Saturday, February 28, 2009

Export of a Toxoplasma gondii Rhoptry Neck Protein Complex at the Host Cell Membrane to Form the Moving Junction during Invasion

PLoS Pathog. 2009 Feb;5(2):e1000309. Epub 2009 Feb 27

Export of a Toxoplasma gondii Rhoptry Neck Protein Complex at the Host Cell Membrane to Form the Moving Junction during Invasion

Besteiro S, Michelin A, Poncet J, Dubremetz JF, Lebrun M.

UMR 5235 CNRS, Université de Montpellier 2, Montpelier, France.

One of the most conserved features of the invasion process in Apicomplexa parasites is the formation of a moving junction (MJ) between the apex of the parasite and the host cell membrane that moves along the parasite and serves as support to propel it inside the host cell. The MJ was, up to a recent period, completely unknown at the molecular level. Recently, proteins originated from two distinct post-Golgi specialised secretory organelles, the micronemes (for AMA1) and the neck of the rhoptries (for RON2/RON4/RON5 proteins), have been shown to form a complex. AMA1 and RON4 in particular, have been localised to the MJ during invasion. Using biochemical approaches, we have identified RON8 as an additional member of the complex. We also demonstrated that all RON proteins are present at the MJ during invasion. Using metabolic labelling and immunoprecipitation, we showed that RON2 and AMA1 were able to interact in the absence of the other members. We also discovered that all MJ proteins are subjected to proteolytic maturation during trafficking to their respective organelles and that they could associate as non-mature forms in vitro. Finally, whereas AMA1 has previously been shown to be inserted into the parasite membrane upon secretion, we demonstrated, using differential permeabilization and loading of RON-specific antibodies into the host cell, that the RON complex is targeted to the host cell membrane, where RON4/5/8 remain associated with the cytoplasmic face. Globally, these results point toward a model of MJ organization where the parasite would be secreting and inserting interacting components on either side of the MJ, both at the host and at its own plasma membranes.

PMID: 19247437 [PubMed - in process]

Manipulative parasites in the world of veterinary science: Implications for epidemiology and pathology

Vet J. 2009 Feb 23. [Epub ahead of print]

Manipulative parasites in the world of veterinary science: Implications for epidemiology and pathology

Lagrue C, Poulin R.

Department of Zoology, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand.

One of the most complex and least understood transmission strategies displayed by pathogenic parasites is that of manipulation of host behaviour. A wide variety of parasites alter their host's behaviour, including species of medical and veterinary importance, such as Diplostomum spathaceum, Echinococcus spp. and Toxoplasma gondii. The manipulative ability of these parasites has implications for pathology and transmission dynamics. Domestic animals are hosts for manipulative pathogens, either by being the target host and acquiring the parasite as a result of vector-host manipulation, or by having their behaviour changed by manipulative parasites. This review uses several well-known pathogens to demonstrate how host manipulation by parasites is potentially important in epidemiology.

PMID: 19243982 [PubMed - as supplied by publisher]

Thursday, February 26, 2009

Guanidine hydrochloride- and urea- induced unfolding of Toxoplasma gondii Ferredoxin- NADP+ reductase

J Biochem. 2009 Feb 23. [Epub ahead of print]

Guanidine hydrochloride- and urea- induced unfolding of Toxoplasma gondii Ferredoxin- NADP+ reductase: stabilization of a functionally inactive holo-intermediate.

Singh K, Bhakuni V.

Division of Molecular and Structural Biology Central Drug Research Institute Lucknow- 226 001, India.

