Sunday, March 29, 2009

Structural studies of PNP from Toxoplasma gondii

Int J Bioinform Res Appl. 2009;5(2):154-62.

Structural studies of PNP from Toxoplasma gondii

Vivan AL, Caceres RA, Basso LA, Santos DS; Walter F. de Azevedo Jr.

Genetics and Molecular Biology, Federal University of Rio Grande do Sul, Av. Bento Goncalves, 9500, Porto Alegre, RS, 91501-970 Brazil.

Toxoplasmosis is a chronic infection that affects approximately 30% of the human population and is caused by Toxoplasma gondii. Determination of the three dimensional structure of PNP from T. gondii could provide new insights into the purine binding site and sub-strate binding, and could be used for future rational design of new drugs against toxoplasmosis. This work describes the molecular model for three dimensional structure of PNP from T.gondii using, as a template, PNP from Plasmodium falciparum. Molecular dynamics showed that this model is stable during a trajectory of 3 ns.

PMID: 19324601 [PubMed - in process]

Waterborne toxoplasmosis - recent developments

Exp Parasitol. 2009 Mar 23. [Epub ahead of print]

Waterborne toxoplasmosis - recent developments

Jones JL, Dubey JP.

Division of Parasitic Diseases, National Center for Zoonotic, Vectorborne and Enteric Diseases, Coordinating Center for Infectious Diseases, Centers for Disease Control and Prevention, 4770 Buford Highway, MS: F22, Chamblee, Georgia 30341,USA.

Humans become infected with Toxoplasma gondii mainly by ingesting uncooked meat containing viable tissue cysts or by ingesting food or water contaminated with oocysts from the feces of infected cats. Circumstantial evidence suggests that oocyst-induced infections in humans are clinically more severe than tissue cyst-acquired infections. Until recently, waterborne transmission of T. gondii was considered uncommon, but a large human outbreak linked to contamination of a municipal water reservoir in Canada by wild felids and the widespread infection of marine mammals in the U.S.A. provided reasons to question this view. The present paper examines the possible importance of T. gondii transmission by water.

PMID: 19324041 [PubMed - as supplied by publisher]

Saturday, March 28, 2009

In vitro effect of TNF-alpha and IFN-gamma in retinal cell infection with Toxoplasma

Invest Ophthalmol Vis Sci. 2009 Apr;50(4):1754-60

In vitro effect of TNF-alpha and IFN-gamma in retinal cell infection with Toxoplasma gondii

Delair E, Creuzet C, Dupouy-Camet J, Roisin MP.

Université Paris Descartes, Faculté de Medicine, Hôpital Cochin, Service d'ophtalmologie, Paris, France. emmanuelle.delair@cch.aphp.fr

PURPOSE: Toxoplasma gondii is an intracellular protozoan parasite and the most common cause of infectious uveitis. This study was conducted to evaluate the in vitro effect of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma in rat retinal cells infected with T. gondii. METHODS: Rat retinal cells, retinal pigment epithelial (RPE) cells, and retinal Müller glial (RMG) cells were in vitro infected with T. gondii RH strain tachyzoites. Cultured cells were stimulated with various concentrations of TNF-alpha and IFN-gamma. The effect of TNF-alpha and IFN-gamma in T. gondii invasion and replication between retinal cells was determined through two different methods: measuring [(3)H]-uracil incorporation and counting infected cells by microscopic examination. RESULTS: Infection by T. gondii was lesser within RPE cells than within RMG cells. IFN-gamma significantly inhibits [(3)H]-uracil incorporation in RMG and RPE cells (respectively, 35%, 83%, and 87% inhibition at 0.1, 1, and 10 ng/mL for RMG cells and 0%, 30%, and 75% for RPE cells). TNF-alpha significantly inhibits [(3)H]-uracil incorporation in RPE cells (23% and 38% inhibition at 1 and 10 ng/mL), but not in RMG cells. These results were confirmed by confocal microscopic data. The percentage of infected cells decreased from 20% to 7% after IFN-gamma stimulation. CONCLUSIONS: Both cytokines IFN-gamma and TNF-alpha inhibited T. gondii replication in the RPE cells, whereas only IFN-gamma had an anti-Toxoplasma activity within the RMG cells. The differences in cytokine response may be the reason that RPE cells are less efficiently infected by T. gondii than are RMG cells.

PMID: 19321794 [PubMed - in process]

Thursday, March 26, 2009

MIC6 associates with aldolase in host cell invasion by Toxoplasma

Parasitol Res. 2009 Mar 24. [Epub ahead of print]

MIC6 associates with aldolase in host cell invasion by Toxoplasma gondii

Zheng B, He A, Gan M, Li Z, He H, Zhan X.

Department of Parasitology, Zhongshan School of Medicine, SunYat-sen University, Guangzhou, 510080, People's Republic of China.

The transmembrane microneme protein MIC6 and its partner MIC1, MIC4 comprise an adhesive complex that play important roles in host cell attachment by the obligate intracellular parasite Toxoplasma gondii. Successful penetration of host cells by T. gondii depends on coordinated interactions between MICs complex and the parasite's cytoskeleton. We have identified that the carboxy-terminal cytoplasmic domain (C domain) of MIC6 interacts with aldolase and the parasite cytoskeleton. Our finding uncovers new features regarding MIC6-aldolase interactions in host cell invasion by T. gondii.

