Thursday, April 29, 2010

Use of two novel approaches to discriminate between closely related host microRNAs that are manipulated by Toxoplasma gondii during infection

RNA. 2010 Apr 27. [Epub ahead of print]

Use of two novel approaches to discriminate between closely related host microRNAs that are manipulated by Toxoplasma gondii during infection

Zeiner GM, Boothroyd JC.

Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305, USA.

Abstract
MicroRNAs (miRNAs) are a class of small, endogenously encoded regulatory RNAs that function to post-transcriptionally regulate gene expression in a wide variety of eukaryotes. Within organisms, some mature miRNAs, such as paralogous miRNAs, have nearly identical nucleotide sequences, which makes them virtually indistinguishable from one another by conventional hybridization-based approaches. Here we describe two inexpensive, sensitive methods for rapidly discriminating between paralogous miRNAs or other closely related miRNAs and for quantifying their abundance. The first approach is a sequential ribonuclease-protection and primer-extension assay; the second approach is a primer-extension assay that employs short oligonucleotide probes to exacerbate the instability of mismatched probe:miRNA hybrids. Both approaches are rapid and can be easily performed in their entirety using common laboratory equipment. As a proof of concept, we have used these methods to determine the exact identities of the human miR-17 family members that are increased by infection with the intracellular parasite Toxoplasma gondii. These methods can be used to rapidly and inexpensively discriminate between any closely related miRNAs in any organism.

PMID: 20423977 [PubMed - as supplied by publisher]

Acute pericarditis and myocarditis by Toxoplasma gondii in an immunocompetent young man: a case report

Infez Med. 2010 Mar;18(1):48-52.

Acute pericarditis and myocarditis by Toxoplasma gondii in an immunocompetent young man: a case report

Pergola G, Cascone A, Russo M.

Department of Infectious Diseases, Second University of Naples, Italy.

Abstract
Infection due to protozoan parasite Toxoplasma gondii is highly prevalent among humans throughout the world. Acquired primary infection is seldom severe in immunocompetent people while it can be life-threatening in immunodeficient ones. We report a case of acquired toxoplasmosis in an immunocompetent healty 32-year-old man, presenting as acute pericarditis and myocarditis. The patient complained of intense chest pain, asthenia, arthralgia, low-grade fever, neck lymphadenopathy. Increased seric cardiac enzymes, electrocardiografic anomalies of repolarization and the presence of pericardic effusion on echocardiogram needed anti-inflammatory and anti-arrhythmic drugs and a close monitoring. The aetiological diagnosis, supported by serological tests positive for toxoplasmosis, recommended an antibiotic therapy as additional treatment (spiramycin 9MU/day for one month). Full symptoms remission and normalization of serological values suggested, however, that no more effective anti-protozoan treatment was needed. Thus, the infection by Toxoplasma gondii should be taken into account in the aetiology of either acute pericarditis or myocarditis, because a specific treatment is available, which can improve on the prognosis of the disease.

PMID: 20424527 [PubMed - in process]

Wednesday, April 28, 2010

Rhomboid 4 (ROM4) Affects the Processing of Surface Adhesins and Facilitates Host Cell Invasion by Toxoplasma gondii

PLoS Pathog. 2010 Apr 22;6(4):e1000858.

Rhomboid 4 (ROM4) Affects the Processing of Surface Adhesins and Facilitates Host Cell Invasion by Toxoplasma gondii

Buguliskis JS, Brossier F, Shuman J, Sibley LD.

Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, United States of America.

Abstract
Host cell attachment by Toxoplasma gondii is dependent on polarized secretion of apical adhesins released from the micronemes. Subsequent translocation of these adhesive complexes by an actin-myosin motor powers motility and host cell invasion. Invasion and motility are also accompanied by shedding of surface adhesins by intramembrane proteolysis. Several previous studies have implicated rhomboid proteases in this step; however, their precise roles in vivo have not been elucidated. Using a conditional knockout strategy, we demonstrate that TgROM4 participates in processing of surface adhesins including MIC2, AMA1, and MIC3. Suppression of TgROM4 led to decreased release of the adhesin MIC2 into the supernatant and concomitantly increased the surface expression of this and a subset of other adhesins. Suppression of TgROM4 resulted in disruption of normal gliding, with the majority of parasites twirling on their posterior ends. Parasites lacking TgROM4 bound better to host cells, but lost the ability to apically orient and consequently most failed to generate a moving junction; hence, invasion was severely impaired. Our findings indicate that TgROM4 is involved in shedding of micronemal proteins from the cell surface. Down regulation of TgROM4 disrupts the normal apical-posterior gradient of adhesins that is important for efficient cell motility and invasion of host cells by T. gondii.

PMID: 20421941 [PubMed - in process]

Molecular and biological characteristics of Toxoplasma gondii isolates from wildlife in France

Vet Parasitol. 2010 Apr 1. [Epub ahead of print]

Molecular and biological characteristics of Toxoplasma gondii isolates from wildlife in France

Aubert D, Ajzenberg D, Richomme C, Gilot-Fromont E, Terrier ME, de Gevigney C, Game Y, Maillard D, Gibert P, Dardé ML, Villena I.