Usually during the folding/unfolding of flavoproteins an apo-intermediate is stabilized before global unfolding of the enzymes occurs. However, stabilization of a holo-intermediate has also been reported for a few flavoproteins. We have studied the unfolding of Toxoplasma gondii Ferredoxin- NADP(+) reductase (TgFNR) using GdnHCl and urea. A functionally inactive holo-intermediate of the enzyme was found to be stabilized during this unfolding process. The intermediate species had cofactor FAD bound to it, but it showed free movement due to which the stabilized intermediates were functionally inactive. The native TgFNR behaves cooperatively with the two structural domains interacting strongly with each other. The denaturants GdnHCl and urea, at low concentrations were found to interact selectively with the NADP(+)- binding domain of TgFNR and induce structural modifications in it. These selective modifications in the protein molecule lead to loss of interactions between two domains and the enzyme behaved non-cooperatively resulting in stabilization of an intermediate species. Significant differences in the structural properties of the GdnHCl- and urea-stabilized holo-intermediates of TgFNR were observed. Comparison of the unfolding pathway of TgFNR (a plant-type FNR) with that of FprA (a GR-type FNR) demonstrates that they follow very different pathways of unfolding.

PMID: 19237441 [PubMed - as supplied by publisher]

Friday, February 20, 2009

GRA2 and ROP1 recombinant antigens as potential markers for detection of Toxoplasma gondii-specific immunoglobulin G in human with acute toxoplasmosis

Clin Vaccine Immunol. 2009 Feb 18. [Epub ahead of print]

GRA2 and ROP1 recombinant antigens as potential markers for detection of Toxoplasma gondii-specific immunoglobulin G in human with acute toxoplasmosis

Holec-Gasior L, Kur J, Hiszczynska-Sawicka E.

Gdask University of Technology, Chemical Faculty, Department of Microbiology, Gdask, POLAND.

A goal of the current study was to evaluate serological applications of Toxoplasma gondii GRA2 and ROP1 antigens. Soluble recombinant GRA2 and ROP1 antigens as fusion proteins containing six histidyl residues at N- and C-terminal were obtained using an Escherichia coli expression system. Purification by a one-step metal affinity chromatography allowed recovery of milligram amounts of pure recombinant proteins per litre of culture. The usefulness of these antigens for diagnosis of human infections was tested on 167 serum samples obtained during routine diagnostic tests. A panel of 37 sera from patients with acute toxoplasmosis was compared to a panel of 90 sera from individuals with past infection. The results indicate that both GRA2 and ROP1 recombinant antigens detected antibodies more frequently from acute (100% and 94.6%, respectively) than from chronic infections (22.5% and 15.5% respectively). These results suggest that IgG antibodies against GRA2 and ROP1 antigens are produced during the acute stage of toxoplasmosis but are uncommon in the chronic phase of the infection. Hence, these recombinant proteins can be used as specific molecular markers to differentiate between acute and chronic infections.

PMID: 19225074 [PubMed - as supplied by publisher]

Wednesday, February 18, 2009

A Dynamin Is Required for the Biogenesis of Secretory Organelles in Toxoplasma gondii

Curr Biol. 2009 Feb 11. [Epub ahead of print]

A Dynamin Is Required for the Biogenesis of Secretory Organelles in Toxoplasma gondii

Breinich MS, Ferguson DJ, Foth BJ, van Dooren GG, Lebrun M, Quon DV, Striepen B, Bradley PJ, Frischknecht F, Carruthers VB, Meissner M.

Hygiene Institute, Department of Parasitology, Heidelberg University School of Medicine, Heidelberg 69120, Germany.

BACKGROUND: Apicomplexans contain only a core set of factors involved in vesicular traffic. Yet these obligate intracellular parasites evolved a set of unique secretory organelles (micronemes, rhoptries, and dense granules) that are required for invasion and modulation of the host cell. Apicomplexa replicate by budding from or within a single mother cell, and secretory organelles are synthesized de novo at the final stage of division. To date, the molecular basis for their biogenesis is unknown. RESULTS: We demonstrate that the apicomplexan dynamin-related protein B (DrpB) belongs to an alveolate specific family of dynamins that is expanded in ciliates. DrpB accumulates in a cytoplasmic region close to the Golgi that breaks up during replication and reforms after assembly of the daughter cells. Conditional ablation of DrpB function results in mature daughter parasites that are devoid of micronemes and rhoptries. In the absence of these organelles, invasion-related secretory proteins are mistargeted to the constitutive secretory pathway. Mutant parasites are able to replicate but are unable to escape from or invade into host cells. CONCLUSIONS: DrpB is the essential mechanoenzyme for the biogenesis of secretory organelles in Apicomplexa. We suggest that DrpB is required during replication to generate vesicles for the regulated secretory pathway that form the unique secretory organelles. Our study supports a role of an alveolate-specific dynamin that was required for the evolution of novel, secretory organelles. In the case of Apicomplexa, these organelles further evolved to enable a parasitic lifestyle.

