also suggests that calcium-activated host proteases called calpains aid parasite exit, possibly by acting upon the host cytoskeleton. Together these discoveries reveal important new molecular players involved in the principal steps of egress by apicomplexans.
PMID: 19614666 [PubMed - as supplied by publisher]
New roles for perforins and proteases in apicomplexan egress
Roiko MS, Carruthers VB.
Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan 48109 U.S.A.
Summary Egress is a pivotal step in the lifecycle of intracellular pathogens initiating the transition from an expiring host cell to a fresh target cell. While much attention has been focused on understanding cell invasion by intracellular pathogens, recent work is providing a new appreciation of mechanisms and therapeutic potential of microbial egress. This review highlights recent insight into cell egress by apicomplexan parasites and emerging contributions of membranolytic and proteolytic secretory products, along with host proteases. New findings suggest that Toxoplasma gondii secretes a pore-forming protein, TgPLP1, during egress that facilitates parasite escape from the cell by perforating the parasitophorous membrane. Also, in a cascade of proteolytic events, Plasmodium falciparum late stage schizonts activate and secrete a subtilisin, PfSUB1, which processes enigmatic putative proteases called SERAs that contribute to merozoite egress. A new report also suggests that calcium-activated host proteases called calpains aid parasite exit, possibly by acting upon the host cytoskeleton. Together these discoveries reveal important new molecular players involved in the principal steps of egress by apicomplexans.
PMID: 19614666 [PubMed - as supplied by publisher]
Host-derived glucose and its transporter in the obligate intracellular pathogen Toxoplasma are dispensable by glutaminolysis
Proc Natl Acad Sci U S A. 2009 Jul 17. [Epub ahead of print]
Host-derived glucose and its transporter in the obligate intracellular pathogen Toxoplasma gondii are dispensable by glutaminolysis
Blume M, Rodriguez-Contreras D, Landfear S, Fleige T, Soldati-Favre D, Lucius R, Gupta N.
Department of Molecular Parasitology, Humboldt University, Philippstrasse 13, 10115, Berlin, Germany;
Toxoplasma gondii, as an obligate intracellular and promiscuous pathogen of mammalian cells, utilizes host sugars for energy and to generate glycoconjugates that are important to its survival and virulence. Here, we report that T. gondii glucose transporter (TgGT1) is proficient in transporting mannose, galactose, and fructose besides glucose, and serves as a major hexose transporter at its plasma membrane. Toxoplasma harbors 3 additional putative sugar transporters (TgST1-3), of which TgST2 is expressed at its surface, whereas TgST1 and TgST3 are intracellular. Surprisingly, TgGT1 and TgST2 are nonessential to the parasite as their ablations inflict only a 30% or no defect in its intracellular growth, respectively. Indeed, Toxoplasma can also tolerate the deletion of both genes while incurring no further growth phenotype. Unlike Deltatgst2, the modest impairment in Deltatggt1 and Deltatggt1/Deltatgst2 mutants is because of a minor delay in their intracellular replication, which is a direct consequence of the abolished import of glucose. The Deltatggt1 displays an attenuated motility in defined minimal media that is rescued by glutamine. TgGT1-complemented parasites show an entirely restored growth, motility, and sugar import. The lack of exogenous glucose in Deltatggt1 culture fails to accentuate its intrinsic growth defect and prompts it to procure glutamine to sustain its metabolism. Unexpectedly, in vivo virulence of Deltatggt1 in mice remains unaffected. Taken together, our data demonstrate that glucose is nonessential for T. gondii tachyzoites, underscore glutamine is a complement substrate, and provide a basis for understanding the adaptation of T. gondii to diverse host cells.
PMID: 19617561 [PubMed - as supplied by publisher]
Host-derived glucose and its transporter in the obligate intracellular pathogen Toxoplasma gondii are dispensable by glutaminolysis
Blume M, Rodriguez-Contreras D, Landfear S, Fleige T, Soldati-Favre D, Lucius R, Gupta N.
Department of Molecular Parasitology, Humboldt University, Philippstrasse 13, 10115, Berlin, Germany;
Toxoplasma gondii, as an obligate intracellular and promiscuous pathogen of mammalian cells, utilizes host sugars for energy and to generate glycoconjugates that are important to its survival and virulence. Here, we report that T. gondii glucose transporter (TgGT1) is proficient in transporting mannose, galactose, and fructose besides glucose, and serves as a major hexose transporter at its plasma membrane. Toxoplasma harbors 3 additional putative sugar transporters (TgST1-3), of which TgST2 is expressed at its surface, whereas TgST1 and TgST3 are intracellular. Surprisingly, TgGT1 and TgST2 are nonessential to the parasite as their ablations inflict only a 30% or no defect in its intracellular growth, respectively. Indeed, Toxoplasma can also tolerate the deletion of both genes while incurring no further growth phenotype. Unlike Deltatgst2, the modest impairment in Deltatggt1 and Deltatggt1/Deltatgst2 mutants is because of a minor delay in their intracellular replication, which is a direct consequence of the abolished import of glucose. The Deltatggt1 displays an attenuated motility in defined minimal media that is rescued by glutamine. TgGT1-complemented parasites show an entirely restored growth, motility, and sugar import. The lack of exogenous glucose in Deltatggt1 culture fails to accentuate its intrinsic growth defect and prompts it to procure glutamine to sustain its metabolism. Unexpectedly, in vivo virulence of Deltatggt1 in mice remains unaffected. Taken together, our data demonstrate that glucose is nonessential for T. gondii tachyzoites, underscore glutamine is a complement substrate, and provide a basis for understanding the adaptation of T. gondii to diverse host cells.
PMID: 19617561 [PubMed - as supplied by publisher]
Tuesday, July 21, 2009
Toxoplasma H2A Variants Reveal Novel Insights into Nucleosome Composition and Functions for this Histone Family
J Mol Biol. 2009 Jul 13. [Epub ahead of print]
Toxoplasma H2A Variants Reveal Novel Insights into Nucleosome Composition and Functions for this Histone Family
Dalmasso MC, Onyango DO, Naguleswaran A, Sullivan WJ Jr, Angel SO.
Laboratorio de Parasitología Molecular, Instituto de Investigaciones Biotecnológicas-Instituto Tecnologico Chascomús (IIB-INTECH), UNSAM-CONICET, Chascomus (7130), Argentina;
Toxoplasma gondii is an obligate intracellular parasite. Toxoplasmosis is incurable because of its ability to differentiate from the rapidly replicating tachyzoite stage into a latent cyst form (bradyzoite stage). Gene regulation pertinent to Toxoplasma differentiation involves histone modification, but very little is known about the histone proteins in this early branching eukaryote. Here we report the characterization of three H2A histones, a canonical H2A1 and variants H2AX and H2AZ. H2AZ is the minor parasite H2A member. H2A1 and H2AX both have an SQ motif, but only H2AX has a complete SQ(E/D)varphi (varphi denotes a hydrophobic residue) known to be phosphorylated in response to DNA damage. We also show that a novel H2B variant interacts with H2AZ and H2A1 but not with H2AX. Chromatin immunoprecipitation (ChIP) revealed that H2AZ and H2Bv are enriched at active genes while H2AX is enriched at repressed genes as well as the silent TgIRE repeat element. During DNA damage, we detected an increase in H2AX phosphorylation as well as increases in h2a1 and h2ax transcription. We also found that h2ax expression, but not h2a1 and h2az, increases in bradyzoites generated in vitro. Similar analysis performed on mature bradyzoites generated in vivo, which are arrested in G0, showed that h2az and h2ax are actively expressed and h2a1 is not, consistent with the idea that h2a1 is the canonical histone orthologue in the parasite. The increase of H2AX, which localizes to silenced areas during bradyzoite differentiation, is consistent with the quiescent nature of this life cycle stage. Our results indicate that the early-branching eukaryotic parasite Toxoplasma contains nucleosomes of novel composition, which is likely to impact multiple facets of parasite biology, including the clinically important process of bradyzoite differentiation.
PMID: 19607843 [PubMed - as supplied by publisher]
Toxoplasma H2A Variants Reveal Novel Insights into Nucleosome Composition and Functions for this Histone Family
Dalmasso MC, Onyango DO, Naguleswaran A, Sullivan WJ Jr, Angel SO.