Laboratoire de Parasitologie-Mycologie, EA 3800, IFR53, Centre National de Référence (CNR) Toxoplasmose/Toxoplasma Biological Resource Center (BRC), Centre Hospitalier-Universitaire de Reims, 45 Rue Cognacq Jay, F-51092 Reims, France.

Abstract
Toxoplasma gondii isolates have been classified into 3 genetic types. Little is known about genotypes of T. gondii isolates in wild animals in Europe. In this report, genotypes of T. gondii isolates from wildlife in France are described. Sera from wildlife were tested for antibodies to T. gondii with the modified agglutination test, and the hearts from animals with titers superior or equal to 1:6 were bioassayed individually in mice. T.gondii was isolated from 9 of 14 seropositive red foxes (Vulpes vulpes), 12 of 33 roe deer (Capreolus capreolus), 1 of 4 deer (Cervus elaphus), 1 of 7 mouflons (Ovis gmelini musimon) and 1 of 2 common mallards (Anas platyrhynchos). No isolate was obtained by bioassay in mice of 1 fallow deer (Dama dama) and of 3 European brown hares (Lepus europaeus). Genotyping of the 24 isolates using PCR-RFLP and microsatellite markers indicated that all were type II and none of these Toxoplasma isolates was virulent for mice. Copyright © 2010 Elsevier B.V. All rights reserved.

PMID: 20417034 [PubMed - as supplied by publisher]

Post-translational membrane sorting of the Toxoplasma gondii GRA6 protein into the parasite-containing vacuole is driven by its N-terminal domain

Int J Parasitol. 2010 Apr 23. [Epub ahead of print]

Post-translational membrane sorting of the Toxoplasma gondii GRA6 protein into the parasite-containing vacuole is driven by its N-terminal domain

Gendrin C, Bittame A, Mercier C, Cesbron-Delauw MF.

Laboratoire Adaptation et Pathogénie des Micro-organismes, CNRS UMR 5163, Université Joseph Fourier GRENOBLE 1, Institut Jean Roget, BP 170, 38042 Grenoble cedex 9, France.; Present address: Laboratoire des Protéines Membranaires, Institut de Biologie Structurale, 41 rue Jules Horowitz, 38027 Grenoble, France.

Abstract
How eukaryotic pathogens export and sort membrane-bound proteins destined for host-cell compartments is still poorly understood. The dense granules of the intracellular protozoan Toxoplasma gondii constitute an unusual secretory pathway that allows soluble export of the GRA proteins which become membrane-associated within the parasite replicative vacuole. This process relies on both the segregation of the proteins routed to the dense granules from those destined to the parasite plasma membrane and on the sorting of the secreted GRA proteins to their proper final membranous system. Here, we provide evidence that the soluble trafficking of GRA6 to the dense granules relies on the N-terminal domain of the protein, which is sufficient to prevent GRA6 targeting to the parasite plasma membrane. We also show that the GRA6 N-terminal domain, possibly by interacting with negatively charged lipids, is fundamental for proper GRA6 association with the vacuolar membranous network of nanotubes. These results support our emerging model: sorting of transmembrane GRA proteins to the host cell vacuole is mainly driven by the dual role of their N-terminal hydrophilic domain and is compartmentally regulated. Copyright © 2010. Published by Elsevier Ltd.

PMID: 20420842 [PubMed - as supplied by publisher]

Monday, April 26, 2010

Recrudescence of Toxoplasma gondii infection in chronically infected rats (Rattus norvegicus)

Exp Parasitol. 2010 Apr 19. [Epub ahead of print]

Recrudescence of Toxoplasma gondii infection in chronically infected rats (Rattus norvegicus)

da Silva RC, da Silva AV, Langoni H.

School of Veterinary Medicine and Animal Science, São Paulo State University, Botucatu, São Paulo State, 18618-000, Brazil.

Abstract
The kinetics of T. gondii infection reactivation in the brain and muscles was analyzed in this study to determine the preferred tissue by the parasite during immunosuppression. Two groups of Wistar rats (G1 and G2) were inoculated with 10(4) bradyzoites of BTU10 strain (genotype I), p.o., and other two groups (G3 and G4) were inoculated with 0.9% saline solution. G2 and G4 were immunosuppressed with dexamethasone (DXM) and hydrocortisone sodium succinate (HSS). The presence of antibodies was researched in all groups through modified agglutination test (MAT) on days 0 and 21 p.i., and brain and muscle tissues of the rats were bioassayed in mice. G2 rats died at approximately 19.2 days after drug treatment, while G1 rats survived. The reactivation was initially observed in G1 brain and G2 muscles. Thus, the initial reactivation in muscles after immunosuppression allows doctors to save precious time to control the evolution of reactivated infection, preventing brain damage to the host. Copyright © 2010. Published by Elsevier Inc.