PMID: 19308454 [PubMed - as supplied by publisher]

Detection of Toxoplasma gondii in cerebrospinal fluid from AIDS patients by nested PCR and rapid identification of type I allele at B1 gene by RFLP

Exp Parasitol. 2009 Mar 21. [Epub ahead of print]

Detection of Toxoplasma gondii in cerebrospinal fluid from AIDS patients by nested PCR and rapid identification of type I allele at B1 gene by RFLP analysis

Alfonso Y, Fraga J, Jiménez N, Fonseca C, Dorta-Contreras AJ, Cox R, Capó V, Bandera F, Pomier O, Ginorio D.

Parasitology Department. Institute of Tropical Medicine Pedro Kourí . PO Box 601. Marianao 13. Ciudad de La Habana. Cuba.

Highly active antiretroviral therapy (HAART) has decreased the incidence of opportunistic infections in the central nervous system (CNS) in AIDS patients. However, toxoplasmic encephalitis (TE) still represents the most common cerebral mass lesion in patients infected with human immunodeficiency virus (HIV). The aim of this study was to evaluate nested PCR-B1 using cerebrospinal fluid (CSF) to detect Toxoplasma gondii DNA for the diagnosis of TE. A total of 114 samples were evaluated, and 33/44 samples from patients with TE were positive by PCR (sensitivity 75%), demonstrating the diagnostic usefulness of PCR technique. PCR B1 products were analyzed by restriction fragment length polymorphism (RFLP) in 30 samples. Only type I allele at B1 was identified in these samples according banding patterns. This is the first report of evaluation of S1-AS1/S2-AS2 set of primers in more than 100 clinical samples as well as the first genotyping study of T. gondii in Cuba.

PMID: 19318095 [PubMed - as supplied by publisher]

Laboratory diagnosis of Toxoplasma gondii infection

Int J Med Sci. 2009;6(3):135-6. Epub 2009 Mar 19

Laboratory diagnosis of Toxoplasma gondii infection

Calderaro A, Peruzzi S, Piccolo G, Gorrini C, Montecchini S, Rossi S, Chezzi C, Dettori G.

Department of Pathology and Laboratory Medicine, Section of Microbiology, University of Parma, Parma, Italy.

PMID: 19319234 [PubMed - in process]

Wednesday, March 25, 2009

Intracellular parasitism with Toxoplasma stimulates mTOR-dependent host cell growth despite impaired signalling to S6K1 and 4E-BP1

Cell Microbiol. 2009 Feb 27. [Epub ahead of print]

Intracellular parasitism with Toxoplasma gondii stimulates mammalian-target-of-rapamycin-dependent host cell growth despite impaired signalling to S6K1 and 4E-BP1

Wang Y, Weiss LM, Orlofsky A.

Department of Pathology, Albery Einstein College of Medicine, Bronx, New York 10461, USA.

The Ser/Thr kinase mammalian-target-of-rapamycin (mTOR) is a central regulator of anabolism, growth and proliferation. We investigated the effects of Toxoplasma gondii on host mTOR signalling. Toxoplasma invasion of multiple cell types rapidly induced sustained mTOR activation that was restricted to infected cells, as determined by rapamycin-sensitive phosphorylation of ribosomal protein S6; however, phosphorylation of the growth-associated mTOR substrates 4E-BP1 and S6K1 was not detected. Infected cells still phosphorylated S6K1 and 4E-BP1 in response to insulin, although the S6K1 response was blunted. Parasite-induced S6 phosphorylation was independent of S6K1 and did not require activation of canonical mTOR-inducing pathways mediated by phosphatidylinositol 3-kinase-Akt and ERK. Host mTOR was localized in a vesicular pattern surrounding the parasitophorous vacuole, suggesting potential activation by phosphatidic acid in the vacuolar membrane. In spite of a failure to phosphorylate 4E-BP1 and S6K1, intracellular T. gondii triggered host cell cycle progression in an mTOR-dependent manner and progression of infected cells displayed increased sensitivity to rapamycin. Moreover, normal cell growth was maintained during parasite-induced cell cycle progression, as indicated by total cellular S6 levels. The Toxoplasma-infected cell provides a unique example of non-canonical mTOR activation supporting growth that is independent of signalling through either S6K1 or 4E-BP1.

PMID: 19302577 [PubMed - as supplied by publisher]

Toxoplasma-induced autophagy: A window into nutritional futile cycles in mammalian cells?

Autophagy. 2009 Apr 9;5(3). [Epub ahead of print]

Toxoplasma-induced autophagy: A window into nutritional futile cycles in mammalian cells?

Orlofsky A.

Department of Pathology, Albert Einstein College of Medicine, Bronx, NY, USA.

The regulation and function of autophagy in response to metabolic signals is not yet well understood. A recent study from our laboratory indicates that an intracellular parasite, Toxoplasma gondii, derives nutritive benefit from the upregulation of host cell autophagy. We discuss this and related findings suggesting that autophagy in infected cells functions as part of a metabolic futile cycle. The hypothesis is presented that endogenous autophagy-based futile cycles may operate in normal mammalian cells, providing a substrate for manipulation by pathogens.

PMID: 19305153 [PubMed - as supplied by publisher]

Tuesday, March 24, 2009

Postdoctoral positions available

POST-DOCTORAL POSITIONS

Post-doctoral fellow positions are available to study transcription, epigenetics, and post-transcriptional control in stress-induced differentiation of Toxoplasma gondii, a protozoan parasite related to malaria that causes opportunistic infections in AIDS patients (see www.sullivanlab.com). This cross-disciplinary research involving parasitology, biochemistry and molecular biology, will be performed in the laboratories of Dr. William Sullivan and Dr. Ronald Wek (Departments of Pharmacology and Biochemistry & Molecular Biology, Indiana University School of Medicine, Indianapolis, USA). Applicants must possess a Ph.D. and have record of productivity as evidenced by publications in international journals. Interested candidates should send their CV and the names/emails of three references electronically to wjsulliv@iupui.edu.