PMID: 19217293 [PubMed - as supplied by publisher]

A Novel Dynamin-Related Protein Has Been Recruited for Apicoplast Fission in Toxoplasma gondii

Curr Biol. 2009 Feb 11. [Epub ahead of print]

A Novel Dynamin-Related Protein Has Been Recruited for Apicoplast Fission in Toxoplasma gondii

van Dooren GG, Reiff SB, Tomova C, Meissner M, Humbel BM, Striepen B.

Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA 30602, USA.

Background: Apicomplexan parasites cause numerous important human diseases, including malaria and toxoplasmosis. Apicomplexa belong to the Alveolata, a group that also includes ciliates and dinoflagellates. Apicomplexa retain a plastid organelle (the apicoplast) that was derived from an endosymbiotic relationship between the alveolate ancestor and a red alga. Apicoplasts are essential for parasite growth and must correctly divide and segregate into daughter cells upon cytokinesis. Apicoplast division depends on association with the mitotic spindle, although little is known about the molecular machinery involved in this process. Apicoplasts lack the conserved machinery that divides chloroplasts in plants and red algae, suggesting that these mechanisms are unique. Results: Here, we demonstrate that a dynamin-related protein in Toxoplasma gondii (TgDrpA) localizes to punctate regions on the apicoplast surface. We generate a conditional dominant-negative TgDrpA cell line to disrupt TgDrpA functions and demonstrate that TgDrpA is essential for parasite growth and apicoplast biogenesis. Fluorescence recovery after photobleaching and time-lapse imaging studies provide evidence for a direct role for TgDrpA in apicoplast fission. Conclusions: Our data suggest that DrpA was likely recruited from the alveolate ancestor to function in fission of the symbiont and ultimately replaced the conserved division machinery of that symbiont.

PMID: 19217294 [PubMed - as supplied by publisher]

Toxoplasma gondii: congenital transmission in a hamster model

Exp Parasitol. 2009 Feb 12. [Epub ahead of print]

Toxoplasma gondii: congenital transmission in a hamster model

Freyre A, Fialho CG, Bigatti LE, Araujo FA, Falcón JD, Mendez J, González M.

Laboratorio de Toxoplasmosis, Departamento de Parasitologi a, Facultad de Veterinaria, Montevideo, Uruguay.

The objective of the research was to test the hamster for a model of transmission of congenital toxoplasmosis. A non invasive method for the diagnosis of pregnancy in hamsters was designed, with a specificity and a sensitivity of 70.2 and 94.7%, respectively (n=168). Of 32 females with a chronic toxoplasma infection, 3 transmitted Toxoplasma congenitally during their first pregnancy, but not during the subsequent pregnancy. Congenital transmission rates of infections initiated during pregnancy with 2 stages of 2 strains of Toxoplasma were in the range of 33 to 100% of the 76 females inoculated . Only 1 of 17 females transmitted the parasite exclusively via milk. It was concluded that the hamster is a promising species for a model of transmission of congenital toxoplasmosis.

PMID: 19217907 [PubMed - as supplied by publisher]

Toxoplasmosis: a history of clinical observations

Int J Parasitol. 2009 Feb 12. [Epub ahead of print]

Toxoplasmosis: a history of clinical observations

Weiss LM, Dubey JP.

Departments of Medicine (Division of Infectious Diseases) and Pathology (Division of Parasitology),Albert Einstein College of Medicine,,1300 Morris Park Avenue, Forchheimer 504, Bronx, NY 10461, USA.