Laboratorio de Parasitología Molecular, Instituto de Investigaciones Biotecnológicas-Instituto Tecnologico Chascomús (IIB-INTECH), UNSAM-CONICET, Chascomus (7130), Argentina;
Toxoplasma gondii is an obligate intracellular parasite. Toxoplasmosis is incurable because of its ability to differentiate from the rapidly replicating tachyzoite stage into a latent cyst form (bradyzoite stage). Gene regulation pertinent to Toxoplasma differentiation involves histone modification, but very little is known about the histone proteins in this early branching eukaryote. Here we report the characterization of three H2A histones, a canonical H2A1 and variants H2AX and H2AZ. H2AZ is the minor parasite H2A member. H2A1 and H2AX both have an SQ motif, but only H2AX has a complete SQ(E/D)varphi (varphi denotes a hydrophobic residue) known to be phosphorylated in response to DNA damage. We also show that a novel H2B variant interacts with H2AZ and H2A1 but not with H2AX. Chromatin immunoprecipitation (ChIP) revealed that H2AZ and H2Bv are enriched at active genes while H2AX is enriched at repressed genes as well as the silent TgIRE repeat element. During DNA damage, we detected an increase in H2AX phosphorylation as well as increases in h2a1 and h2ax transcription. We also found that h2ax expression, but not h2a1 and h2az, increases in bradyzoites generated in vitro. Similar analysis performed on mature bradyzoites generated in vivo, which are arrested in G0, showed that h2az and h2ax are actively expressed and h2a1 is not, consistent with the idea that h2a1 is the canonical histone orthologue in the parasite. The increase of H2AX, which localizes to silenced areas during bradyzoite differentiation, is consistent with the quiescent nature of this life cycle stage. Our results indicate that the early-branching eukaryotic parasite Toxoplasma contains nucleosomes of novel composition, which is likely to impact multiple facets of parasite biology, including the clinically important process of bradyzoite differentiation.
PMID: 19607843 [PubMed - as supplied by publisher]
Friday, July 17, 2009
Inconsistencies of genome annotations in apicomplexan parasites revealed by 5'-end-one-pass and full-length sequences of oligo-capped cDNAs
BMC Genomics. 2009 Jul 15;10(1):312. [Epub ahead of print]
Inconsistencies of genome annotations in apicomplexan parasites revealed by 5'-end-one-pass and full-length sequences of oligo-capped cDNAs
Wakaguri H, Suzuki Y, Sasaki M, Sugano S, Watanabe J.
ABSTRACT: BACKGROUND: Apicomplexan parasites are causative agents of various diseases including malaria and have been targets of extensive genomic sequencing. We generated 5'-EST collections for six apicomplexa parasites using our full-length oligo-capping cDNA library method. To improve upon the current genome annotations, as well as to validate the importance for physical cDNA clone resources, we generated a large-scale collection of full-length cDNAs for several apicomplexa parasites. RESULTS: In this study, we used a total of 61,056 5'-end-single-pass cDNA sequences from Plasmodium falciparum, P. vivax, P. yoelii, P. berghei, Cryptosporidium parvum, and Toxoplasma gondii. We compared these partially sequenced cDNA sequences with the currently annotated gene models and observed significant inconsistencies between the two datasets. In particular, we found that on average 14% of the exons in the current gene models were not supported by any cDNA evidence, and that 16% of the current gene models may contain at least one mis-annotation and should be re-evaluated. We also identified a large number of transcripts that had been previously unidentified. For 732 cDNAs in T. gondii, the entire sequences were determined in order to evaluate the annotated gene models at the complete full-length transcript level. We found that 41% of the T. gondii gene models contained at least one inconsistency. We also identified and confirmed by RT-PCR 140 previously unidentified transcripts found in the intergenic regions of the current gene annotations. We show that the majority of these discrepancies are due to questionable predictions of one or two extra exons in the upstream or downstream regions of the genes. CONCLUSIONS: Our data indicates that the current gene models are likely to still be incomplete and have much room for improvement. Our unique full-length cDNA information is especially useful for further refinement of the annotations for the genomes of apicomplexa parasites.
PMID: 19602295 [PubMed - as supplied by publisher]
Inconsistencies of genome annotations in apicomplexan parasites revealed by 5'-end-one-pass and full-length sequences of oligo-capped cDNAs
Wakaguri H, Suzuki Y, Sasaki M, Sugano S, Watanabe J.
ABSTRACT: BACKGROUND: Apicomplexan parasites are causative agents of various diseases including malaria and have been targets of extensive genomic sequencing. We generated 5'-EST collections for six apicomplexa parasites using our full-length oligo-capping cDNA library method. To improve upon the current genome annotations, as well as to validate the importance for physical cDNA clone resources, we generated a large-scale collection of full-length cDNAs for several apicomplexa parasites. RESULTS: In this study, we used a total of 61,056 5'-end-single-pass cDNA sequences from Plasmodium falciparum, P. vivax, P. yoelii, P. berghei, Cryptosporidium parvum, and Toxoplasma gondii. We compared these partially sequenced cDNA sequences with the currently annotated gene models and observed significant inconsistencies between the two datasets. In particular, we found that on average 14% of the exons in the current gene models were not supported by any cDNA evidence, and that 16% of the current gene models may contain at least one mis-annotation and should be re-evaluated. We also identified a large number of transcripts that had been previously unidentified. For 732 cDNAs in T. gondii, the entire sequences were determined in order to evaluate the annotated gene models at the complete full-length transcript level. We found that 41% of the T. gondii gene models contained at least one inconsistency. We also identified and confirmed by RT-PCR 140 previously unidentified transcripts found in the intergenic regions of the current gene annotations. We show that the majority of these discrepancies are due to questionable predictions of one or two extra exons in the upstream or downstream regions of the genes. CONCLUSIONS: Our data indicates that the current gene models are likely to still be incomplete and have much room for improvement. Our unique full-length cDNA information is especially useful for further refinement of the annotations for the genomes of apicomplexa parasites.
PMID: 19602295 [PubMed - as supplied by publisher]
Membrane Contact Sites between Apicoplast and ER in Toxoplasma gondii Revealed by Electron Tomography
Traffic. 2009 Jun 9. [Epub ahead of print]
Membrane Contact Sites between Apicoplast and ER in Toxoplasma gondii Revealed by Electron Tomography
Tomova C, Humbel BM, Geerts WJ, Entzeroth R, Holthuis JC, Verkleij AJ.
Electron Microscopy and Structural Analysis, Department of Biology, Faculty of Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Toxoplasma gondii is an obligate intracellular parasite from the phylum Apicomplexa. A hallmark of these protozoans is the presence of a unique apical complex of organelles that includes the apicoplast, a plastid acquired by secondary endosymbiosis. The apicoplast is indispensible for parasite viability. It harbours a fatty acid biosynthesis type II (FAS II) pathway and plays a key role in the parasite lipid metabolism. Possibly, the apicoplast provides components for the establishment and the maturation of the parasitophorous vacuole, ensuring the successful infection of the host cell. This implies the presence of a transport mechanism for fast and accurate allocation of lipids between the apicoplast and other membrane-bound compartments in the parasite cell. Using a combination of high-pressure freezing, freeze-substitution and electron tomography, we analysed the ultrastructural organization of the apicoplast of T. gondii in relation with the endoplasmic reticulum (ER). This allowed us to clearly show the presence of four continuous membranes surrounding the apicoplast. We present, for the first time, the existence of membrane contact sites between the apicoplast outermost membrane and the ER. We describe the morphological characteristics of these structures and discuss their potential significance for the subcellular distribution of lipids in the parasite.
PMID: 19602198 [PubMed - as supplied by publisher]
Membrane Contact Sites between Apicoplast and ER in Toxoplasma gondii Revealed by Electron Tomography
Tomova C, Humbel BM, Geerts WJ, Entzeroth R, Holthuis JC, Verkleij AJ.
Electron Microscopy and Structural Analysis, Department of Biology, Faculty of Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Toxoplasma gondii is an obligate intracellular parasite from the phylum Apicomplexa. A hallmark of these protozoans is the presence of a unique apical complex of organelles that includes the apicoplast, a plastid acquired by secondary endosymbiosis. The apicoplast is indispensible for parasite viability. It harbours a fatty acid biosynthesis type II (FAS II) pathway and plays a key role in the parasite lipid metabolism. Possibly, the apicoplast provides components for the establishment and the maturation of the parasitophorous vacuole, ensuring the successful infection of the host cell. This implies the presence of a transport mechanism for fast and accurate allocation of lipids between the apicoplast and other membrane-bound compartments in the parasite cell. Using a combination of high-pressure freezing, freeze-substitution and electron tomography, we analysed the ultrastructural organization of the apicoplast of T. gondii in relation with the endoplasmic reticulum (ER). This allowed us to clearly show the presence of four continuous membranes surrounding the apicoplast. We present, for the first time, the existence of membrane contact sites between the apicoplast outermost membrane and the ER. We describe the morphological characteristics of these structures and discuss their potential significance for the subcellular distribution of lipids in the parasite.