PMID: 20412799 [PubMed - as supplied by publisher]

Toxoplasma gondii: flat-mounting of retina as a new tool for the observation of ocular infection in mice

Exp Parasitol. 2010 Apr 19. [Epub ahead of print]

Toxoplasma gondii: flat-mounting of retina as a new tool for the observation of ocular infection in mice

Escoffier P, Jeanny JC, Marinach-Patrice C, Jonet L, Raoul W, Behar-Cohen F, Paris L, Danis M, Dubremetz JF, Mazier D.

Université Pierre et Marie Curie-Paris6, UMR S 945, Paris F-75013, France; INSERM, UMR S 945, Paris F-75013, France.

Abstract
Ocular toxoplasmosis is the principal cause of posterior uveitis and a leading cause of blindness. Animal models are required to improve our understanding of the pathogenesis of this disease. The method currently used for the detection of retinal cysts in animals involves the observation, under a microscope, of all the sections from infected eyes. However, this method is time-consuming and lacks sensitivity. We have developed a rapid, sensitive method for observing retinal cysts in mice infected with Toxoplasma gondii. This method involves combining the flat-mounting of retina - a compromise between macroscopic observation and global analysis of this tissue - and the use of an avirulent recombinant strain of T. gondii expressing the Escherichia coli beta-galactosidase gene, visually detectable at the submacroscopic level. Single cyst unilateral infection was found in 6 out of 17 mice killed within 28 days of infection, whereas a bilateral infection was found in only one mouse. There was no correlation between brain cysts number and ocular infection. Copyright © 2010. Published by Elsevier Inc.

PMID: 20412796 [PubMed - as supplied by publisher]

Wednesday, April 21, 2010

Characterization of a novel organelle in Toxoplasma gondii with similar composition and function to the plant vacuole

Mol Microbiol. 2010 Apr 14. [Epub ahead of print]

Characterization of a novel organelle in Toxoplasma gondii with similar composition and function to the plant vacuole

Miranda K, Pace DA, Cintron R, Rodrigues JC, Fang J, Smith A, Rohloff P, Coelho E, de Haas F, de Souza W, Coppens I, Sibley LD, Moreno SN.

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

Abstract
Abstract Toxoplasma gondii belongs to the phylum Apicomplexa and is an important cause of congenital disease and infection in immunocompromised patients. Like most apicomplexans, Toxoplasma gondii possesses several plant-like features, such as the chloroplast-like organelle, the apicoplast. We describe and characterize a novel organelle in T. gondii tachyzoites, which is visible by light microscopy, and possesses a broad similarity to the plant vacuole. High resolution electron tomography shows the interaction of this vacuole with other organelles. The presence of a plant-like vacuolar proton pyrophosphatase (TgVP1), a vacuolar proton ATPase, a cathepsin L-like protease (TgCPL), an aquaporin (TgAQP1), as well as Ca(2+)/H(+) and Na(+)/H(+) exchange activities, supports similarity to the plant vacuole. Biochemical characterization of TgVP1 in enriched fractions shows a functional similarity to the respective plant enzyme. The organelle is a Ca(2+) store and appears to have protective effects against salt stress potentially linked to its sodium transport activity. In intracellular parasites, the organelle fragments, with some markers co-localizing with the late endosomal marker, Rab7, suggesting its involvement with the endocytic pathway. Studies on the characterization of this novel organelle will be relevant to the identification of novel targets for chemotherapy against T. gondii and other apicomplexan parasites as well.

PMID: 20398214 [PubMed - as supplied by publisher]

The Hsp90 co-chaperone p23 of Toxoplasma gondii: Identification, functional analysis and dynamic interactome determination

Mol Biochem Parasitol. 2010 Apr 16. [Epub ahead of print]

The Hsp90 co-chaperone p23 of Toxoplasma gondii: Identification, functional analysis and dynamic interactome determination

Echeverria PC, Figueras MJ, Vogler M, Kriehuber T, de Miguel N, Deng B, Dalmasso MC, Matthews DE, Matrajt M, Haslbeck M, Buchner J, Angel SO.

Laboratorio de Parasitología Molecular, UB2, IIB-INTECH, CONICET-UNSAM, Camino de Circunvalación Laguna Km. 6, C.C 164, (B7130IIWA) Chascomús, Prov. Buenos Aires, Argentina.

Abstract
Toxoplasma gondii is among the most successful parasites, with nearly half of the human population chronically infected. Recently a link between the T. gondii Hsp90 chaperone machinery and parasite development was observed. Here, the T. gondii Hsp90 co-chaperones p23 and Hip were identified mining the Toxoplasma- database (www.toxodb.org). Their identity was confirmed by domain structure and blast analysis. Additionally, analysis of the secondary structure and studies on the chaperone function of the purified protein verified the p23 identity. Studies of co-immunoprecipitation (co-IP) identified two different types of complexes, one comprising at least Hip-Hsp70-Hsp90 and another containing at least p23-Hsp90. Indirect immunofluorescence assays showed that Hip is localized in the cytoplasm in tachyzoites and as well in bradyzoites. For p23 in contrast, a solely cytoplasmic localization was only observed in the tachyzoite stage whereas nuclear and cytosolic distribution and colocalization with Hsp90 was observed in bradyzoites. These results indicate that the T. gondii Hsp90-heterocomplex cycle is similar to the one proposed for higher eukaryotes, further highlighting the implication of the Hsp90/p23 in parasite development. Furthermore, co-IP experiments of tachyzoite/bradyzoite lysates with anti-p23 antiserum and identification of the complexed proteins together with the use of the curated interaction data available from different source (orthologs and Plasmodium databases) allowed us to construct an interaction network (interactome) covering the dynamics of the Hsp90 chaperone machinery. Copyright © 2010. Published by Elsevier B.V.