It has been 100 years since Toxoplasma gondii was initially described in Tunis by Nicolle and Manceaux (1908) in the tissues of the gundi (Ctenodoactylus gundi) and in Brazil by Splendore (1908) in the tissues of a rabbit. Toxoplasma gondii is a ubiquitous, Apicomplexan parasite of warm-blooded animals that can cause several clinical syndromes including encephalitis, chorioretinitis, congenital infection and neonatal mortality. Fifteen years after the description of T. gondii by Nicolle and Manceaux a fatal case of toxoplasmosis in a child was reported by Janků. In 1939 Wolf, Cowen and Paige were the first to conclusively identify T. gondii as a cause of human disease. This review examines the clinical manifestations of infection with T. gondii and the history of the discovery of these manifestations.

PMID: 19217908 [PubMed - as supplied by publisher]

Sexual recombination punctuated by outbreaks and clonal expansions predicts Toxoplasma gondii population genetics

Int J Parasitol. 2009 Feb 12. [Epub ahead of print]

Sexual recombination punctuated by outbreaks and clonal expansions predicts Toxoplasma gondii population genetics

Grigg ME, Sundar N.

Molecular Parasitology Unit, Laboratory of Parasitic Diseases, NIAID, NIH, Bethesda MD, 20815, USA.

The cosmopolitan parasitic pathogen Toxoplasma gondii is capable of infecting essentially any warm-blooded vertebrate worldwide, including most birds and mammals, and establishes chronic infections in one-third of the globe's human population. The success of this highly prevalent zoonosis is largely the result of its ability to propagate both sexually and clonally. Frequent genetic exchanges via sexual recombination among extant parasite lineages that mix in the definitive felid host produces new lines that emerge to expand the parasite's host range and cause outbreaks. Highly successful lines spread clonally via carnivorism and in some cases sweep to pandemic levels. The extent to which sexual reproduction versus clonal expansion shapes Toxoplasma's current, global population genetic structure is the central question this review will attempt to answer.

PMID: 19217909 [PubMed - as supplied by publisher]

Efficient gene replacements in Toxoplasma gondii strains deficient for nonhomologous end-joining

Eukaryot Cell. 2009 Feb 13. [Epub ahead of print]

Efficient gene replacements in Toxoplasma gondii strains deficient for nonhomologous end-joining

Fox BA, Ristuccia JG, Gigley JP, Bzik DJ.

Department of Microbiology and Immunology, Dartmouth Medical School, 1 Medical Center Drive, Lebanon, NH 03756 USA.

A high frequency of nonhomologous recombination has hampered gene targeting approaches in the model Apicomplexan parasite Toxoplasma gondii. To address whether the nonhomologous end-joining (NHEJ) DNA repair pathway could be disrupted in this obligate intracellular parasite, putative KU proteins were identified and a predicted KU80 gene was deleted. The efficiency of gene targeting via double cross-over homologous recombination at several genetic loci was found to be greater than 97% of the total transformants in KU80 knockouts. Gene replacement efficiency was markedly increased (300 to 400-fold) in KU80 knockouts compared to wild-type strains. Target DNA flanks of only approximately 500 bp were found to be sufficient for efficient gene replacements in KU80 knockouts. KU80 knockouts stably retained a normal growth rate in vitro and the high virulence phenotype of Type I strains, but exhibited an increased sensitivity to double-strand DNA breaks induced by treatment with phleomycin or gamma-irradiation. Collectively, these results revealed that a significant KU-dependent NHEJ DNA repair pathway was present in Toxoplasma gondii. Integration essentially occurs only at the homologous targeted sites in the KU80 knockout background, making this genetic background an efficient host for gene targeting to speed post-genome functional analysis and genetic dissection of parasite biology.