Monday, March 23, 2009

Anti-CD25 antibody-mediated depletion of effector T cell populations enhances susceptibility of mice to acute but not chronic Toxoplasma

J Immunol. 2009 Apr 1;182(7):3985-94

Anti-CD25 antibody-mediated depletion of effector T cell populations enhances susceptibility of mice to acute but not chronic Toxoplasma gondii infection

Couper KN, Lanthier PA, Perona-Wright G, Kummer LW, Chen W, Smiley ST, Mohrs M, Johnson LL.

Trudeau Institute, Saranac Lake, NY 12983, USA. kevin.couper@lshtm.ac.uk

Natural regulatory T cells (Tregs) constitutively express the IL-2R alpha-chain (CD25) on their surface. Consequently, administration of anti-CD25 Abs is a commonly used technique to deplete Treg populations in vivo. However, activated effector T cells may also transiently express CD25, and are thus also potential targets for anti-CD25 Abs. In this study using Toxoplasma gondii as a model proinflammatory infection, we have examined the capacity of anti-CD25 Abs to target effector T cell populations during an inflammatory episode, to determine to what extent that this action may modulate the outcome of disease. Anti-CD25 Ab-treated C57BL/6 mice displayed significantly reduced CD4(+) T cell IFN-gamma production during acute T. gondii infection and exhibited reduced weight loss and liver pathology during early acute infection; aspects of infection previously associated with effector CD4(+) T cell responses. In agreement, anti-CD25 Ab administration impaired parasite control and caused mice to succumb to infection during late acute/early chronic stages of infection with elevated tissue parasite burdens. In contrast, anti-CD25 Ab treatment of mice with established chronic infections did not markedly affect brain parasite burdens, suggesting that protective T cell populations do not express CD25 during chronic stages of T. gondii infection. In summary, we have demonstrated that anti-CD25 Abs may directly abrogate effector T cell responses during an inflammatory episode, highlighting important limitations of the use of anti-CD25 Ab administration to examine Treg function during inflammatory settings.

Publication Types:
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

PMID: 19299696 [PubMed - in process]

Thursday, March 19, 2009

Disruption of a mitochondrial MutS DNA repair enzyme homologue confers drug resistance

Mol Microbiol. 2009 Mar 6. [Epub ahead of print]

Disruption of a mitochondrial MutS DNA repair enzyme homologue confers drug resistance in the parasite Toxoplasma gondii

Garrison EM, Arrizabalaga G.

Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Life Sciences South Room 142, Moscow, ID 83844, USA.

MutS homologues (MSHs) are critical components of the eukaryotic mismatch repair machinery. In addition to repairing mismatched DNA, mismatch repair enzymes are known in higher eukaryotes to directly signal cell cycle arrest and apoptosis in response to DNA-damaging agents. Accordingly, mammalian cells lacking certain MSHs are resistant to chemotherapeutic drugs. Interestingly, we have discovered that the disruption of TgMSH-1, an MSH in the pathogenic parasite, Toxoplasma gondii, confers drug resistance. Through a genetic selection for T. gondii mutants resistant to the antiparasitic drug monensin, we have isolated a strain that is resistant not only to monensin but also to salinomycin and the alkylating agent, methylnitrosourea. We have shown that this phenotype is due to the disruption of TgMSH-1 as the multidrug-resistance phenotype is complemented by a wild-type copy of TgMSH-1 and is recapitulated by a directed disruption of this gene in a wild-type strain. We have also shown that, unlike previously described MSHs involved in signalling, TgMSH-1 localizes to the parasite mitochondrion. These results provide the first example of a mitochondrial MSH that is involved in drug sensitivity and implicate the induction of mitochondrial stress as a mode of action of the widely used drug, monensin.

PMID: 19291232 [PubMed - as supplied by publisher]

Tracking Transmission of the Zoonosis Toxoplasma gondii

Adv Parasitol. 2009;68:139-59

Chapter 6 Tracking Transmission of the Zoonosis Toxoplasma gondii

Smith JE.

Toxoplasma gondii is a highly successful parasite that infects many host species and has colonised a wide range of habitats. Review of the parasite's life cycle demonstrates that it has become adapted to exploit multiple routes of transmission through a sexual cycle in the definitive host and asexually, through carnivory, and by vertical transmission. These alternative routes may operate synergistically to enhance transmission, but they might also provide a vehicle for selection leading to partitioning of strains in the environment. Genetic analysis has shown that parasite population structure varies globally. In South America, there is high strain diversity while in North America, Europe and Africa three clonal strain types predominate. This may imply a shift from sexual to asexual transmission. Mapping of the parasite genome has provided a wealth of markers for strain characterisation. Close genotyping of isolates gives evidence of multiple infection and recombination in natural populations and reveals differences in both the distribution and the phenotype of strains. More intensive epidemiological studies are now required to unravel the networks of transmission operating within defined habitats.

PMID: 19289193 [PubMed - in process]

Destruction and Control of Toxoplasma gondii Tachyzoites Using Gold Nanosphere/Antibody Conjugates

Small. 2009 Mar 16. [Epub ahead of print]

Destruction and Control of Toxoplasma gondii Tachyzoites Using Gold Nanosphere/Antibody Conjugates

Pissuwan D, Valenzuela SM, Miller CM, Killingsworth MC, Cortie MB.

Institute for Nanoscale Technology University of Technology Sydney P.O. Box 123, Broadway, Sydney, NSW 2007 (Australia).