PMID: 20403389 [PubMed - as supplied by publisher]

Saturday, April 17, 2010

The effect of prolactin (PRL) on the growth of Toxoplasma gondii tachyzoites in vitro

Parasitol Res. 2010 Apr 16. [Epub ahead of print]

The effect of prolactin (PRL) on the growth of Toxoplasma gondii tachyzoites in vitro

Dzitko K, Gatkowska J, Płociński P, Dziadek B, Długońska H.

Department of Immunoparasitology, Institute of Microbiology and Immunology, University of Łodź, ul. Banacha 12/16, 90-237, Łodź, Poland, dzika@biol.uni.lodz.pl.

Abstract
During the development and effector phases of the anti-Toxoplasma response, the immunological system of a host is involved in several complex interactions with the endocrine system, and prolactin (PRL) is one of the most important hormones involved in immunoregulation. In this work, the influence of the recombinant human prolactin (rhPRL) on the viability, penetration, and intensity of intracellular proliferation of Toxoplasma gondii BK strain in vitro was evaluated. Using one murine (L929) and two human cell lines (Hs27 and HeLa), no toxic effect of the rhPRL on host cells was found (by determining cellular viability using MTT assay). A similar lack of rhPRL cytotoxic activity was found in the case of the extracellular tachyzoites of T. gondii BK. Replication of parasites in the presence of rhPRL was analyzed first by simultaneous addition of the hormone and the parasites into a microculture of the host cells (treatment during infection). No statistically significant changes in the intensity of parasite proliferation in all used host cells were found for a wide range of the hormone concentrations. However, pre-incubation of the tachyzoites with rhPRL resulted in a significant reduction (up to 36.15%) in the replication abilities of the parasite. Further experiments revealed that in fact, the inhibition of replication was caused by a limited capacity of the parasites to penetrate host's cells as demonstrated by the reduced number of infected cells.

PMID: 20397028 [PubMed - as supplied by publisher]

Thursday, April 15, 2010

The P-glycoprotein inhibitor GF120918 modulates Ca2+-dependent processes and lipid metabolism in Toxoplasma gondii

PLoS One. 2010 Apr 8;5(4):e10062.

The P-glycoprotein inhibitor GF120918 modulates Ca2+-dependent processes and lipid metabolism in Toxoplasma gondii

Bottova I, Sauder U, Olivieri V, Hehl AB, Sonda S.

Institute of Parasitology, University of Zurich, Zurich, Switzerland.

Abstract
Up-regulation of the membrane-bound efflux pump P-glycoprotein (P-gp) is associated with the phenomenon of multidrug-resistance in pathogenic organisms, including protozoan parasites. In addition, P-gp plays a role in normal physiological processes, however our understanding of these P-gp functions remains limited. In this study we investigated the effects of the P-gp inhibitor GF120918 in Toxoplasma gondii, a model apicomplexan parasite and an important human pathogen. We found that GF120918 treatment severely inhibited parasite invasion and replication. Further analyses of the molecular mechanisms involved revealed that the P-gp inhibitor modulated parasite motility, microneme secretion and egress from the host cell, all cellular processes known to depend on Ca2+ signaling in the parasite. In support of a potential role of P-gp in Ca2+-mediated processes, immunoelectron and fluorescence microscopy showed that T. gondii P-gp was localized in acidocalcisomes, the major Ca2+ storage in the parasite, at the plasma membrane, and in the intravacuolar tubular network. In addition, metabolic labeling of extracellular parasites revealed that inhibition or down-regulation of T. gondii P-gp resulted in aberrant lipid synthesis. These results suggest a crucial role of T. gondii P-gp in essential processes of the parasite biology and further validate the potential of P-gp activity as a target for drug development.

PMID: 20386707 [PubMed - in process]

Mic1-3 Knockout Toxoplasma gondii is a good candidate for a vaccine against T. gondii-induced abortion in sheep

Vet Res. 2010 Apr 13;41(4):49. [Epub ahead of print]

Mic1-3 Knockout Toxoplasma gondii is a good candidate for a vaccine against T. gondii-induced abortion in sheep

Mévélec MN, Ducournau C, Bassuny Ismael A, Olivier M, Sèche E, Lebrun M, Bout D, Dimier-Poisson I.