PMID: 19602198 [PubMed - as supplied by publisher]
Investigation of folding of purified recombinant GRA1 protein using web based protein disorder servers and trypsin digestion
Protein Pept Lett. 2009;16(7):834-41
Investigation of folding of purified recombinant GRA1 protein using web based protein disorder servers and trypsin digestion
Döşkaya M, Caner A, Değirmenci A, Jurnak F, Gürüz Y.
Department of Parasitology, Ege University Medical School, Bornova/Izmir 35100, Turkey. mert.doskaya@ege.edu.tr
The successful folding of a recombinant protein after expression and purification is essential for structural, biochemical and vaccination studies. Toxoplasma gondii recombinant GRA1 protein is a promising vaccine candidate against toxoplasmosis. In the present study, the folding of recombinant GRA1 protein has been evaluated by web based bioinformatics tools that predict protein folding. Subsequently, trypsin digestion, which is a simple indication of proper protein folding, has been used to determine whether recombinant GRA1 protein is likely to be folded. The results indicate that the recombinant GRA1 protein is predicted to be folded by most of the web based bioinformatics predictors. Moreover, in protease digestion experiments, the recombinant GRA1, which was purified to homogeneity without the use of denaturants, gives rise to a discrete band pattern that is indicative of a folded protein. Together, the results suggest that recombinant GRA1 protein is in a folded conformation, suitable for structural, biochemical and vaccination studies.
PMID: 19601915 [PubMed - in process]
Investigation of folding of purified recombinant GRA1 protein using web based protein disorder servers and trypsin digestion
Döşkaya M, Caner A, Değirmenci A, Jurnak F, Gürüz Y.
Department of Parasitology, Ege University Medical School, Bornova/Izmir 35100, Turkey. mert.doskaya@ege.edu.tr
The successful folding of a recombinant protein after expression and purification is essential for structural, biochemical and vaccination studies. Toxoplasma gondii recombinant GRA1 protein is a promising vaccine candidate against toxoplasmosis. In the present study, the folding of recombinant GRA1 protein has been evaluated by web based bioinformatics tools that predict protein folding. Subsequently, trypsin digestion, which is a simple indication of proper protein folding, has been used to determine whether recombinant GRA1 protein is likely to be folded. The results indicate that the recombinant GRA1 protein is predicted to be folded by most of the web based bioinformatics predictors. Moreover, in protease digestion experiments, the recombinant GRA1, which was purified to homogeneity without the use of denaturants, gives rise to a discrete band pattern that is indicative of a folded protein. Together, the results suggest that recombinant GRA1 protein is in a folded conformation, suitable for structural, biochemical and vaccination studies.
PMID: 19601915 [PubMed - in process]
Wednesday, July 15, 2009
Toxoplasma gondii cathepsin l is the primary target of the invasion inhibitory compound LHVS
J Biol Chem. 2009 Jul 13. [Epub ahead of print]
Toxoplasma gondii cathepsin l is the primary target of the invasion inhibitory compound LHVS
Larson ET, Parussini F, Huynh MH, Giebel JD, Kelley AM, Zhang L, Boygo M, Merritt EA, Carruthers VB.
University of Washington, United States;
The protozoan parasite Toxoplasma gondii relies on post-translational modification, including proteolysis, of proteins required for recognition and invasion of host cells. We have characterized the T. gondii cysteine protease cathepsin L (TgCPL), one of five cathepsins found in the T. gondii genome. We show that TgCPL is the primary target of the compound LHVS, which was previously shown to inhibit parasite invasion by blocking the release of invasion proteins from microneme secretory organelles. As shown by fluorescently-labeled LHVS and TgCPL-specific antibodies, TgCPL is associated with a discrete vesicular structure in the apical region of extracellular parasites, but is found in multiple puncta throughout the cytoplasm of intracellular replicating parasites. LHVS fails to label cells lacking TgCPL due to targeted disruption of the TgCPL gene in two different parasite strains. We present a structural model for the inhibition of TgCPL by LHVS based on a 2.0 Angstrom resolution crystal structure of TgCPL in complex with its propeptide. Possible roles for TgCPL as a protease involved in the degradation or limited proteolysis of parasite proteins involved in invasion are discussed.
PMID: 19596863 [PubMed - as supplied by publisher]
Toxoplasma gondii cathepsin l is the primary target of the invasion inhibitory compound LHVS
Larson ET, Parussini F, Huynh MH, Giebel JD, Kelley AM, Zhang L, Boygo M, Merritt EA, Carruthers VB.
University of Washington, United States;
The protozoan parasite Toxoplasma gondii relies on post-translational modification, including proteolysis, of proteins required for recognition and invasion of host cells. We have characterized the T. gondii cysteine protease cathepsin L (TgCPL), one of five cathepsins found in the T. gondii genome. We show that TgCPL is the primary target of the compound LHVS, which was previously shown to inhibit parasite invasion by blocking the release of invasion proteins from microneme secretory organelles. As shown by fluorescently-labeled LHVS and TgCPL-specific antibodies, TgCPL is associated with a discrete vesicular structure in the apical region of extracellular parasites, but is found in multiple puncta throughout the cytoplasm of intracellular replicating parasites. LHVS fails to label cells lacking TgCPL due to targeted disruption of the TgCPL gene in two different parasite strains. We present a structural model for the inhibition of TgCPL by LHVS based on a 2.0 Angstrom resolution crystal structure of TgCPL in complex with its propeptide. Possible roles for TgCPL as a protease involved in the degradation or limited proteolysis of parasite proteins involved in invasion are discussed.
PMID: 19596863 [PubMed - as supplied by publisher]
Detailed insights from microarray and crystallographic studies into carbohydrate recognition by microneme protein 1 (MIC1) of Toxoplasma
Protein Sci. 2009 Jul 10. [Epub ahead of print]
Detailed insights from microarray and crystallographic studies into carbohydrate recognition by microneme protein 1 (MIC1) of Toxoplasma gondii
Garnett JA, Liu Y, Leon E, Allman S, Friedrich N, Saouros S, Curry S, Soldati-Favre D, Davis B, Feizi T, Matthews S.
Division of Molecular Biosciences, Imperial College London, Biochemistry Building, South Kensington, London SW7 2AZ, United Kingdom.
The intracellular protozoan Toxoplasma gondii is among the most widespread parasites. The broad host cell range of the parasite can be explained by carbohydrate microarray screening analyses that have demonstrated the ability of the T. gondii adhesive protein, TgMIC1, to bind to a wide spectrum of sialyl oligosaccharide ligands. Here, we investigate by further microarray analyses in a dose-response format the differential binding of TgMIC1 to 2-3- and 2-6-linked sialyl carbohydrates. Interestingly, two novel synthetic fluorinated analogs of 3'siaLacNAc(1-4) and 3'siaLacNAc(1-3) were identified as highly potent ligands. To understand the structural basis of the carbohydrate binding specificity of TgMIC1, we have determined the crystal structures of TgMIC1 micronemal adhesive repeat (MAR)-region (TgMIC1-MARR) in complex with the five sialyl-N-acetyllactosamine analogs. These crystal structures have revealed a specific, water-mediated hydrogen bond network that accounts for the preferential binding of TgMIC1-MARR to arrayed 2-3-linked sialyl oligosaccharides and the high potency of the fluorinated analogs. Furthermore, we provide strong evidence for the first observation of a C-F H-O hydrogen bond within a lectin-carbohydrate complex. Finally, detailed comparison with other oligosaccharide-protein complexes in the Protein Data Bank (PDB) reveals a new family of sialic-acid binding sites from lectins in parasites, bacteria and viruses.
PMID: 19593815 [PubMed - as supplied by publisher]
Detailed insights from microarray and crystallographic studies into carbohydrate recognition by microneme protein 1 (MIC1) of Toxoplasma gondii
Garnett JA, Liu Y, Leon E, Allman S, Friedrich N, Saouros S, Curry S, Soldati-Favre D, Davis B, Feizi T, Matthews S.
Division of Molecular Biosciences, Imperial College London, Biochemistry Building, South Kensington, London SW7 2AZ, United Kingdom.