PMID: 19291731 [PubMed - as supplied by publisher]

Wednesday, March 18, 2009

Host cell entry by apicomplexa parasites requires actin polymerization in the host cell

Cell Host Microbe. 2009 Mar 19;5(3):259-72

Host cell entry by apicomplexa parasites requires actin polymerization in the host cell

Gonzalez V, Combe A, David V, Malmquist NA, Delorme V, Leroy C, Blazquez S, Ménard R, Tardieux I.

Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France; INSERM U567, Université Paris Descartes, CNRS (UMR 8104), 22 rue Méchain, 75014 Paris, France.

Apicomplexa are obligate intracellular parasites that actively invade host cells using their membrane-associated, actin-myosin motor. The current view is that host cell invasion by Apicomplexa requires the formation of a parasite-host cell junction, which has been termed the moving junction, but does not require the active participation of host actin. Using Toxoplasma gondii tachyzoites and Plasmodium berghei sporozoites, we show that host actin participates in parasite entry. Parasites induce the formation of a ring-shaped F-actin structure in the host cell at the parasite-cell junction, which remains stable during parasite entry. The Arp2/3 complex, an actin-nucleating factor, is recruited at the ring structure and is important for parasite entry. We propose that Apicomplexa invasion of host cells requires not only the parasite motor but also de novo polymerization of host actin at the entry site for anchoring the junction on which the parasite pulls to penetrate the host cell.

PMID: 19286135 [PubMed - in process]

Infection and stillbirth

Semin Fetal Neonatal Med. 2009 Mar 11. [Epub ahead of print]

Infection and stillbirth

McClure EM, Goldenberg RL.

Department of Epidemiology, UNC Global School of Public Health, Chapel Hill, North Carolina, USA.

Infection may cause stillbirth by several mechanisms, including direct infection, placental damage, and severe maternal illness. Various organisms have been associated with stillbirth, including many bacteria, viruses, and protozoa. In developed countries, between 10% and 25% of stillbirths may be caused by an infection, whereas in developing countries, which have much higher stillbirth rates, the contribution of infection is much greater. In developed countries, ascending bacterial infection, both before and after membrane rupture, with organisms such as Escherichia coli, group B streptococci, and Ureaplasma urealyticum is usually the most common infectious cause of stillbirth. However, in areas where syphilis is prevalent, up to half of all stillbirths may be caused by this infection alone. Malaria may be an important cause of stillbirth in women infected for the first time in pregnancy. The two most important viral causes of stillbirth are parvovirus and Coxsackie virus, although a number of other viral infections appear to be causal. Toxoplasma gondii, Listeria monocytogenes, and the organisms that cause leptospirosis, Q fever, and Lyme disease have all been implicated as etiologic for stillbirth. In certain developing countries, the stillbirth rate is high and the infection-related component so great that achieving a substantial reduction in stillbirth should be possible by reducing maternal infections. However, because infection-related stillbirth is uncommon in developed countries, and because those that do occur are caused by a wide variety of organisms, reducing this etiologic component of stillbirth much further will be difficult.

PMID: 19285457 [PubMed - as supplied by publisher]

The type II NADH dehydrogenase inhibitor HDQ leads to {Delta}{Psi}m collapse and ATP depletion in Toxoplasma

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

The type II NADH dehydrogenase inhibitor HDQ leads to {Delta}{Psi}m collapse and ATP depletion in Toxoplasma gondii

Lin SS, Gross U, Bohne W.

Institute of Medical Microbiology, University of Göttingen, Kreuzbergring 57, Göttingen D-37075; GERMANY.

The apicomplexan parasite Toxoplasma gondii expresses type II NADH dehydrogenases (NDH2s) instead of a canconial complex I at the inner mitochondrial membrane. These non-proton pumping enzymes are considered as promising drug targets, due to their absence in mammalian cells. We recently showed by inhibition kinetics that TgNDH2-I is a target of the quinolone-like compound HDQ, which inhibits T. gondii replication in the nanomolar range. In this study, the cationic fluorescent probes Mitotracker and DiOC6(3) were used to monitor the influence of HDQ on DeltaPsim in T. gondii. Real-time imaging revealed that nanomolar HDQ concentrations led to a DeltaPsim collapse within minutes, which is followed by a severe ATP depletion of 30% after 1 h and 70% after 24 h. The DeltaPsim depolarization was attenuated when substrates for other dehydrogenases which can donate electrons to ubiquinone were added to digitonin-permeabilized cells, or when infected cultures were treated with the F0-ATPase inhibitor oligomycin. A prolonged treatment with sublethal concentrations of HDQ induced differentiation into bradyzoites. This dormant stage is likely to be less dependent on DeltaPsim, since DeltaPsim-positive parasites were found at a significant lower frequency in alkaline pH induced bradyzoites than in tachyzoites. Together, our studies reveal that oxidative phosphorylation is essential for maintaining the ATP level in the fast growing tachyzoite stage and that HDQ interferes with this pathway by inhibiting the electron transport chain at the level of ubiquinone reduction.

PMID: 19286986 [PubMed - as supplied by publisher]

Evaluation of protective effect of multi-epitope DNA vaccine encoding six antigen segments of Toxoplasma gondii in mice

Parasitol Res. 2009 Mar 14. [Epub ahead of print]

Evaluation of protective effect of multi-epitope DNA vaccine encoding six antigen segments of Toxoplasma gondii in mice

Liu S, Shi L, Cheng YB, Fan GX, Ren HX, Yuan YK.