Abstract
This study assessed the effectiveness of a mutant strain of Toxoplasma gondii (RH strain) lacking the mic1 and mic3 genes (Mic1-3KO) against Toxoplasma abortion in sheep. Ewes were inoculated subcutaneously with 105 Mic1-3KO tachyzoïtes in three independent experiments. Following vaccination, Mic1-3KO induced a mild febrile response and serum IgG antibodies, which persisted throughout the experiments. Tissue cysts formed in the sheep, but were not, under our experimental conditions, infectious when given orally. Ewes were mated two months after vaccination and were orally challenged with the PRU strain of T. gondii at mid-gestation (400 oocysts in experiments 1 and 2, 100 oocysts in experiment 3). Challenge of vaccinated pregnant ewes resulted in a slight febrile response, whereas unvaccinated ewes developed a more severe, characteristic febrile response of longer duration. After challenge, all unvaccinated ewes aborted whereas 62%, 91% and 64% (experiments 1, 2 and 3 respectively) of the lambs from vaccinated ewes were viable, with no clinical signs of infection. Mic1-3KO was as effective as S48, the strain used as a live vaccine for sheep (Toxovax(R)). A dose of 105 Mic1-3KO tachyzoites was sufficient to induce protection (versus a dose of 2 x 106). Both subcutaneous and intraperitoneal injections were effective. Moreover, preliminary results showed the potential of Mic1-3KO to reduce the development of tissue cysts in lambs born to vaccinated ewes. This study demonstrates that Mic1-3KO is a potent vaccine candidate.

PMID: 20385082 [PubMed - as supplied by publisher]

Using quantitative reverse transcriptase PCR and cell culture plaque assays to determine resistance of Toxoplasma gondii oocysts to chemical sanitizer

J Microbiol Methods. 2010 Apr 9. [Epub ahead of print]

Using quantitative reverse transcriptase PCR and cell culture plaque assays to determine resistance of Toxoplasma gondii oocysts to chemical sanitizers

Villegas EN, Augustine SA, Villegas LF, Ware MW, See MJ, Lindquist HD, Schaefer FW 3rd, Dubey JP.

National Exposure Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH 45268; Department of Biological Sciences, McMicken School of Arts and Sciences, University of Cincinnati, Cincinnati, OH 45220.

Abstract
Toxoplasma gondii oocysts are highly resistant to many chemical sanitizers. Methods used to determine oocyst infectivity have relied primarily on mouse, chicken, and feline bioassays. Although considered gold standards, they only provide a qualitative assessment of oocyst viability. In this study, two alternative approaches were developed to quantitate viable T. gondii oocysts following treatment with several common sanitizers. The first is a quantitative reverse-transcriptase real-time PCR (RT-qPCR) assay targeting the ACT1 and SporoSAG genes to enumerate viable T. gondii oocysts. RT-qPCR C(T) values between Wescodyne((R)), acidified ethanol, or heat treated oocysts were not significantly different as compared with untreated controls. By contrast, treatment with formalin or Clorox((R)) resulted in a 2-log(10) reduction in C(T) values. An in vitro T. gondii oocyst plaque assay (TOP-assay) was also developed to measure oocyst viability. This assay used a combination of bead milling and bile digestion, followed by culturing the excysted sporozoites in a confluent fibroblast cell monolayer. Results showed that no significant reduction in sporozoite viability was detected in acidified ethanol or Wescodyne((R)) treated oocysts while at least a 2-log(10) reduction in plaques formed was observed with Clorox((R)) treated oocysts. Moreover, formalin or heat treatment of oocysts resulted in at least a 5-log(10) reduction in plaques formed. This study demonstrates that an mRNA-based PCR viability assay targeting the ACT1 or SporoSAG genes is a relatively rapid technique compared to in vitro and in vivo assays. In addition, the TOP-assay proved very effective and sensitive at quantifying oocyst viability than animal bioassays. Published by Elsevier B.V.

PMID: 20385175 [PubMed - as supplied by publisher]

An assay for selection of sera with circulating toxoplasma gondii antigens

J Immunoassay Immunochem. 2010 Jan;31(1):79-91.

An assay for selection of sera with circulating toxoplasma gondii antigens

Emelia O, Zeehaida M, Sulaiman O, Rohela M, Saadatnia G, Yeng C, Rahmah N.

Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Minden, Penang, Malaysia.

Abstract
We have developed an ELISA that employs monoclonal anti-Toxoplasma SAG1 (p30) as the capture antibody to detect T. gondii circulating antigens in patients' serum samples. Using serum spiked with Toxoplasma soluble and with SAG1 recombinant proteins, the detection limits were 31.25 ng/mL and 62.50 ng/mL, respectively. We obtained positive results in 28% (21/75) and 11% (23/206) of probable active and chronic toxoplasmosis serum samples, respectively. Western blot analysis on pooled antigen-positive serum samples showed antigenic bands of molecular weights 25 and 75 kDa from sera of probable active infection and five antigenic bands ranging in size from 26 to 33 kDa from chronic infection sera. This assay would be useful as an initial serum selection step in developing a Toxoplasma antigen detection test and for characterization studies.

PMID: 20391020 [PubMed - in process]

Wednesday, April 14, 2010

Is there a relationship between Toxoplasma gondii infection and idiopathic Parkinson's disease?