The intracellular protozoan Toxoplasma gondii is among the most widespread parasites. The broad host cell range of the parasite can be explained by carbohydrate microarray screening analyses that have demonstrated the ability of the T. gondii adhesive protein, TgMIC1, to bind to a wide spectrum of sialyl oligosaccharide ligands. Here, we investigate by further microarray analyses in a dose-response format the differential binding of TgMIC1 to 2-3- and 2-6-linked sialyl carbohydrates. Interestingly, two novel synthetic fluorinated analogs of 3'siaLacNAc(1-4) and 3'siaLacNAc(1-3) were identified as highly potent ligands. To understand the structural basis of the carbohydrate binding specificity of TgMIC1, we have determined the crystal structures of TgMIC1 micronemal adhesive repeat (MAR)-region (TgMIC1-MARR) in complex with the five sialyl-N-acetyllactosamine analogs. These crystal structures have revealed a specific, water-mediated hydrogen bond network that accounts for the preferential binding of TgMIC1-MARR to arrayed 2-3-linked sialyl oligosaccharides and the high potency of the fluorinated analogs. Furthermore, we provide strong evidence for the first observation of a C-F H-O hydrogen bond within a lectin-carbohydrate complex. Finally, detailed comparison with other oligosaccharide-protein complexes in the Protein Data Bank (PDB) reveals a new family of sialic-acid binding sites from lectins in parasites, bacteria and viruses.
PMID: 19593815 [PubMed - as supplied by publisher]
Protective immune response in BALB/c mice induced by a suicidal DNA vaccine of the MIC3 gene of Toxoplasma
Vet Parasitol. 2009 Jun 21. [Epub ahead of print]
Protective immune response in BALB/c mice induced by a suicidal DNA vaccine of the MIC3 gene of Toxoplasma gondii
Fang R, Nie H, Wang Z, Tu P, Zhou D, Wang L, He L, Zhou Y, Zhao J.
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, Hubei, PR China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei, PR China.
To evaluate the protective efficiency of a suicidal DNA vaccine against protozoal parasite Toxoplasma gondii, the microneme protein 3 (MIC3) gene was cloned into suicidal vector pSCA1 and conventional DNA vaccine vector pcDNA3.1+ respectively, their protection against T. gondii challenge were assessed in this study. The recombinant plasmids named pSCA/MIC3 and pcDNA/MIC3 were transfected into BHK-21 cells. The expression of MIC3 in BHK-21 cells was confirmed by RT-PCR and indirect immunofluorescence test. Then BALB/c mice were immunized with pSCA/MIC3 or pcDNA/MIC3. Anti-Tg-MIC3 antibodies were detected by indirect ELISA and the cell immune response were examined by lymphocyte proliferation assay and real time RT-PCR. The results showed that the titre of anti-Tg-MIC3 antibodies, stimulation index (SI) of lymphocyte proliferation response and IFN-gamma expression level induced by pSCA/MIC3 and pcDNA/MIC3 were significantly higher than controls (P<0.05), whereas IL-4 expression level in BALB/c mice immunized with either pSCA/MIC3 or pcDNA/MIC3 was lower than that in control group. After a lethal challenge against T. gondii, survival time of the mice immunized with this suicidal DNA vaccine pSCA/MIC3 and conventional DNA vaccine pcDNA/MIC3 were significantly prolonged in comparison with the control groups (P<0.05), but the difference of protective immune response in BALB/c mice between pSCA/MIC3 and pcDNA/MIC3 was not statistically significant (P>0.05). The findings demonstrated that like conventional DNA vaccine pcDNA/MIC3, suicidal DNA vaccine pSCA/MIC3 also provided favourable efficacy, but it could improve the biosafety of conventional vaccines. This result suggested that suicidal DNA vaccine pSCA/MIC3 is a potential candidate vaccine against toxoplasmosis.
PMID: 19592172 [PubMed - as supplied by publisher]
Protective immune response in BALB/c mice induced by a suicidal DNA vaccine of the MIC3 gene of Toxoplasma gondii
Fang R, Nie H, Wang Z, Tu P, Zhou D, Wang L, He L, Zhou Y, Zhao J.
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, Hubei, PR China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, Hubei, PR China.
To evaluate the protective efficiency of a suicidal DNA vaccine against protozoal parasite Toxoplasma gondii, the microneme protein 3 (MIC3) gene was cloned into suicidal vector pSCA1 and conventional DNA vaccine vector pcDNA3.1+ respectively, their protection against T. gondii challenge were assessed in this study. The recombinant plasmids named pSCA/MIC3 and pcDNA/MIC3 were transfected into BHK-21 cells. The expression of MIC3 in BHK-21 cells was confirmed by RT-PCR and indirect immunofluorescence test. Then BALB/c mice were immunized with pSCA/MIC3 or pcDNA/MIC3. Anti-Tg-MIC3 antibodies were detected by indirect ELISA and the cell immune response were examined by lymphocyte proliferation assay and real time RT-PCR. The results showed that the titre of anti-Tg-MIC3 antibodies, stimulation index (SI) of lymphocyte proliferation response and IFN-gamma expression level induced by pSCA/MIC3 and pcDNA/MIC3 were significantly higher than controls (P<0.05), whereas IL-4 expression level in BALB/c mice immunized with either pSCA/MIC3 or pcDNA/MIC3 was lower than that in control group. After a lethal challenge against T. gondii, survival time of the mice immunized with this suicidal DNA vaccine pSCA/MIC3 and conventional DNA vaccine pcDNA/MIC3 were significantly prolonged in comparison with the control groups (P<0.05), but the difference of protective immune response in BALB/c mice between pSCA/MIC3 and pcDNA/MIC3 was not statistically significant (P>0.05). The findings demonstrated that like conventional DNA vaccine pcDNA/MIC3, suicidal DNA vaccine pSCA/MIC3 also provided favourable efficacy, but it could improve the biosafety of conventional vaccines. This result suggested that suicidal DNA vaccine pSCA/MIC3 is a potential candidate vaccine against toxoplasmosis.
PMID: 19592172 [PubMed - as supplied by publisher]
Identification of faecal transmission of Toxoplasma gondii: Small science, large characters
Int J Parasitol. 2009 Jul 1;39(8):871-875. Epub 2009 Feb 4
Identification of faecal transmission of Toxoplasma gondii: Small science, large characters
Ferguson DJ.
Nuffield Department of Clinical Laboratory Science, Oxford University, John Radcliffe Hospital, Oxford, OX3 9DU, UK.
The first clue to the elucidation of the complete life cycle of Toxoplasma gondii was the identification of an infectious form in cat faeces that could be transmitted orally and could survive in the external environment for extended periods. This personal review describes the scientist (W.M. Hutchison) and the background to the initial discovery and covers the period to the complete elucidation of the life cycle of T. gondii.
PMID: 19596630 [PubMed - as supplied by publisher]
Identification of faecal transmission of Toxoplasma gondii: Small science, large characters
Ferguson DJ.
Nuffield Department of Clinical Laboratory Science, Oxford University, John Radcliffe Hospital, Oxford, OX3 9DU, UK.
The first clue to the elucidation of the complete life cycle of Toxoplasma gondii was the identification of an infectious form in cat faeces that could be transmitted orally and could survive in the external environment for extended periods. This personal review describes the scientist (W.M. Hutchison) and the background to the initial discovery and covers the period to the complete elucidation of the life cycle of T. gondii.
PMID: 19596630 [PubMed - as supplied by publisher]
Tuesday, July 14, 2009
Postdoctoral Position
Postdoctoral Position in Molecular and Cellular Parasitology
A postdoctoral position is available immediately in the laboratory of Anthony Sinai PhD. at the University of Kentucky. The laboratory’s interests are in the area of the Cell Biology of the Host-Pathogen interaction using Toxoplasma gondii as a model system. We are particularly interested in the modulation of host functions surrounding the apoptotic cascade and the NFkappaB pathway by the parasite. In addition following collaborative proteomic we have identified a large number of proteins in the vacuolar membrane surrounding the intracellular parasites many of which are fertile targets for investigation. Among targets we a re interested in are glycoproteins likely involved in tissue cyst formation. Finally, in a new direction for the lab we are beginning to investigate the relationship between host energy metabolism and parasite growth and differentiation. The approaches used in the laboratory include state of the art cell biological (fluorescence, confocal and electron microscopy, live cell imaging, flow cytometry, subcellular fractionation), biochemical (protein biology and purification, proteomics, assay development) and molecular biological (genetic screens, molecular cloning, quantitative RT-PCR, bioinformatics). The laboratory is currently supported by an RO1 grant from the NIH with additional support pending. The successful candidate should have a background in molecular and/or cell biology and an interest in microbial pathogenesis. Salary and benefits will be commiserate with experience. The Department of Microbiology, Immunology and Molecular Genetics, offers a highly interactive environment with an emphasis on training. The University is located in Lexington, Kentucky and offers an excellent quality of life. The University of Kentucky is committed to Equal Employment Opportunity and Affirmative Action.