Department of Immunology and Microbiology, School of Medicine, Xi'an Jiaotong University, Xi'an, 710061, China.

To investigate the vaccine potential of multi-epitope vaccines against toxoplasmosis, a multi-epitope DNA vaccine, eukaryotic plasmid pcDNA3.1/T-ME expressing six antigen segments (SAG1(238-256), SAG1(281-320), GRA1(170-193), GRA4(331-345), GRA4(229-245), and GRA2(171-185)) of Toxoplasma gondii was constructed. We investigated the efficacy of pcDNA3.1/T-ME with or without co-administration of a CpG-oligodeoxynucleotide (CpG-ODN) as an adjuvant to protect mice (BALB/c and C57BL/6) against toxoplasmosis. High survival rates were observed in mice immunized with pcDNA3.1/T-ME when challenged with T. gondii RH strain. Lymphocyte proliferation assays, cytokine, and antibody determinations show that mice immunized with pcDNA3.1/T-ME produced stronger humoral and Th1-type cellular immune responses compared to untreated mice or those immunized with empty plasmids. However, co-immunization with CpG-ODN resulted in impaired immune responses. Our data demonstrates that multi-epitope DNA vaccination is a potential strategy for the control of toxoplasmosis and paves the way for further investigations into producing a multi-epitope anti-T. gondii DNA vaccine.

PMID: 19288132 [PubMed - as supplied by publisher]

Thursday, March 12, 2009

A unique dual activity amino acid hydroxylase in Toxoplasma gondii

PLoS ONE. 2009;4(3):e4801. Epub 2009 Mar 11

A unique dual activity amino acid hydroxylase in Toxoplasma gondii

Gaskell EA, Smith JE, Pinney JW, Westhead DR, McConkey GA.

Institute of Integrative and Comparative Biology, University of Leeds, Leeds, United Kingdom.

The genome of the protozoan parasite Toxoplasma gondii was found to contain two genes encoding tyrosine hydroxylase; that produces L-DOPA. The encoded enzymes metabolize phenylalanine as well as tyrosine with substrate preference for tyrosine. Thus the enzymes catabolize phenylalanine to tyrosine and tyrosine to L-DOPA. The catalytic domain descriptive of this class of enzymes is conserved with the parasite enzyme and exhibits similar kinetic properties to metazoan tyrosine hydroxylases, but contains a unique N-terminal extension with a signal sequence motif. One of the genes, TgAaaH1, is constitutively expressed while the other gene, TgAaaH2, is induced during formation of the bradyzoites of the cyst stages of the life cycle. This is the first description of an aromatic amino acid hydroxylase in an apicomplexan parasite. Extensive searching of apicomplexan genome sequences revealed an ortholog in Neospora caninum but not in Eimeria, Cryptosporidium, Theileria, or Plasmodium. Possible role(s) of these bi-functional enzymes during host infection are discussed.

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

PMID: 19277211 [PubMed - in process]

Wednesday, March 11, 2009

A hypothesis for the evolution of nuclear-encoded, plastid-targeted glyceraldehyde-3-phosphate dehydrogenase genes in "chromalveolate" members

PLoS ONE. 2009;4(3):e4737. Epub 2009 Mar 9

A hypothesis for the evolution of nuclear-encoded, plastid-targeted glyceraldehyde-3-phosphate dehydrogenase genes in "chromalveolate" members

Takishita K, Yamaguchi H, Maruyama T, Inagaki Y.

Japan Agency for Marine-Earth Science and Technology, Yokosuka, Kanagawa, Japan. takishitak@jamstec.go.jp

Eukaryotes bearing red alga-derived plastids--photosynthetic alveolates (dinoflagellates plus the apicomplexan Toxoplasma gondii plus the chromerid Chromera velia), photosynthetic stramenopiles, haptophytes, and cryptophytes--possess unique plastid-targeted glyceraldehyde-3-phosphate dehydrogenases (henceforth designated as "GapC1"). Pioneering phylogenetic studies have indicated a single origin of the GapC1 enzymes in eukaryotic evolution, but there are two potential idiosyncrasies in the GapC1 phylogeny: Firstly, the GapC1 tree topology is apparently inconsistent with the organismal relationship among the "GapC1-containing" groups. Secondly, four stramenopile GapC1 homologues are consistently paraphyletic in previously published studies, although these organisms have been widely accepted as monophyletic. For a closer examination of the above issues, in this study GapC1 gene sampling was improved by determining/identifying nine stramenopile and two cryptophyte genes. Phylogenetic analyses of our GapC1 dataset, which is particularly rich in the stramenopile homologues, prompt us to propose a new scenario that assumes multiple, lateral GapC1 gene transfer events to explain the incongruity between the GapC1 phylogeny and the organismal relationships amongst the "GapC1-containing" groups. Under our new scenario, GapC1 genes uniquely found in photosynthetic alveolates, photosynthetic stramenopiles, haptophytes, and cryptopyhytes are not necessarily a character vertically inherited from a common ancestor.

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

PMID: 19270733 [PubMed - in process]

Molecular diagnosis of Toxoplasma gondii infection in cerebrospinal fluid from AIDS patients

Cerebrospinal Fluid Res. 2009 Mar 6;6(1):2. [Epub ahead of print]

Molecular diagnosis of Toxoplasma gondii infection in cerebrospinal fluid from AIDS patients

Alfonso Y, Fraga J, Fonseca C, Jimenez N, Pinillos T, Dorta-Contreras AJ, Cox R, Capo V, Pomier O, Bandera F, Ginorio D.