PMID: 19218423 [PubMed - as supplied by publisher]

Tagging of endogenous genes in a Toxoplasma gondii strain lacking Ku80

Eukaryot Cell. 2009 Feb 13. [Epub ahead of print]

Tagging of endogenous genes in a Toxoplasma gondii strain lacking Ku80

Huynh MH, Carruthers VB.

Department of Microbiology and Immunology, University of Michigan School of Medicine, 1150 W. Medical Center Dr., Ann Arbor, MI 48109 USA.

As with other organisms with a completed genome sequence, opportunities for performing large scale studies, such as expression and localization, on Toxoplasma gondii are now much more feasible. We present a system for tagging genes endogenously with yellow fluorescent protein (YFP) in a Deltaku80 strain. Ku80 is involved in DNA strand repair and non-homologous DNA end-joining; previous studies in other organisms have shown that in its absence, random integration is eliminated, allowing the insertion of constructs with homologous sequences into the proper loci. We generated a vector consisting of YFP and a DHFR-TS selectable marker. The YFP is preceded by a LIC (ligation-independent cloning) cassette, which allows the insertion of PCR products containing complementary LIC sequences. We demonstrate that the Deltaku80 strain is more effective and efficient in integrating the YFP-tagged genes into the correct locus compared to the wild-type strain RH. We then selected several hypothetical proteins that were identified by a proteomic screen of excreted-secreted antigens (ESA) and that displayed microarray expression profiles similar to known micronemal proteins, with the thought that these could potentially be new proteins with roles in cell invasion. We localized these hypothetical proteins by YFP fluorescence and show expression by immunoblotting. Our findings demonstrate that the combination of the Deltaku80 strain and the pYFP.LIC constructs reduces both the time and cost required to determine localization of a new gene of interest. This should open the opportunity for performing larger scale studies of novel T. gondii genes.

PMID: 19218426 [PubMed - as supplied by publisher]

Comparison of indirect fluorescent antibody test and modified agglutination test

Parasitol Res. 2009 Feb 17. [Epub ahead of print]

Comparison of indirect fluorescent antibody test and modified agglutination test for detecting Toxoplasma gondii immunoglobulin G antibodies in dog and cat

Macrì G, Sala M, Linder AM, Pettirossi N, Scarpulla M.

Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana (IZS LT), Rome, Italy,

The present study describes the comparison between a modified agglutination test (MAT) and the indirect fluorescent antibody test (IFAT) for the detection of Toxoplasma specific IgG antibodies in dog and cat sera. MAT showed an "almost perfect" agreement with IFAT in detecting positive and negative results in cat sera, where as only a "substantial" agreement was observed in dog sera due to false negative results. Differences relative to sample dilution were recorded and serological titres obtained by MAT and IFAT are not directly comparable in cat and dog sera.

PMID: 19221795 [PubMed - as supplied by publisher]

Friday, February 13, 2009

Neuropsychiatric disease and Toxoplasma gondii infection

Neuroimmunomodulation. 2009;16(2):122-33. Epub 2009 Feb 11

Neuropsychiatric disease and Toxoplasma gondii infection

Henriquez SA, Brett R, Alexander J, Pratt J, Roberts CW.

Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow, UK.

Toxoplasma gondii infects approximately 30% of the world's population, but causes overt clinical symptoms in only a small proportion of people. In recent years, the ability of the parasite to manipulate the behaviour of infected mice and rats and alter personality attributes of humans has been reported. Furthermore, a number of studies have now suggested T. gondii infection as a risk factor for the development of schizophrenia and depression in humans. As T. gondii forms cysts that are located in various anatomical sites including the brain during a chronic infection, it is well placed anatomically to mediate these effects directly. The T. gondii genome is known to contain 2 aromatic amino acid hydroxylases that potentially could directly affect dopamine and/or serotonin biosynthesis. However, stimulation of the immune response has also recently been associated with mood and behavioural alterations in humans, and compounds designed to alter mood, such as fluoxetine, have been demonstrated to alter aspects of immune function. Herein, the evidence for T.-gondii-induced behavioural changes relevant to schizophrenia and depression is reviewed. Potential mechanisms responsible for these changes in behaviour including the role of tryptophan metabolism and the hypothalamic-pituitary-adrenal axis are discussed. Copyright (c) 2009 S. Karger AG, Basel.