Scand J Infect Dis. 2010 Apr 9. [Epub ahead of print]

Is there a relationship between Toxoplasma gondii infection and idiopathic Parkinson's disease?

Celik T, Kamişli O, Babür C, Cevik MO, Oztuna D, Altinayar S.

School of Health, Adiyaman University, Adiyaman.

Abstract
Abstract Idiopathic Parkinson's disease defines a group of Parkinson's disease (PD) of which the aetiology is unknown but an underlying brain disease is suspected. We selected patients of this subgroup of PD and investigated the seropositivity rate for anti-Toxoplasma IgG antibody by Sabin-Feldman dye test (SFDT). By measuring seropositivity in PD patients, we searched for a probable relationship between Toxoplasma gondii infection and idiopathic PD incidence. Fifty patients diagnosed with idiopathic PD and 50 healthy volunteers were included in the study. Blood samples were taken from all 100 participants and anti-T. gondii antibody titres were investigated using SFDT. Anti-T. gondii antibodies were detected at a titre of >/=1/16 in 25 of the 50 patients (50%) and in 20 of the control group (40%). No higher antibody titre was found in the control group. In conclusion, despite the emerging literature on a possible relationship between T. gondii infection and neurological disease, and the high anti-T. gondii seropositivity found in our PD patients, we did not detect any statistically significant association between T. gondii and idiopathic PD.

PMID: 20380545 [PubMed - as supplied by publisher]

Monday, April 12, 2010

Immunological reactions in response to apicomplexan glycosylphosphatidylinositols

Glycobiology. 2010 Apr 8. [Epub ahead of print]

Immunological reactions in response to apicomplexan glycosylphosphatidylinositols

Debierre-Grockiego F, Schwarz RT.

UMR Université-INRA 0483, Immunologie Parasitaire Vaccinologie et Biothérapies anti-infectieuses, UFR Sciences Pharmaceutiques, 31 avenue Monge, 37200 Tours, France.

Abstract
Apicomplexan protozoa are a phylum of parasites that includes pathogens such as Plasmodium, the causative agent of the most severe form of malaria responsible for almost 1 million deaths per year and Toxoplasma gondii causing toxoplasmosis, a disease leading to cerebral meningitis in immuno-compromised individuals or to abortion in farm animals or in women that are infected for the first time during pregnancy. The initial immune reactions developed by the host are similar in response to an infection with Plasmodium and Toxoplasma in the sense that the same cells of the innate immune system are stimulated to produce inflammatory cytokines. The glycosylphosphatidylinositol (GPI) anchor is the major carbohydrate modification in parasite proteins and the GPIs are essential for parasite survival. Two immediate GPI precursors with the structures ethanolamine phosphate-6(Manalpha1-2)Manalpha1-2Manalpha1-6Manalpha1-4GlcN-PI and ethanolamine-phosphate-6Manalpha1-2Manalpha1-6Man-alpha1-4-GlcN-PI are synthesized by P. falciparum. Two main structures are synthesized by T. gondii: ethanolamine phosphate-6Manalpha1-2Manalpha1-6(GalNAcbeta1-4)Manalpha1-4GlcN-PI and ethanolamine phosphate-6Manalpha1-2Manalpha1-6(Glcalpha1-4GalNAcbeta1-4)Manalpha1-4GlcN-PI. This review describes the biosynthesis of the apicomplexan GPIs and their role in the activation of the host immune system.

PMID: 20378610 [PubMed - as supplied by publisher]

Transnuclear mice with predefined T cell receptor specificities against Toxoplasma gondii obtained via SCNT

Science. 2010 Apr 9;328(5975):243-8.

Transnuclear mice with predefined T cell receptor specificities against Toxoplasma gondii obtained via SCNT

Kirak O, Frickel EM, Grotenbreg GM, Suh H, Jaenisch R, Ploegh HL.

Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA. kirak@wi.mit.edu

Abstract
Mice that are transgenic for rearranged antigen-specific T cell receptors (TCRs) are essential tools to study T cell development and function. Such TCRs are usually isolated from the relevant T cells after long-term culture, often after repeated antigen stimulation, which unavoidably skews the T cell population used. Random genomic integration of the TCR alpha and beta chain and expression from nonendogenous promoters represent additional drawbacks of transgenics. Using epigenetic reprogramming via somatic cell nuclear transfer, we demonstrated that T cells with predefined specificities against Toxoplasma gondii can be used to generate mouse models that express the TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

PMID: 20378817 [PubMed - in process]

Saturday, April 10, 2010

Mitochondrial translation in absence of local tRNA aminoacylation and methionyl tRNA formylation in Apicomplexa

Mol Microbiol. 2010 Mar 31. [Epub ahead of print]

Mitochondrial translation in absence of local tRNA aminoacylation and methionyl tRNA formylation in Apicomplexa

Pino P, Aeby E, Foth BJ, Sheiner L, Soldati T, Schneider A, Soldati-Favre D.