ABSTRACT: BACKGROUND: Toxoplasmic encephalitis (TE) is one of the most common opportunistic infections in immunocompromised patients. In Cuba, despite the highly active antiretroviral therapy, TE is still the most important cause of cerebral mass lesions in patients infected with the human immunodeficiency virus (HIV). The detection of Toxoplasma gondii by PCR may facilitate the diagnosis and follow-up of TE in acquired immunodeficiency syndrome (AIDS) patients by direct identification of parasite DNA in clinical samples. The aim of the present study was to evaluate a rapid PCR method using the B1 gene to detect T. gondii in cerebrospinal fluid (CSF) samples from patients with suspected TE. METHODS: CSF samples from AIDS and HIV-negative patients were analyzed. Patients were divided into two groups according to the Centre for Disease Control and Prevention (CDC) criteria for AIDS-related TE: AIDS patients with suspected neurotoxoplasmosis and AIDS and HIV-negative patients with other confirmed neurological diseases but no suspicions of TE. Predictive values, diagnostic accuracy, sensitivity and specificity of the PCR B1 method were calculated. RESULTS: The results obtained from 190 patients showed that this assay has a good sensitivity and specificity (83.3% and 95.7%, respectively) for the diagnosis of TE in AIDS patients. CONCLUSIONS: PCR using the B1 gene and B22/B23 set of primers is a single, rapid and reliable method that may be valuable for discrimination between toxoplasmosis and other central nervous system (CNS) diseases.

PMID: 19267913 [PubMed - as supplied by publisher]

Monday, March 09, 2009

Selection at a Single Locus Leads to Widespread Expansion of Toxoplasma gondii Lineages That Are Virulent in Mice

PLoS Genet. 2009 Mar;5(3):e1000404. Epub 2009 Mar 6

Selection at a Single Locus Leads to Widespread Expansion of Toxoplasma gondii Lineages That Are Virulent in Mice

Khan A, Taylor S, Ajioka JW, Rosenthal BM, Sibley LD.

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

Pathogenicity differences among laboratory isolates of the dominant clonal North American and European lineages of Toxoplasma gondii are largely controlled by polymorphisms and expression differences in rhoptry secretory proteins (ROPs). However, the extent to which such differences control virulence in natural isolates of T. gondii, including those from more diverse genetic backgrounds, is uncertain. We elucidated the evolutionary history and functional consequences of diversification in the serine/threonine kinase ROP18, a major virulence determinant in the mouse model. We characterized the extent of sequence polymorphism and the evolutionary forces acting on ROP18 and several antigen-encoding genes within a large collection of natural isolates, comparing them to housekeeping genes and introns. Surprisingly, despite substantial genetic diversity between lineages, we identified just three principal alleles of ROP18, which had very ancient ancestry compared to other sampled loci. Expression and allelic differences between these three alleles of ROP18 accounted for much of the variation in acute mouse virulence among natural isolates. While the avirulent type III allele was the most ancient, intermediate virulent (type II) and highly virulent (type I) lineages predominated and showed evidence of strong selective pressure. Out-group comparison indicated that historical loss of an upstream regulatory element increased ROP18 expression, exposing it to newfound diversifying selection, resulting in greatly enhanced virulence in the mouse model and expansion of new lineages. Population sweeps are evident in many genomes, yet their causes and evolutionary histories are rarely known. Our results establish that up-regulation of expression and selection at ROP18 in T. gondii has resulted in three distinct alleles with widely different levels of acute virulence in the mouse model. Preservation of all three alleles in the wild indicates they are likely adaptations for different niches. Our findings demonstrate that sweeping changes in population structure can result from alterations in a single gene.

PMID: 19266027 [PubMed - in process]

Genotype of 88 Toxoplasma gondii Isolates Associated with Toxoplasmosis in Immunocompromised Patients and Correlation with Clinical Findings

J Infect Dis. 2009 Mar 5. [Epub ahead of print]

Genotype of 88 Toxoplasma gondii Isolates Associated with Toxoplasmosis in Immunocompromised Patients and Correlation with Clinical Findings

Ajzenberg D, Yera H, Marty P, Paris L, Dalle F, Menotti J, Aubert D, Franck J, Bessières MH, Quinio D, Pelloux H, Delhaes L, Desbois N, Thulliez P, Robert-Gangneux F, Kauffmann-Lacroix C, Pujol S, Rabodonirina M, Bougnoux ME, Cuisenier B, Duhamel C, Duong TH, Filisetti D, Flori P, Gay-Andrieu F, Pratlong F, Nevez G, Totet A, Carme B, Bonnabau H, Dardé ML, Villena I.

Centre Hospitalier Universitaire, Amiens, 2Centre Hospitalier Universitaire, Bordeaux, 3Centre Hospitalier Universitaire, Brest, 4Centre Hospitalier Universitaire, Caen, 5Centre Hospitalier Universitaire, Dijon, 6Centre Hospitalier Universitaire, Grenoble, 7Centre Hospitalier Universitaire, Lille, 8Laboratoire de Parasitologie-Mycologie, EA 3174, Faculté de Médecine, Université de Limoges, and 9Centre National de Référence Toxoplasmose, Centre Hospitalier Universitaire, and 10Unité Fonctionnelle de Recherche Clinique et Biostatistique UFRCB, Centre Hospitalier Universitaire, Limoges, Université de Limoges, Limoges, 11Hospices civils de Lyon, Hôpital de la Croix-Rousse, Lyon, 12Centre Hospitalier Universitaire, Marseille, 13Centre Hospitalier Universitaire, Montpellier, 14Centre Hospitalier Universitaire, Nantes, 15Centre Hospitalier Universitaire, Nice, 16Institut de Puériculture, 17Hôpital Cochin and 18Hôpital Necker-Enfants Malades, 19Hôpital Pitié-Salpêtrière, and 20Hôpital Saint-Louis, Assistance Publique-Hôpitaux de Paris, Paris, 21Centre Hospitalier Universitaire, Poitiers, 22Centre Hospitalier Universitaire, Reims, 23Centre Hospitalier Universitaire, Rennes, 24Centre Hospitalier Universitaire, Saint-Etienne, 25Hôpitaux Universitaires de Strasbourg, Strasbourg, 26Centre Hospitalier Universitaire, Toulouse, 27Centre Hospitalier Universitaire, Tours, France; 28Centre Hospitalier Général, Cayenne, French Guiana; 29Centre Hospitalier Universitaire, Fort de France, Martinique.