PMID: 19212132 [PubMed - in process]

Wednesday, February 11, 2009

The Toxoplasma gondii-Shuttling Function of Dendritic Cells is Linked to the Parasite Genotype

Infect Immun. 2009 Feb 9. [Epub ahead of print]

The Toxoplasma gondii-Shuttling Function of Dendritic Cells is Linked to the Parasite Genotype

Lambert H, Vutova PP, Adams WC, Loré K, Barragan A.

Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, SE-141 86 Stockholm, Sweden; Department of Parasitology, Mycology and Environmental Microbiology, and Department of Virology, Swedish Institute for Infectious Disease Control, SE-171 82 Stockholm, Sweden.

Following intestinal invasion, the processes leading to systemic dissemination of the obligate intracellular protozoan Toxoplasma gondii remain poorly understood. Recently, tachyzoites representative of type I, II and III T. gondii populations were shown to differ with respect to their ability to transmigrate across cellular barriers. In this process of active parasite motility, type I strains exhibit a superior migratory capacity than the type II and type III strains. Data also suggest that tachyzoites rely on migrating dendritic cells (DC) as shuttling leukocytes to disseminate in tissue, e.g. the brain where cysts develop. In this study, T. gondii tachyzoites sampled from the three populations were allowed to infect primary human blood DC, murine intestinal DC or in vitro-derived DC, and compared for different phenotypic traits. All three archetypical lineages of T. gondii induced a hypermigratory phenotype in DC shortly after infection in vitro. Type II (and III) strains induced higher migratory frequency and intensity in DC compared to type I. Additionally, adoptive transfer of infected DC favored dissemination of type II and type III parasites compared to type I parasites in syngeneic mice. Type II parasites exhibited stronger intracellular association to both CD11c(+) DC and other leukocytes in vivo compared to type I. Altogether, these findings suggest that infected DC contribute to parasite propagation in a strain type specific manner, and that the parasite genotype (type II) most frequently associated with toxoplasmosis in humans efficiently exploits DC migration for parasite dissemination.

PMID: 19204091 [PubMed - as supplied by publisher]

Toxoplasma gondii glycosylphosphatidylinositols up-regulate MHC

Innate Immun. 2009 Feb;15(1):25-32.

Toxoplasma gondii glycosylphosphatidylinositols up-regulate major histocompatibility complex (MHC) molecule expression on primary murine macrophages

Debierre-Grockiego F, Molitor N, Schwarz RT, Lüder CG.

Institut für Virologie, AG Parasitologie, Philipps Universität Marburg, Marburg, Germany.

Toxoplasma gondii is an obligatory intracellular parasite able to block the IFN-gamma-induced up-regulation of major histocompatibility complex (MHC) class I and class II molecules. This facilitates parasite-mediated evasion of T-cell responses. Glycosylphosphatidylinositols (GPIs) are involved in the pathogenicity of protozoan parasites and we investigated if GPIs are responsible for inhibition of MHC expression on macrophages. In contrast to the blockade observed in cells infected with viable tachyzoites, T. gondii GPIs up-regulated MHC class I and class II molecules on the surface of both unstimulated and IFN-gamma-stimulated primary murine macrophages. This effect was correlated to the ability of GPIs to increase the antigen presentation to CD8(+) lymphocytes. T. gondii GPIs did not activate STAT1, one of the factors involved in the transcription of MHC class I and class II genes. However, the GPI-induced MHC class I up-regulation was abrogated by SN50, a specific NF-KB inhibitor. Up-regulation of surface MHC molecules by GPIs may lead to the elimination of non-infected cells of the host immune system, contributing to the immune escape strategy of T. gondii.