Department of Microbiology and Molecular Medicine, CMU, University of Geneva, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland.

Abstract
Summary Apicomplexans possess three translationally active compartments: the cytosol, a single tubular mitochondrion, and a vestigial plastid organelle called apicoplast. Mitochondrion and apicoplast are of bacterial evolutionary origin and therefore depend on a bacterial-like translation machinery. The minimal mitochondrial genome contains only three ORFs, and in Toxoplasma gondii the absence of mitochondrial tRNA genes is compensated for by the import of cytosolic eukaryotic tRNAs. Although all compartments require a complete set of charged tRNAs, the apicomplexan nuclear genomes do not hold sufficient aminoacyl-tRNA synthetase (aaRSs) genes to be targeted individually to each compartment. This study reveals that aaRSs are either cytosolic, apicoplastic or shared between the two compartments by dual targeting but are absent from the mitochondrion. Consequently, tRNAs are very likely imported in their aminoacylated form. Furthermore, the unexpected absence of tRNA(Met) formyltransferase and peptide deformylase implies that the requirement for a specialized formylmethionyl-tRNA(Met) for translation initiation is bypassed in the mitochondrion of Apicomplexa.

PMID: 20374492 [PubMed - as supplied by publisher]

Thiazole, Oxadiazole, and Carboxamide Derivatives of Artemisinin are Highly Selective and Potent Inhibitors of Toxoplasma

J Med Chem. 2010 Apr 7. [Epub ahead of print]

Thiazole, Oxadiazole, and Carboxamide Derivatives of Artemisinin are Highly Selective and Potent Inhibitors of Toxoplasma gondii

Hencken CP, Jones-Brando L, Bordón C, Stohler R, Mott BT, Yolken R, Posner GH, Woodard LE.

Department of Chemistry, The Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218.

Abstract
We have prepared 23 new dehydroartemisinin (DART) trioxane derivatives (11 thiazoles, 2 oxadiazoles, and 10 carboxamides) and have screened them for in vitro activity in the Toxoplasma lytic cycle. Fifteen (65%) of the derivatives were noncytotoxic to host cells (TD(50) >/= 320 muM). Eight thiazole derivatives and two carboxamide derivatives displayed effective inhibition of Toxoplasma growth (IC(50) = 0.25-0.42 muM), comparable in potency to artemether (IC(50) = 0.31 muM) and >100 times more inhibitory than the currently employed front-line drug trimethoprim (IC(50) = 46 muM). The thiazoles as a group were more effective than the other derivatives at inhibiting growth of extracellular as well as intracellular parasites. Unexpectedly, two thiazole trioxanes (5 and 6) were parasiticidal; both inhibited parasite replication irreversibly after parasite exposure to 10 muM of drug for 24 h, whereas the standard trioxane drugs artemisinin and artemether were not parasiticidal. Some of the new derivatives of artemisinin described here represent effective anti-Toxoplasma trioxanes as well as molecular probes for elucidating the mechanism of action of the DART class of artemisinin derivatives.

PMID: 20373807 [PubMed - as supplied by publisher]

Wednesday, April 07, 2010

Design, synthesis, biological evaluation and computational investigation of novel inhibitors of dihydrofolate reductase of opportunistic pathogens

Bioorg Med Chem. 2010 Mar 18. [Epub ahead of print]

Design, synthesis, biological evaluation and computational investigation of novel inhibitors of dihydrofolate reductase of opportunistic pathogens

Bag S, Tawari NR, Degani MS, Queener SF.

Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, Matunga (E), Mumbai 400 019, India.

Abstract
The present work deals with design, synthesis and biological evaluation of novel, diverse compounds as potential inhibitors of dihydrofolate reductase (DHFR) from opportunistic microorganisms; Pneumocystis carinii (pc), Toxoplasma gondii (tg) and Mycobacteriumavium (ma). A set of 14 structurally diverse compounds were designed with varying key pharmacophoric features of DHFR inhibitors, bulky distal substitutions and different bridges joining the distal part and 2,4-diaminopyrimidine nucleus. The designed compounds were synthesized and evaluated in enzyme assay against pc, tg and ma DHFR. The rat liver (rl) DHFR was used as mammalian standard. As the next logical step of the project, flexible molecular docking studies were carried out to predict the binding modes of these compounds in pcDHFR active site and the obtained docked poses were post processed using MM-GBSA protocol for prediction of relative binding affinity. The predicted binding modes were able to rationalize the experimental results in most cases. Of particular interest, both the docking scores and MM-GBSA predicted DeltaG(bind) were able to distinguish between the active and low active compounds. Furthermore, good correlation coefficient of 0.797 was obtained between the IC(50) values and MM-GBSA predicted DeltaG(bind). Taken together, the current work provides not only a novel scaffold for further optimization of DHFR inhibitors but also an understanding of the specific interactions of inhibitors with DHFR and structural modifications that improve selectivity. Copyright © 2010 Elsevier Ltd. All rights reserved.