We report the genotyping analysis of Toxoplasma gondii isolates in samples collected from 88 immunocompromised patients, along with clinical and epidemiological data. Most of these samples were collected in France during the current decade by the Toxoplasma Biological Resource Center. Lack of specific anti-Toxoplasma treatment, pulmonary toxoplasmosis, and involvement of multiple organs were the 3 main risk factors associated with death for this patient group. Genotyping results with 6 microsatellite markers showed that type II isolates were predominant among patients who acquired toxoplasmic infection in Europe. Non-type II isolates included 13 different genotypes and were mainly collected from patients who acquired toxoplasmosis outside Europe. Type III was the second most common genotype recovered from patients, whereas type I was rare in our population. Three nonarchetypal genotypes were repeatedly recovered from different patients who acquired the infection in sub-Saharan Africa (genotypes Africa 1 and Africa 2) and in the French West Indies (genotype Caribbean 1). The distribution of genotypes (type II vs. non-type II) was not significantly different when patients were stratified by underlying cause of immunosuppression, site of infection, or outcome. We conclude that in immunocompromised patients, host factors are much more involved than parasite factors in patients' resistance or susceptibility to toxoplasmosis.

PMID: 19265484 [PubMed - as supplied by publisher]

Virulent Toxoplasma gondii Evade Immunity-Related GTPase-Mediated Parasite Vacuole Disruption within Primed Macrophages

J Immunol. 2009 Mar 15;182(6):3775-81

Virulent Toxoplasma gondii Evade Immunity-Related GTPase-Mediated Parasite Vacuole Disruption within Primed Macrophages

Zhao Y, Ferguson DJ, Wilson DC, Howard JC, Sibley LD, Yap GS.

Department of Medicine and Center for Immunity and Inflammation, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, NJ 07101.

Cytokine-activated macrophages restrain the replication of intracellular parasites and disrupt the integrity of vacuolar pathogens. In this study, we show that inducible nitric oxide synthase and the immunity-related GTPase (IRG) family member Irgm3, respectively, are required for the ability of in vivo primed macrophages to restrain the growth of Toxoplasma gondii and to destroy the parasite's intracellular niche. Remarkably, virulent Type I strains of T. gondii evade IRG-dependent vacuolar disruption, while remaining susceptible to iNOS-dependent restriction. The ability of virulent T. gondii to escape killing by macrophages is controlled at the level of the individual vacuole and is associated with differential permissiveness for association of the IRG proteins Irga6 (IIGP1) and Irgb6 (TGTP) to the vacuolar membrane. Surprisingly, expression of the Type I ROP-18 virulence determinant in an avirulent strain did not confer the evasive phenotype. These results pinpoint evasion of vacuolar disruption by IRG proteins as a new determinant of pathogen virulence.

PMID: 19265156 [PubMed - in process]

Friday, March 06, 2009

A4D12 monoclonal antibody recognizes a new linear epitope from SAG2A Toxoplasma gondii tachyzoites, identified by phage display bioselection

Immunobiology. 2009 Mar 2. [Epub ahead of print]

A4D12 monoclonal antibody recognizes a new linear epitope from SAG2A Toxoplasma gondii tachyzoites, identified by phage display bioselection

Cunha-Júnior JP, Silva DA, Silva NM, Souza MA, Souza GR, Prudencio CR, Pirovani CP, Cezar M Cascardo J, Barbosa BF, Goulart LR, Mineo JR.

Laboratory of Immunoparasitology, Institute of Biomedical Sciences, Federal University of Uberlândia, Brazil.

Toxoplasma gondii surface is coated by closely related antigens that belong to SRS (SAG-1 related sequences) superfamily. Two tachyzoite-specific SRS antigens, SAG1 and SAG2, are immunodominant proteins that apparently modulate the virulence of infection by inducing the host immune response against tachyzoites during the acute phase. In this study, we described a conformationally insensitive monoclonal antibody (A4D12mAb) that recognizes a linear epitope shared by two isoforms of p22 that is expressed in the surface of T. gondii tachyzoites. By using phage display approach and production of recombinant proteins, we clearly demonstrated that the A4D12mAb recognizes an epitope within C-terminal region of SAG2A. This mAb reacts with both T. gondii genotypes (I and II) but not with a closely related parasite, Neospora caninum. Also, the pretreatment of tachyzoites with A4D12 mAb did not inhibit T. gondii infection, suggesting that the epitope herein mapped is not crucial for tachyzoite invasion. However, a panel of human T. gondii positive sera showed significant degree of inhibition of A4D12 mAb reactivity against T. gondii native antigens, indicating that both A4D12 mAb and human sera recognize an overlapping immunodominant epitope within C-terminal region of SAG2A. To our knowledge, this is the first evidence using bioselection by phage display that identifies a T. gondii linear epitope recognized by a mAb specific to SAG2A. In conclusion, the results here presented add a new piece of information concerning T. gondii SAG2A molecule, emphasizing two dissimilar biological roles of this molecule, particularly for A4D12 epitope, suggesting that these characteristics may be important for parasite survival, since it is part of parasite components able to induce a strong immune response enough to allow host survival and establish long-term chronic infection.