PMID: 19201822 [PubMed - in process]

Monday, February 09, 2009

Rab11A-controlled assembly of the inner membrane complex is required for completion of apicomplexan cytokinesis

PLoS Pathog. 2009 Jan;5(1):e1000270. Epub 2009 Jan 23

Rab11A-controlled assembly of the inner membrane complex is required for completion of apicomplexan cytokinesis

Agop-Nersesian C, Naissant B, Ben Rached F, Rauch M, Kretzschmar A, Thiberge S, Menard R, Ferguson DJ, Meissner M, Langsley G

Department of Parasitology, Hygieneinstitut, University Hospital Heidelberg, Heidelberg, Germany.

The final step during cell division is the separation of daughter cells, a process that requires the coordinated delivery and assembly of new membrane to the cleavage furrow. While most eukaryotic cells replicate by binary fission, replication of apicomplexan parasites involves the assembly of daughters (merozoites/tachyzoites) within the mother cell, using the so-called Inner Membrane Complex (IMC) as a scaffold. After de novo synthesis of the IMC and biogenesis or segregation of new organelles, daughters bud out of the mother cell to invade new host cells. Here, we demonstrate that the final step in parasite cell division involves delivery of new plasma membrane to the daughter cells, in a process requiring functional Rab11A. Importantly, Rab11A can be found in association with Myosin-Tail-Interacting-Protein (MTIP), also known as Myosin Light Chain 1 (MLC1), a member of a 4-protein motor complex called the glideosome that is known to be crucial for parasite invasion of host cells. Ablation of Rab11A function results in daughter parasites having an incompletely formed IMC that leads to a block at a late stage of cell division. A similar defect is observed upon inducible expression of a myosin A tail-only mutant. We propose a model where Rab11A-mediated vesicular traffic driven by an MTIP-Myosin motor is necessary for IMC maturation and to deliver new plasma membrane to daughter cells in order to complete cell division.

PMID: 19165333 [PubMed - in process]

Sunday, February 08, 2009

Novel structural and regulatory features of rhoptry secretory kinases in Toxoplasma

EMBO J. 2009 Feb 5. [Epub ahead of print]

Novel structural and regulatory features of rhoptry secretory kinases in Toxoplasma gondii

Qiu W, Wernimont A, Tang K, Taylor S, Lunin V, Schapira M, Fentress S, Hui R, Sibley LD.

Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.

Serine/threonine kinases secreted from rhoptry organelles constitute important virulence factors of Toxoplasma gondii. Rhoptry kinases are highly divergent and their structures and regulatory mechanism are hitherto unknown. Here, we report the X-ray crystal structures of two related pseudokinases named ROP2 and ROP8, which differ primarily in their substrate-binding site. ROP kinases contain a typical bilobate kinase fold and a novel N-terminal extension that both stabilizes the N-lobe and provides a unique means of regulation. Although ROP2 and ROP8 were catalytically inactive, they provided a template for homology modelling of the active kinase ROP18, a major virulence determinant of T. gondii. Autophosphorylation of key residues in the N-terminal extension resulted in ROP18 activation, which in turn phosphorylated ROP2 and ROP8. Mutagenesis and mass spectrometry experiments revealed that ROP18 was maximally activated when this phosphorylated N-terminus relieved autoinhibition resulting from extension of aliphatic side chains into the ATP-binding pocket. This novel means of regulation governs ROP kinases implicated in parasite virulence.

PMID: 19197235 [PubMed - as supplied by publisher]

Disruption of the Toxoplasma gondii parasitophorous vacuole by IFNgamma-inducible immunity-related GTPases (IRG proteins) triggers necrotic cell death

PLoS Pathog. 2009 Feb;5(2):e1000288. Epub 2009 Feb 6

Disruption of the Toxoplasma gondii parasitophorous vacuole by IFNgamma-inducible immunity-related GTPases (IRG proteins) triggers necrotic cell death

Zhao YO, Khaminets A, Hunn JP, Howard JC.

Institute for Genetics, University of Cologne, Cologne, Germany.