PMID: 20363634 [PubMed - as supplied by publisher]

SDS-coated atovaquone nanosuspensions

J Drug Target. 2010 Apr 1. [Epub ahead of print]

SDS-coated atovaquone nanosuspensions show improved therapeutic efficacy against experimental acquired and reactivated toxoplasmosis by improving passage of gastrointestinal and blood-brain barriers

Shubar HM, Lachenmaier S, Heimesaat MM, Lohman U, Mauludin R, Mueller RH, Fitzner R, Borner K, Liesenfeld O.

Institute of Microbiology and Hygiene, Campus Benjamin Franklin, Charité-University Medicine Berlin, Berlin, Germany.

Abstract
Toxoplasmic encephalitis (TE) is the most common clinical manifestation of reactivated infection with Toxoplasma gondii in immunocompromised patients that is lethal if untreated. The combination of pyrimethamine plus sulfadiazine or clindamycin is the standard therapy for the treatment of TE, but these combinations are associated with hematologic toxicity and/or life-threatening allergic reactions. Therefore, alternative treatment options are needed. Atovaquone is safe and highly effective against T. gondii in vitro, but the oral micronized solution shows poor bioavailability. We synthesized atovaquone nanosuspensions (ANSs) coated with poloxamer 188 (P188) and sodium dodecyl sulfate (SDS) to improve oral bioavailability and passage through the blood-brain barrier (BBB). Coating of ANSs with SDS resulted in enhanced oral bioavailability and enhanced brain uptake of atovaquone compared to Wellvone((R)) in murine models of acute and reactivated toxoplasmosis as measured by high performance liquid chromatography (HPLC). Parasite loads and inflammatory changes in brains of mice treated with SDS-coated ANS were significantly reduced compared to untreated controls and to Wellvone((R))-treated mice. In conclusion, nanosuspensions coated with SDS may ultimately lead to improvements in the treatment of TE and other cerebral diseases.

PMID: 20367080 [PubMed - as supplied by publisher]

Sunday, April 04, 2010

Processing and secretion of ROP13: A unique Toxoplasma effector protein

Int J Parasitol. 2010 Mar 29. [Epub ahead of print]

Processing and secretion of ROP13: A unique Toxoplasma effector protein

Turetzky JM, Chu DK, Hajagos BE, Bradley PJ.

Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles CA 90095-1489 USA.

Like most intracellular pathogens, Toxoplasma synthesizes and secretes an arsenal of proteins to successfully invade its host cell and hijack host functions for intracellular survival. The rhoptries are key secretory organelles that inject proteins into the host cell where they are positioned to co-opt host processes, although little is known regarding how these proteins exert their functions. We show here that the rhoptry protein ROP13 is synthesized as a pre-pro-protein that is processed in the parasite. Processing occurs at a conserved SphiXE cleavage site as mutagenesis of glutamic acid to alanine at the P1 position disrupts ROP13 maturation. We also demonstrate that processing of the prodomain is not necessary for rhoptry targeting and secretion. While gene disruption reveals that ROP13 is not essential for growth in fibroblasts in vitro or for virulence in vivo, we find that ROP13 is a soluble effector protein that can access the cytoplasm of host cells. Exogenously expressed ROP13 in human cells remains cytosolic but also appears toxic, suggesting that over-expression of this effector protein is disrupting some function within the host cell. Copyright © 2010. Published by Elsevier Ltd.

PMID: 20359481 [PubMed - as supplied by publisher]

Toxoplasma tachyzoites from cell culture are more appropriate in some situations

J Clin Pathol. 2010 Apr 1. [Epub ahead of print]

Toxoplasma tachyzoites from cell culture are more appropriate in some situations

Chatterton JM, McDonagh S, Ho-Yen DO.

Scottish Toxoplasma Reference Laboratory, Microbiology Department, Raigmore Hospital, Inverness, UK.

Background Laboratories traditionally culture toxoplasma tachyzoites in animals for testing and experimental use. This article considers why available cell culture methods are not used more often. Aim To compare HeLa cell culture and animal culture for production of toxoplasma tachyzoites. Methods In 2000 HeLa culture replaced animal culture for continuous production of toxoplasma tachyzoites in the Scottish Toxoplasma Reference Laboratory. The performance of animal culture (1994-1998) was compared with HeLa culture (2004-2008). A PubMed search was carried out for 1998 and 2008 to assess the culture methods used in laboratories. Results Animal culture was able to produce higher yields of tachyzoites (10(9) from a cotton rat peritoneal harvest compared to 10(7) from a 75 cm(2) cell culture flask) but significantly more HeLa cultures were successful (93% versus 84%; p=0.025). There was no difference in the quality of tachyzoites from animal and HeLa cultures as demonstrated by the high levels of success in the dye test. HeLa culture offered significant advantages in flexibility and control. A review of the literature showed no significant change in the method of culture used in laboratories between 1998 and 2008 (p=0.36). Conclusion The availability of cell culture methods and the increasingly stringent regulations on the use of animals have not resulted in a decline in the use of animal culture. Animals are necessary for certain experiments but many studies could use cell-culture-derived parasites.

PMID: 20360140 [PubMed - as supplied by publisher]