Please submit a curriculum vitae, a 1-2 page outline of research interests and professional goals and the names of 3 professional references. Information should be submitted electronically (as pdf files) to Anthony Sinai PhD. E-mail : sinai@uky.edu. Web site: http://www.mc.uky.edu/microbiology/sinai.asp
A postdoctoral position is available immediately in the laboratory of Anthony Sinai PhD. at the University of Kentucky. The laboratory’s interests are in the area of the Cell Biology of the Host-Pathogen interaction using Toxoplasma gondii as a model system. We are particularly interested in the modulation of host functions surrounding the apoptotic cascade and the NFkappaB pathway by the parasite. In addition following collaborative proteomic we have identified a large number of proteins in the vacuolar membrane surrounding the intracellular parasites many of which are fertile targets for investigation. Among targets we a re interested in are glycoproteins likely involved in tissue cyst formation. Finally, in a new direction for the lab we are beginning to investigate the relationship between host energy metabolism and parasite growth and differentiation. The approaches used in the laboratory include state of the art cell biological (fluorescence, confocal and electron microscopy, live cell imaging, flow cytometry, subcellular fractionation), biochemical (protein biology and purification, proteomics, assay development) and molecular biological (genetic screens, molecular cloning, quantitative RT-PCR, bioinformatics). The laboratory is currently supported by an RO1 grant from the NIH with additional support pending. The successful candidate should have a background in molecular and/or cell biology and an interest in microbial pathogenesis. Salary and benefits will be commiserate with experience. The Department of Microbiology, Immunology and Molecular Genetics, offers a highly interactive environment with an emphasis on training. The University is located in Lexington, Kentucky and offers an excellent quality of life. The University of Kentucky is committed to Equal Employment Opportunity and Affirmative Action.
Please submit a curriculum vitae, a 1-2 page outline of research interests and professional goals and the names of 3 professional references. Information should be submitted electronically (as pdf files) to Anthony Sinai PhD. E-mail : sinai@uky.edu. Web site: http://www.mc.uky.edu/microbiology/sinai.asp
Postdoctoral Opportunities
Opportunities for postdoctoral research in cell & molecular biology of human parasites
NIH funded postdoctoral position(s) are available in the Striepen lab at the Center for Tropical & Emerging Global Diseases at the University of Georgia. We are using a broad array of modern approaches to study the biology of apicomplexan parasites using Toxoplasma gondii as our main experimental model. Current work in the lab is focused on the function and cell biology of a unique parasite endosymbiont (e.g. Proc. Natl. Acad. Sci. 103: 13192, Proc. Natl. Acad. Sci. U.S.A. 105:13574, Current Biol., 19:267‐276), the mechanisms of parasite cell division and cell cycle control (e.g. PLoS Path. 3: e78, J. Cell Sci. 119, 2236, PLoS Path. 4: e36) and the development of novel agents for the treatment of cryptosporidiosis (e.g. Proc. Natl. Acad. Sci. USA. 101: 3154‐3159, Chemistry & Biology 15: 70).
We are currently looking for a highly motivated postdoctoral fellow to spearhead new genetic and mechanistic studies into parasite cell division or parasite metabolism. The lab is part of the one of the largest and most vibrant centers for molecular parasitology in the U.S. bringing together 20 highly interactive labs studying a wide variety of parasitic diseases (http://www.ctegd.uga.edu). The Center recently moved into newly constructed laboratory space and provides outstanding working conditions. The lab and the Center have in house access to state of the art research equipment and expertise particularly in the areas of cell imaging, cell sorting, bioinformatics, biochemistry and immunology.
Athens is a hip college town close to Atlanta and boasts a large research university (34,000 students), a famous music scene with uncounted bands and live venues, great weather almost year round and reasonable cost of living (http://flagpole.com/Guide/).
Please contact:
Boris Striepen
Center for Tropical and Emerging Global Diseases
University of Georgia Paul D. Coverdell Center
Athens, GA 30602
U.S.A.
Tel: 706‐583‐0588
e‐mail: striepen@cb.uga.edu
web: http://webs.cb.uga.edu/~striepen/
NIH funded postdoctoral position(s) are available in the Striepen lab at the Center for Tropical & Emerging Global Diseases at the University of Georgia. We are using a broad array of modern approaches to study the biology of apicomplexan parasites using Toxoplasma gondii as our main experimental model. Current work in the lab is focused on the function and cell biology of a unique parasite endosymbiont (e.g. Proc. Natl. Acad. Sci. 103: 13192, Proc. Natl. Acad. Sci. U.S.A. 105:13574, Current Biol., 19:267‐276), the mechanisms of parasite cell division and cell cycle control (e.g. PLoS Path. 3: e78, J. Cell Sci. 119, 2236, PLoS Path. 4: e36) and the development of novel agents for the treatment of cryptosporidiosis (e.g. Proc. Natl. Acad. Sci. USA. 101: 3154‐3159, Chemistry & Biology 15: 70).
We are currently looking for a highly motivated postdoctoral fellow to spearhead new genetic and mechanistic studies into parasite cell division or parasite metabolism. The lab is part of the one of the largest and most vibrant centers for molecular parasitology in the U.S. bringing together 20 highly interactive labs studying a wide variety of parasitic diseases (http://www.ctegd.uga.edu). The Center recently moved into newly constructed laboratory space and provides outstanding working conditions. The lab and the Center have in house access to state of the art research equipment and expertise particularly in the areas of cell imaging, cell sorting, bioinformatics, biochemistry and immunology.
Athens is a hip college town close to Atlanta and boasts a large research university (34,000 students), a famous music scene with uncounted bands and live venues, great weather almost year round and reasonable cost of living (http://flagpole.com/Guide/).
Please contact:
Boris Striepen
Center for Tropical and Emerging Global Diseases
University of Georgia Paul D. Coverdell Center
Athens, GA 30602
U.S.A.
Tel: 706‐583‐0588
e‐mail: striepen@cb.uga.edu
web: http://webs.cb.uga.edu/~striepen/
Wednesday, July 08, 2009
Mechanisms controlling glideosome function in apicomplexans
Curr Opin Microbiol. 2009 Jul 3. [Epub ahead of print]
Mechanisms controlling glideosome function in apicomplexans
Daher W, Soldati-Favre D.
Department of Microbiology and Molecular Medicine, CMU, University of Geneva, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland.
The glideosome is a unique attribute of the Apicomplexa phylum. This myosin-based machine powers parasite motility, migration across biological barriers, host cell invasion and egress from infected cells. The timing, duration and orientation of gliding motility are tightly regulated to assure establishment of infection. Control of glideosome function occurs at several levels. The assembly of the molecular motor complex is governed by posttranslational modifications resulting from a calcium-dependent signalling cascade. The spatially controlled polymerization of actin filaments crucially impacts motility. The relocation of glycolytic enzymes in close proximity of the glideosome may enhance the local production of energy to sustain movement.
PMID: 19577950 [PubMed - as supplied by publisher]
Mechanisms controlling glideosome function in apicomplexans
Daher W, Soldati-Favre D.
Department of Microbiology and Molecular Medicine, CMU, University of Geneva, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland.
The glideosome is a unique attribute of the Apicomplexa phylum. This myosin-based machine powers parasite motility, migration across biological barriers, host cell invasion and egress from infected cells. The timing, duration and orientation of gliding motility are tightly regulated to assure establishment of infection. Control of glideosome function occurs at several levels. The assembly of the molecular motor complex is governed by posttranslational modifications resulting from a calcium-dependent signalling cascade. The spatially controlled polymerization of actin filaments crucially impacts motility. The relocation of glycolytic enzymes in close proximity of the glideosome may enhance the local production of energy to sustain movement.
PMID: 19577950 [PubMed - as supplied by publisher]
Comparison of effects of green tea catechins on apicomplexan hexose transporters and mammalian orthologues
Mol Biochem Parasitol. 2009 Jul 2. [Epub ahead of print]
Comparison of effects of green tea catechins on apicomplexan hexose transporters and mammalian orthologues
Slavic K, Derbyshire ET, Naftalin RJ, Krishna S, Staines HM.
Centre for Infection, Division of Cellular and Molecular Medicine, St. George's, University of London, Cranmer Terrace, London SW17 0RE, UK.