Toxoplasma gondii is a natural intracellular protozoal pathogen of mice and other small mammals. After infection, the parasite replicates freely in many cell types (tachyzoite stage) before undergoing a phase transition and encysting in brain and muscle (bradyzoite stage). In the mouse, early immune resistance to the tachyzoite stage is mediated by the family of interferon-inducible immunity-related GTPases (IRG proteins), but little is known of the nature of this resistance. We reported earlier that IRG proteins accumulate on intracellular vacuoles containing the pathogen, and that the vacuolar membrane subsequently ruptures. In this report, live-cell imaging microscopy has been used to follow this process and its consequences in real time. We show that the rupture of the vacuole is inevitably followed by death of the intracellular parasite, shown by its permeability to cytosolic protein markers. Death of the parasite is followed by the death of the infected cell. The death of the cell has features of pyronecrosis, including membrane permeabilisation and release of the inflammatory protein, HMGB1, but caspase-1 cleavage is not detected. This sequence of events occurs on a large scale only following infection of IFNgamma-induced cells with an avirulent strain of T. gondii, and is reduced by expression of a dominant negative mutant IRG protein. Cells infected by virulent strains rarely undergo necrosis. We did not find autophagy to play any role in the key steps leading to the death of the parasite. We conclude that IRG proteins resist infection by avirulent T. gondii by a novel mechanism involving disruption of the vacuolar membrane, which in turn ultimately leads to the necrotic death of the infected cell.

Publication Types:
Research Support, Non-U.S. Gov't

PMID: 19197351 [PubMed - in process]

Vaccination concepts against Toxoplasma gondii

Expert Rev Vaccines. 2009 Feb;8(2):215-25

Vaccination concepts against Toxoplasma gondii

Garcia JL.

Department of Preventive Veterinary Medicine, Londrina State University, Campus Universitário, Rodovia Celso Garcia Cid, Pr 445 Km 380, Cx. Postal 6001, Londrina, PR 86051-990, Brazil.

Toxoplasma gondii is a parasite that infects animals and humans worldwide. Despite the current knowledge of immunology, pathology and genetics related to the parasite, a safe vaccine for prevention of the infection in both humans and animals does not exist. Here, we review some aspects concerning vaccination against T. gondii.

PMID: 19196201 [PubMed - in process]

Wednesday, February 04, 2009

Exosomes are an effective vaccine against congenital toxoplasmosis in mice

Vaccine. 2009 Jan 29. [Epub ahead of print]

Exosomes are an effective vaccine against congenital toxoplasmosis in mice

Beauvillain C, Juste MO, Dion S, Pierre J, Dimier-Poisson I.

Université François-Rabelais de Tours, INRA, 31, Avenue Monge, 37200 Tours, France; UMR 0483 Université-INRA d'Immunologie Parasitaire et Vaccinologie, Bio-Thérapies Anti-Infectieuses, IFR des Agents Transmissibles et Infectiologie, UFR des Sciences Pharmaceutiques, 31, Avenue Monge, 37200 Tours, France.

Toxoplasmosis is a serious disease in humans and may cause abortion or congenital infection if a woman is exposed to the disease for the first time during pregnancy. Infection before pregnancy normally results in immunity protecting the foetus, suggesting that it may be possible to block vertical transmission of the parasite by appropriate vaccination before pregnancy. We found that the vaccination of CBA/J mice, before pregnancy, with exosomes secreted by SRDCs pulsed in vitro with Toxoplasma gondii-derived antigens (TAg) induced a protective response in the pups. Indeed, vaccination resulted in the presence of significantly fewer cysts in pup brains. This protection was associated with strong humoral responses in the serum in vivo. We also observed cellular responses in vivo, with cell proliferation associated with the production of cytokines by the splenocytes. Thus, exosomes are nucleic acid-free vesicles able to induce immune responses correlated with protection against T. gondii infection in a congenital model. They are therefore a potentially useful tool for vaccination against infectious disease.

PMID: 19186199 [PubMed - as supplied by publisher]