PMID: 19261354 [PubMed - as supplied by publisher]

Thursday, March 05, 2009

Development of forward genetics in Toxoplasma gondii

Int J Parasitol. 2009 Feb 27. [Epub ahead of print]

Development of forward genetics in Toxoplasma gondii

Sibley LD.

Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Ave. St. Louis MO 63110 USA.

The development of forward genetics as a functional system in Toxoplasma gondii spanned more than three decades from the mid-1970s until now. The initial demonstration of experimental genetics relied on chemically-induced drug resistant mutants that were crossed by co-infecting cats, collecting oocysts, sporulating and hatching progeny in vitro. To capitalize on this, genetic markers were employed to develop linkage maps by tracking inheritance through experimental crosses. In all, three generations of genetic maps were developed to define the chromosomes, estimate recombination rates, and provide a system for linkage analysis. Ultimately this genetic map would become the foundation for the assembly of the T. gondii genome, which was derived from whole genome shotgun sequencing, into a chromosome-centric view. Finally, application of forward genetics to multigenic biological traits showed the potential to map and identify specific genes that control complex phenotypes including virulence.

PMID: 19254720 [PubMed - as supplied by publisher]

AZITHROMYCIN REDUCES OCULAR INFECTION DURING CONGENITAL TRANSMISSION OF TOXOPLASMOSIS IN THE Calomys Callosus MODEL

J Parasitol. 2009 Mar 2:1. [Epub ahead of print]

AZITHROMYCIN REDUCES OCULAR INFECTION DURING CONGENITAL TRANSMISSION OF TOXOPLASMOSIS IN THE Calomys Callosus MODEL

Lopes CD, Silva NM, Ferro EA, Sousa RA, Firmino ML, Bernardes ES, Roque-Barreira MC, Pena JD.

Toxoplasma gondii is a widely distributed obligatory intracellular parasite that causes severe disease to the fetus when transmitted during pregnancy. Drugs used to avoid congenital transmission have shown side effects and their efficacy is controversial. The most widely used drug for the treatment of acute toxoplasmosis during pregnancy is the association between pyrimethamine and sulfadiazine, which has several side effects. In this work we tested the efficacy of azithromycin in reducing congenital transmission of Toxoplasma in the rodent Calomys callosus. Females of C. callosus were inoculated perorally with 20 cysts of ME49 strain of T. gondii on the day of fertilization and fetuses were collected from the 15th to the 19th day of gestation. Azithromycin (300mg/kg) or association with pyrimethamine (100 or 50 mg/Kg) and sulfadiazine (100 or 75mg/kg) and folinic acid (15mg/kg) (SPAf) or vehicle was administered orally in different days after infection. Brain and ocular tissues were removed and processed for immunohistochemistry using a polyclonal antibody against T. gondii, or processed for parasite DNA quantification. Toxoplasma gondii was detected in the brains of all females and fetuses' eyes when treated with SPAf. On the other hand, in females treated with azithromycin, there was a reduction of T. gondii in the brains of mothers and no parasites were detected in eyes of fetuses, indicating that azithromycin may represent an alternative treatment for toxoplasmosis during pregnancy.

PMID: 19254072 [PubMed - as supplied by publisher]

Tuesday, March 03, 2009

The role played by electron microscopy in advancing our understanding of Toxoplasma gondii and other apicomplexans

Int J Parasitol. 2009 Feb 25. [Epub ahead of print]

The role played by electron microscopy in advancing our understanding of Toxoplasma gondii and other apicomplexans

Dubremetz JF, Ferguson DJ.

UMR CNRS 5235, Bt 24, CC 107, Université de Montpellier 2, Place Eugène Bataillon, 34095 Montpellier cedex 05 France.

In many ways the history of the discovery of the life cycle of Toxoplasma gondii and the development of biological electron microscopy progressed in parallel through the 1950s and 1960s. Although Toxoplasma was discovered in 1908, it was only in the 1950s that the extent of the infection in humans and domestic animals was realised and work was undertaken to elucidate its life cycle (reviewed elsewhere in this edition). The development of ultrastructural techniques and their application to biological systems including Toxoplasma developed over the same period. This resulted in a synergistic effect with the re-classification of previously unrelated parasites within a single phylum, the Apicomplexa, which was based on the ultrastructural appearances of the infectious stages. This review will describe the central role played by electron microscopy and Toxoplasma in the developments associated with this progress.

PMID: 19249305 [PubMed - as supplied by publisher]

The early years of Toxoplasma research: What's past is prologue

Int J Parasitol. 2009 Feb 26. [Epub ahead of print]

The early years of Toxoplasma research: What's past is prologue

Morrissette NS, Ajioka JW.

Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine CA, 92697 USA.

In the century since the first description of Toxoplasma gondii, history and circumstance have led scientists to define this organism in diverse contexts. From its discovery by researchers shaped by early 20th century versions of the germ theory to its more recent roles as an important globally distributed pathogen and a model apicomplexan, our definitions of Toxoplasma are as much a reflection of our frame of reference as they are an absolute definition of this organism. Although these transformations act as portals for new avenues of investigation, the essential questions that inform current research are founded in the work of early investigators who studied Toxoplasma.

PMID: 19250939 [PubMed - as supplied by publisher]