Here we have investigated the inhibitory properties of green tea catechins on the Plasmodium falciparum hexose transporter (PfHT), the Babesia bovis hexose transporter 1 (BboHT1) and the mammalian facilitative glucose transporters, GLUT1 and GLUT5, expressed in Xenopus laevis oocytes. (-)-Epicatechin-gallate (ECG) and (-)-epigallocatechin-gallate (EGCG) inhibited D-glucose transport by GLUT1 and PfHT, and D-fructose transport by GLUT5, with apparent K(i) values between 45 and 117muM. BboHT1 was more potently inhibited by the ungallated catechins (-)-epicatechin (EC) and (-)-epigallocatechin (EGC), with apparent K(i) values of 108 and 168muM, respectively. Site-directed mutagenesis experiments provided little further support for previously reported models of catechin binding to hexose transporters. Furthermore, P. falciparum growth inhibition by catechins was not affected by the external D-glucose concentration. Our results provide new data on the inhibitory action of catechins against sugar transporters but were unable to elucidate the antimalarial mechanism of action of these agents.
PMID: 19577593 [PubMed - as supplied by publisher]
Comparison of effects of green tea catechins on apicomplexan hexose transporters and mammalian orthologues
Slavic K, Derbyshire ET, Naftalin RJ, Krishna S, Staines HM.
Centre for Infection, Division of Cellular and Molecular Medicine, St. George's, University of London, Cranmer Terrace, London SW17 0RE, UK.
Here we have investigated the inhibitory properties of green tea catechins on the Plasmodium falciparum hexose transporter (PfHT), the Babesia bovis hexose transporter 1 (BboHT1) and the mammalian facilitative glucose transporters, GLUT1 and GLUT5, expressed in Xenopus laevis oocytes. (-)-Epicatechin-gallate (ECG) and (-)-epigallocatechin-gallate (EGCG) inhibited D-glucose transport by GLUT1 and PfHT, and D-fructose transport by GLUT5, with apparent K(i) values between 45 and 117muM. BboHT1 was more potently inhibited by the ungallated catechins (-)-epicatechin (EC) and (-)-epigallocatechin (EGC), with apparent K(i) values of 108 and 168muM, respectively. Site-directed mutagenesis experiments provided little further support for previously reported models of catechin binding to hexose transporters. Furthermore, P. falciparum growth inhibition by catechins was not affected by the external D-glucose concentration. Our results provide new data on the inhibitory action of catechins against sugar transporters but were unable to elucidate the antimalarial mechanism of action of these agents.
PMID: 19577593 [PubMed - as supplied by publisher]
Dynamic Imaging of CD8(+) T cells and dendritic cells during infection with Toxoplasma
PLoS Pathog. 2009 Jul;5(7):e1000505. Epub 2009 Jul 3
Dynamic Imaging of CD8(+) T cells and dendritic cells during infection with Toxoplasma gondii
John B, Harris TH, Tait ED, Wilson EH, Gregg B, Ng LG, Mrass P, Roos DS, Dzierszinski F, Weninger W, Hunter CA.
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA.
To better understand the initiation of CD8(+) T cell responses during infection, the primary response to the intracellular parasite Toxoplasma gondii was characterized using 2-photon microscopy combined with an experimental system that allowed visualization of dendritic cells (DCs) and parasite specific CD8(+) T cells. Infection with T. gondii induced localization of both these populations to the sub-capsular/interfollicular region of the draining lymph node and DCs were required for the expansion of the T cells. Consistent with current models, in the presence of cognate antigen, the average velocity of CD8(+) T cells decreased. Unexpectedly, infection also resulted in modulation of the behavior of non-parasite specific T cells. This TCR-independent process correlated with the re-modeling of the lymph node micro-architecture and changes in expression of CCL21 and CCL3. Infection also resulted in sustained interactions between the DCs and CD8(+) T cells that were visualized only in the presence of cognate antigen and were limited to an early phase in the response. Infected DCs were rare within the lymph node during this time frame; however, DCs presenting the cognate antigen were detected. Together, these data provide novel insights into the earliest interaction between DCs and CD8(+) T cells and suggest that cross presentation by bystander DCs rather than infected DCs is an important route of antigen presentation during toxoplasmosis.
Publication Types:
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
PMID: 19578440 [PubMed - in process]
Dynamic Imaging of CD8(+) T cells and dendritic cells during infection with Toxoplasma gondii
John B, Harris TH, Tait ED, Wilson EH, Gregg B, Ng LG, Mrass P, Roos DS, Dzierszinski F, Weninger W, Hunter CA.
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA.
To better understand the initiation of CD8(+) T cell responses during infection, the primary response to the intracellular parasite Toxoplasma gondii was characterized using 2-photon microscopy combined with an experimental system that allowed visualization of dendritic cells (DCs) and parasite specific CD8(+) T cells. Infection with T. gondii induced localization of both these populations to the sub-capsular/interfollicular region of the draining lymph node and DCs were required for the expansion of the T cells. Consistent with current models, in the presence of cognate antigen, the average velocity of CD8(+) T cells decreased. Unexpectedly, infection also resulted in modulation of the behavior of non-parasite specific T cells. This TCR-independent process correlated with the re-modeling of the lymph node micro-architecture and changes in expression of CCL21 and CCL3. Infection also resulted in sustained interactions between the DCs and CD8(+) T cells that were visualized only in the presence of cognate antigen and were limited to an early phase in the response. Infected DCs were rare within the lymph node during this time frame; however, DCs presenting the cognate antigen were detected. Together, these data provide novel insights into the earliest interaction between DCs and CD8(+) T cells and suggest that cross presentation by bystander DCs rather than infected DCs is an important route of antigen presentation during toxoplasmosis.
Publication Types:
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
PMID: 19578440 [PubMed - in process]
Transmembrane domain containing surface protein from Toxoplasma augments replication in activated immune cells and est of chronic infection
Infect Immun. 2009 Jul 6. [Epub ahead of print]
Transmembrane domain containing surface protein from Toxoplasma gondii augments replication in activated immune cells and the establishment of a chronic infection
Pollard AM, Skariah S, Mordue DG, Knoll LJ.
Toxoplasma gondii mutants identified as defective in the establishment of chronic infection were screened to isolate those specifically impaired in their ability to replicate within activated macrophages. One of the identified mutants contains an insertion in hypothetical gene TGME49_111670. Genetic complementation restores the ability of the mutant to replicate in immune cells and produce cysts in the brains of mice. While the mutant is more sensitive to nitric oxide compared to its parental strain, it is not defective in its ability to suppress nitric oxide. The disrupted protein has no significant homology to proteins with known functions, but is predicted to have one transmembrane domain. Immunofluorescence shows the protein on the parasite surface even in activated macrophages, co-localizing with a tachyzoite surface antigen SAG1, and oriented with its C-terminal end external. Western analysis reveals that the protein is down regulated in bradyzoites. Despite the tachyzoite-specificity of this protein, mice infected with the mutant succumb to acute infection similarly to those infected with the parent strain. Serum from mice with a chronic T. gondii infection reacts to a polypeptide from TGME49_11670, indicating that the protein is seen by the immune system during infection. This study is the first to characterize a T. gondii surface protein that contains a transmembrane domain, and show that the protein contributes to parasite replication in activated immune cells and the establishment of chronic infection.
PMID: 19581395 [PubMed - as supplied by publisher]
Transmembrane domain containing surface protein from Toxoplasma gondii augments replication in activated immune cells and the establishment of a chronic infection
Pollard AM, Skariah S, Mordue DG, Knoll LJ.
Toxoplasma gondii mutants identified as defective in the establishment of chronic infection were screened to isolate those specifically impaired in their ability to replicate within activated macrophages. One of the identified mutants contains an insertion in hypothetical gene TGME49_111670. Genetic complementation restores the ability of the mutant to replicate in immune cells and produce cysts in the brains of mice. While the mutant is more sensitive to nitric oxide compared to its parental strain, it is not defective in its ability to suppress nitric oxide. The disrupted protein has no significant homology to proteins with known functions, but is predicted to have one transmembrane domain. Immunofluorescence shows the protein on the parasite surface even in activated macrophages, co-localizing with a tachyzoite surface antigen SAG1, and oriented with its C-terminal end external. Western analysis reveals that the protein is down regulated in bradyzoites. Despite the tachyzoite-specificity of this protein, mice infected with the mutant succumb to acute infection similarly to those infected with the parent strain. Serum from mice with a chronic T. gondii infection reacts to a polypeptide from TGME49_11670, indicating that the protein is seen by the immune system during infection. This study is the first to characterize a T. gondii surface protein that contains a transmembrane domain, and show that the protein contributes to parasite replication in activated immune cells and the establishment of chronic infection.
PMID: 19581395 [PubMed - as supplied by publisher]
Vertical transmission of HIV and toxoplasma by reactivation in a chronically infected woman
Braz J Infect Dis. 2009 Feb;13(1):70-1
Vertical transmission of HIV and toxoplasma by reactivation in a chronically infected woman
Fernandes RC, Vasconcellos VP, Araújo LC, Medina-Acosta E.
Municipal Program for the Surveillance of Sexually Transmitted Diseases and AIDS, Campos dos Goytacazes, Brazil. reg.fernandes@bol.com.br
Prevention of mother-to-child transmission of HIV and Toxoplasma dual infections in the immunocompromised patient remains a healthcare challenge. We report a case of congenital toxoplasmosis resulting from reactivation of latent infection in a severely immunodepressed HIV-infected pregnant woman, who had poor adherence to therapy; this case illustrates the difficulties encountered in management of such a rare condition.
Publication Types:
Research Support, Non-U.S. Gov't
PMID: 19578634 [PubMed - in process]
Vertical transmission of HIV and toxoplasma by reactivation in a chronically infected woman
Fernandes RC, Vasconcellos VP, Araújo LC, Medina-Acosta E.
Municipal Program for the Surveillance of Sexually Transmitted Diseases and AIDS, Campos dos Goytacazes, Brazil. reg.fernandes@bol.com.br
Prevention of mother-to-child transmission of HIV and Toxoplasma dual infections in the immunocompromised patient remains a healthcare challenge. We report a case of congenital toxoplasmosis resulting from reactivation of latent infection in a severely immunodepressed HIV-infected pregnant woman, who had poor adherence to therapy; this case illustrates the difficulties encountered in management of such a rare condition.
Publication Types:
Research Support, Non-U.S. Gov't
PMID: 19578634 [PubMed - in process]
Thursday, July 02, 2009
Post-doctoral position available
A post-doctoral position is available in the laboratory of Silvia Moreno (smoreno@cb.uga.edu) at the Center for Tropical and Emerging Global Diseases, University of Georgia, to study the role of a recently discovered plant-like vacuole from Toxoplasma gondii. This new organelle contains several important transporters usually found in the tonoplast (the membrane of the plant vacuole), such as the vacuolar-proton pyrophosphatase, an aquaporin channel and a cysteine protease similar to aleurain, a plant vacuole protease. Proteomic analysis of a subcellular fraction enriched in this organelle indicates that it contains several transporters and enzymes involved in the homeostasis of calcium and other ions. Preliminary results from our laboratory indicate a
homeostatic role during the survival of the parasite while outside the host cell.
The project will study:
1) The role of the plant-like vacuole in the homeostasis of calcium and other ions in Toxoplasma gondii.
2) Trafficking of proteins to and from this organelle and its relationship with the endocytic pathway
3) Biogenesis and fusion of the plant-like vacuole with other vacuoles such as acidocalcisomes
The laboratory interacts very closely with the laboratory of Dr. Roberto Docampo with several areas of research in common as ion metabolism and cell signaling of Toxoplasma gondii and trypanosomatids (Trypanosoma cruzi, T. brucei and Leishmania spp.). Since the first description of the acidocalcisomes in Trypanosoma brucei we have been studying their function and origin. As the characteristics and features of acidocalcisomes were revealed, it became apparent that they were morphologically and chemically similar to the “granules” described more than 100 years ago as “metachromatic granules”, “volutin granules” or “polyphosphate bodies” in different microorganisms. For more information on our group research interests check our web site: http://docampo_moreno.ctegd.uga.edu/.
Applicants should have a PhD in a biological science field. Preference will be given to applicants with strong background in molecular biology and/or cell biology. Experience working with Toxoplasma gondii is not necessary but is an advantage.
Interested candidates should contact Dr. Silvia N.J. Moreno by email: smoreno@cb.uga.edu and send their CV and the names/emails of three references.
Evaluation of Toxoplasma gondii placental transmission in BALB/c mice model
Exp Parasitol. 2009 Jun 26. [Epub ahead of print]
Evaluation of Toxoplasma gondii placental transmission in BALB/c mice model
Pezerico SB, Langoni H, Silva AV, Silva RC.
Faculdade de Medicina de Botucatu (FMB), Universidade Estadual Paulista (UNESP), 18618-000, Botucatu, SP, Brazil; Faculdade de Medicina Veterinária e Zootecnia(FMVZ), Universidade Estadual Paulista (UNESP), 18618-000, Botucatu, SP, Brazil.
Toxoplasma gondii infection is common worldwide and highly important to pregnant women as it can be transmitted to the fetus via the placenta. This study aimed at evaluating the prevention of placental transmission in two different strains after chronic infection with each one of the strains. A BALB/c mice model was inoculated 30 days before breeding (immunization) and re-infected 12 and 15 days after pregnancy (challenge). Seven experimental groups were assayed: G1: ME49-immunization (type II), M7741-challenge (type III); G2: M7741-immunization, ME49-challenge; G3, ME49-immunization; G4: M7741-immunization; G5: ME49-challenge; G6: M7741-challenge; G7: saline solution inoculation. Serology, mouse bioassay, PCR and RLFP of the uterus, placenta and fetus were performed to determine the congenital transmission of the strains challenged after chronic infection. IgG T. gondii antibodies were detected in G1, G2, G3 and G4, but not in G5, G6 and G7. All animals of G5 and G6 were IgM-positive. Congenital infection was not detected by bioassay and PCR. Nonetheless, placentas from G3 and G4 resulted positive but no corresponding fetal infection was detected. G1 and G2 did not show the genotype of the strain challenged during pregnancy, only those of chronic infection. Thus, the chronically infected BALB/c mice showed no re-infection after inoculation with another strain during pregnancy. Further studies with different parasite loads and different mice lineages are needed.
PMID: 19563804 [PubMed - as supplied by publisher]
Evaluation of Toxoplasma gondii placental transmission in BALB/c mice model
Pezerico SB, Langoni H, Silva AV, Silva RC.
Faculdade de Medicina de Botucatu (FMB), Universidade Estadual Paulista (UNESP), 18618-000, Botucatu, SP, Brazil; Faculdade de Medicina Veterinária e Zootecnia(FMVZ), Universidade Estadual Paulista (UNESP), 18618-000, Botucatu, SP, Brazil.
Toxoplasma gondii infection is common worldwide and highly important to pregnant women as it can be transmitted to the fetus via the placenta. This study aimed at evaluating the prevention of placental transmission in two different strains after chronic infection with each one of the strains. A BALB/c mice model was inoculated 30 days before breeding (immunization) and re-infected 12 and 15 days after pregnancy (challenge). Seven experimental groups were assayed: G1: ME49-immunization (type II), M7741-challenge (type III); G2: M7741-immunization, ME49-challenge; G3, ME49-immunization; G4: M7741-immunization; G5: ME49-challenge; G6: M7741-challenge; G7: saline solution inoculation. Serology, mouse bioassay, PCR and RLFP of the uterus, placenta and fetus were performed to determine the congenital transmission of the strains challenged after chronic infection. IgG T. gondii antibodies were detected in G1, G2, G3 and G4, but not in G5, G6 and G7. All animals of G5 and G6 were IgM-positive. Congenital infection was not detected by bioassay and PCR. Nonetheless, placentas from G3 and G4 resulted positive but no corresponding fetal infection was detected. G1 and G2 did not show the genotype of the strain challenged during pregnancy, only those of chronic infection. Thus, the chronically infected BALB/c mice showed no re-infection after inoculation with another strain during pregnancy. Further studies with different parasite loads and different mice lineages are needed.
PMID: 19563804 [PubMed - as supplied by publisher]
A Toxoplasma type 2C serine-threonine phosphatase is involved in parasite growth in the mammalian host cell
Microbes Infect. 2009 Jun 26. [Epub ahead of print]
A Toxoplasma type 2C serine-threonine phosphatase is involved in parasite growth in the mammalian host cell
Jan G, Delorme V, Saksouk N, Abrivard M, Gonzalez V, Cayla X, Hakimi MA, Tardieux I.
Institut Cochin, Université Paris Descartes, CNRS UMR 8104, Paris, France; Inserm, U567, Paris, France.
Toxoplasma gondii is a human protozoan parasite that belongs to the phylum of Apicomplexa and causes toxoplasmosis. As the other members of this phylum, T. gondii obligatory multiplies within a host cell by a peculiar type of mitosis that leads to daughter cell assembly within a mother cell. Although parasite growth and virulence have been linked for years, few molecules controlling mitosis have been yet identified and they include a couple of kinases but not the counteracting phosphatases. Here, we report that in contrast to other animal cells, type 2C is by far the major type of serine threonine phosphatase activity both in extracellular and in intracellular dividing parasites. Using wild type and transgenic parasites, we characterized the 37kDa TgPP2C molecule as an abundant cytoplasmic and nuclear enzyme with activity being under tight regulation. In addition, we showed that the increase in TgPP2C activity significantly affected parasite growth by impairing cytokinesis while nuclear division still occurred. This study supports for the first time that type 2C protein phosphatase is an important regulator of cell growth in T. gondii.
PMID: 19563907 [PubMed - as supplied by publisher]
A Toxoplasma type 2C serine-threonine phosphatase is involved in parasite growth in the mammalian host cell
Jan G, Delorme V, Saksouk N, Abrivard M, Gonzalez V, Cayla X, Hakimi MA, Tardieux I.
Institut Cochin, Université Paris Descartes, CNRS UMR 8104, Paris, France; Inserm, U567, Paris, France.
Toxoplasma gondii is a human protozoan parasite that belongs to the phylum of Apicomplexa and causes toxoplasmosis. As the other members of this phylum, T. gondii obligatory multiplies within a host cell by a peculiar type of mitosis that leads to daughter cell assembly within a mother cell. Although parasite growth and virulence have been linked for years, few molecules controlling mitosis have been yet identified and they include a couple of kinases but not the counteracting phosphatases. Here, we report that in contrast to other animal cells, type 2C is by far the major type of serine threonine phosphatase activity both in extracellular and in intracellular dividing parasites. Using wild type and transgenic parasites, we characterized the 37kDa TgPP2C molecule as an abundant cytoplasmic and nuclear enzyme with activity being under tight regulation. In addition, we showed that the increase in TgPP2C activity significantly affected parasite growth by impairing cytokinesis while nuclear division still occurred. This study supports for the first time that type 2C protein phosphatase is an important regulator of cell growth in T. gondii.
PMID: 19563907 [PubMed - as supplied by publisher]
Toxoplasma gondii cyclophilin 18-mediated production of nitric oxide induces bradyzoite conversion in a CCR5-dependent manner
Infect Immun. 2009 Jun 29. [Epub ahead of print]
Toxoplasma gondii cyclophilin 18-mediated production of nitric oxide induces bradyzoite conversion in a CCR5-dependent manner
Ibrahim HM, Bannai H, Xuan X, Nishikawa Y.
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan; Zoology Department, Faculty of Science, Minufiya University, Shibin El kom, Egypt.
Toxoplasma gondii modulates pro- and anti-inflammatory responses to regulate parasite multiplication and host survival. Pressure from the immune response causes the conversion of tachyzoites to slowly dividing bradyzoites. The regulatory mechanisms involved in this switch are poorly understood. The aim of this study was to investigate the immunomodulatory role of T. gondii cyclophilin18 (TgCyp18) in macrophages and the consequences of the cellular responses on the conversion machinery. The recombinant TgCyp18 induced the production of nitric oxide (NO), IL-12 and TNF-alpha through its binding with cysteine-cysteine chemokine receptor 5 (CCR5) and the production of IFN-gamma and IL-6 in a CCR5-independent manner. Interestingly, treatment of macrophages with TgCyp18 resulted in the inhibition of parasite growth and enhancement of the conversion into bradyzoites via NO in a CCR5-dependent manner. In conclusion, T. gondii possesses sophisticated mechanisms to manipulate host-cell responses in a TgCyp18-mediated process.
PMID: 19564392 [PubMed - as supplied by publisher]
Toxoplasma gondii cyclophilin 18-mediated production of nitric oxide induces bradyzoite conversion in a CCR5-dependent manner
Ibrahim HM, Bannai H, Xuan X, Nishikawa Y.
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan; Zoology Department, Faculty of Science, Minufiya University, Shibin El kom, Egypt.
Toxoplasma gondii modulates pro- and anti-inflammatory responses to regulate parasite multiplication and host survival. Pressure from the immune response causes the conversion of tachyzoites to slowly dividing bradyzoites. The regulatory mechanisms involved in this switch are poorly understood. The aim of this study was to investigate the immunomodulatory role of T. gondii cyclophilin18 (TgCyp18) in macrophages and the consequences of the cellular responses on the conversion machinery. The recombinant TgCyp18 induced the production of nitric oxide (NO), IL-12 and TNF-alpha through its binding with cysteine-cysteine chemokine receptor 5 (CCR5) and the production of IFN-gamma and IL-6 in a CCR5-independent manner. Interestingly, treatment of macrophages with TgCyp18 resulted in the inhibition of parasite growth and enhancement of the conversion into bradyzoites via NO in a CCR5-dependent manner. In conclusion, T. gondii possesses sophisticated mechanisms to manipulate host-cell responses in a TgCyp18-mediated process.
PMID: 19564392 [PubMed - as supplied by publisher]
Is Toxoplasma gondii a Causal Agent in Migraine?
Am J Med Sci. 2009 Jun 26. [Epub ahead of print]
Is Toxoplasma gondii a Causal Agent in Migraine?
Koseoglu E, Yazar S, Koc I.
From the Departments of Neurology (ek, ik) and Parasitology (sy), Medical Faculty, Erciyes University, Kayseri, Turkey.
BACKGROUND:: Many different tissues may be parasitized by Toxoplasma gondii, particularly, lung, heart, lymphoid organs, and the central nervous tissues. Tissue cysts of this parasite in the brain may spontaneously rupture, releasing parasites that cause antibody titers to rise. In immunocompetent subjects with acquired toxoplasmosis, the most frequent symptoms were lymphadenopathy and headache. In the neurogenic inflammation theory of the pathogenesis of migraine, the cause of initial release of ions and inflammatory agents has not been established. In this study, we aimed to investigate if T. gondii infection is a possible cause of neurogenic inflammation of migraines. METHODS:: The anti-T. gondii antibody status of 104 patients with migraine were studied and compared with those of control groups, 50 healthy subjects and 50 subjects with headache due to rhinosinusitis, by using a micro-enzyme-linked immunosorbent assay technique. RESULTS:: Forty-six (44.2%) patients with migraine, 13 (26.0%) healthy control subjects, and 12 (24%) control subjects with rhinosinusitis were positive for anti-T. gondii IgG antibody. The rate of positivity in the migraine patient group was statistically different from those of the control groups (P < 0.05). CONCLUSIONS:: The results show the presence of chronic Toxoplasma infection in patients with migraine. Toxoplasma infection may contribute to neurogenic inflammation as the pathogenesis of migraine, as many studies in the literature have reported that Toxoplasma infection causes biochemical and immunologic changes.
PMID: 19564786 [PubMed - as supplied by publisher]
Is Toxoplasma gondii a Causal Agent in Migraine?
Koseoglu E, Yazar S, Koc I.
From the Departments of Neurology (ek, ik) and Parasitology (sy), Medical Faculty, Erciyes University, Kayseri, Turkey.
BACKGROUND:: Many different tissues may be parasitized by Toxoplasma gondii, particularly, lung, heart, lymphoid organs, and the central nervous tissues. Tissue cysts of this parasite in the brain may spontaneously rupture, releasing parasites that cause antibody titers to rise. In immunocompetent subjects with acquired toxoplasmosis, the most frequent symptoms were lymphadenopathy and headache. In the neurogenic inflammation theory of the pathogenesis of migraine, the cause of initial release of ions and inflammatory agents has not been established. In this study, we aimed to investigate if T. gondii infection is a possible cause of neurogenic inflammation of migraines. METHODS:: The anti-T. gondii antibody status of 104 patients with migraine were studied and compared with those of control groups, 50 healthy subjects and 50 subjects with headache due to rhinosinusitis, by using a micro-enzyme-linked immunosorbent assay technique. RESULTS:: Forty-six (44.2%) patients with migraine, 13 (26.0%) healthy control subjects, and 12 (24%) control subjects with rhinosinusitis were positive for anti-T. gondii IgG antibody. The rate of positivity in the migraine patient group was statistically different from those of the control groups (P < 0.05). CONCLUSIONS:: The results show the presence of chronic Toxoplasma infection in patients with migraine. Toxoplasma infection may contribute to neurogenic inflammation as the pathogenesis of migraine, as many studies in the literature have reported that Toxoplasma infection causes biochemical and immunologic changes.
PMID: 19564786 [PubMed - as supplied by publisher]
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